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WO2024259353A2 - Methods of generating esophagus basal cells from pluripotent stem cells - Google Patents

Methods of generating esophagus basal cells from pluripotent stem cells Download PDF

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WO2024259353A2
WO2024259353A2 PCT/US2024/034174 US2024034174W WO2024259353A2 WO 2024259353 A2 WO2024259353 A2 WO 2024259353A2 US 2024034174 W US2024034174 W US 2024034174W WO 2024259353 A2 WO2024259353 A2 WO 2024259353A2
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cells
cell
concentration
population
basal
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WO2024259353A3 (en
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Anthony E. Oro
Ying Yang
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • A61K35/37—Digestive system
    • A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
    • A—HUMAN NECESSITIES
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the human esophagus is an endodermal-derived stratified squamous epithelium which serves as a protective barrier from abrasive food friction.
  • the esophageal epithelium is established and maintained by basal cells (BCs), which are adult stem cells named for their proximity to the basement membrane. Underneath the epithelium lies the local mesenchyme, which emanates inductive signals guiding the specification of BCs from columnar progenitors, and wiring the downstream BC differentiation programs.
  • BCs basal cells
  • Such a tissue architecture of esophagus resembles other stratified epithelial like skin, despite them being derived from different germ layers.
  • eBCs are capable of regenerating the entire esophageal epithelium.
  • the disclosure provides a versatile method to accelerate cell replacement therapy for lineage-specific stratified epithelial genetic defects and wounds.
  • the derived esophogeal basal cells are useful for transplantation and tissue regeneration for various purposes, including without limitation rare genetic diseases like recessive or dominant esophagitis, dystrophic epidermolysis bullosa; esophageal injury; cancer; etc.
  • the in vitro differentiation of eBC occurs through sequential stages. At each stage, specific factors are provided, and in some cases, specific signaling pathways are activated or inhibited, to drive differentiation and achieve purified populations of the desired cells at each stage.
  • a method of producing a population of definitive esophogeal basal cells (eBC) from hPSCs is provided.
  • the method provides a means of producing a substantially pure population of eBC in defined culture conditions, in media comprising extracellular signaling agents to guide differentiation, the method comprising: (a) differentiating human pluripotent stem cells into definitive endoderm cells; (b) differentiating definitive endoderm cells into anterior foregut endoderm cells; (c) differentiating anterior foregut endoderm cells into dorsal anterior foregut endoderm cells; (d) differentiating dorsal anterior foregut endoderm cells into early eBC; (e) differentiating early eBC into definitive eBC.
  • the desired eBC cell population is optionally purified for cells of interest, and may be maintained on organoid culture.
  • the eBC are expandable and are capable of self-renewal.
  • greater than 50%, greater than 75%, greater than 80% or more of the final cell definitive eBC population express one, two, three or more of the eBC markers FOXA1, FOXA2, SOX2, SOX9 and P63 with ITGB4, EPCAM, Krt5, and Krt15.
  • the cells express EPCAM and ITG4.
  • the level of purity of a particular purified population will vary depending on various factors and may be achieved through use of the cell derivation methods described herein including or excluding the use of one or more binding agents used to isolate particular cell types.
  • methods are provided for the use of eBCs in screening for cellular responses.
  • methods are provided for treating a subject for a condition requiring esophogeal cells by administration of eBCs or cells derived therefrom, produced by the methods of the disclosure.
  • systems and kits for producing eBC types and/or screening for cellular responses and/or treating subjects with such eBC are provided.
  • a method of screening a substantially pure population of eBC for a cellular response may include contacting a population of substantially pure population of eBC with a pharmacological agent and evaluating the population of cells for a cellular response induced by the pharmacological agent.
  • the screening may be in vitro screening and the contacting may be performed in vitro.
  • the screening may be in vivo screening and the contacting may be performed by administering the pharmacological agent to a host animal that contains the population of cells.
  • aspects of the disclosure relate to methods of treating a subject for a condition through the administration of a population of eBC derived or produced according to the methods described herein.
  • the method of treating a subject for a condition through administration of cells derived according to the methods as described herein may further include co-administration with at least one pro-survival or pro-engraftment factor.
  • the cells administered to a subject are genetically modified in at least one genetic locus.
  • kits for the production, derivation, purification, and use of a derived population of eBC that include one or more induction compositions and/or one or more specific binding agents and/or combinations thereof.
  • kits may or may not include one or more cell types described herein.
  • Aspects of the disclosure include systems for the production, derivation, purification, and use of a substantially pure population of eBC that include one or more components configured to administer one or more induction compositions and/or one or more specific inducing agents and/or one or more specific binding agents and/or combinations thereof.
  • such systems are configured to administer such compositions and/or agents at specific amounts or for specific periods of time according to the methods described herein.
  • methods are provided for the use of machine learning to develop methods of differentiating stem or progenitor cells to a cell type of interest.
  • Such methods may utilize single cell and spatial technologies to generate a spatiotemporal multi-omics cell atlas for development of the cell or tissue of interest.
  • an existing cell atlas such as that disclosed herein may be used.
  • a machine learning algorithm is used to prioritize the combinations of candidate developmental signals for in silico method of differentiating a cell type of interest. Functional validation of the predicted developmental signals can be performed.
  • FIGS. 1A-1B A multi-omics cell atlas of human esophageal development.
  • A Workflow schematic: first integration of scRNA-seq, Visium spatial transcriptomics and CODEX multiplexed immunofluorescent cell imaging to build the multi-omics cell atlas to depict human esophageal development, followed by investigation into intercellular signal dynamics to nominate basal cell specification signals. Then the deep-learning algorithm Manatee predicts the optimal signal combination to drive basal cell specification, which is further used to build the hPSC-based esophageal mucosa manufacturing platform.
  • FIGS.2A-2F The cellular heterogeneity and lineage trajectory of human esophageal epithelium during development.
  • A Epithelial single cells were projected on the UMAP space, and color-coded based on cell type. Inset showing UMAP visualization by developmental timepoint.
  • B Proportions of the identified cell types at each timepoint.
  • C Dot plot showing the expression levels for selected markers of epithelial progenitors (Epi_PG), basal cells (BC), immature ciliated cells (Cil-1) and mature ciliated cells (Cil-2) for CODEX image representation.
  • D-E CODEX images confirming the presence of Epi_PG, BC (D), Cil-1, Cil- 2 (E) using selected markers.
  • Early Representative images from E45 (Early), E120 (Late) and E72 (Mid) samples. Scale bars: 10 ⁇ m.
  • F Monocle2 trajectory of the whole epithelial compartment, color-coded based on cell type. Insets: projection of epithelial cells collected at representative stages on the Monocle2 trajectory.
  • FIGS. 3A-3H Esophageal stromal diversity and mesenchymal lineages.
  • A Stromal single cells were projected on the UMAP space, and color-coded based on cell types/states. Inset showing UMAP visualization by cellular compartment (top right) and developmental timepoint (bottom right).
  • B Proportions of the identified cell types at each timepoint.
  • C Dot plot showing the expression levels for selected markers of different fibroblasts, myofibroblasts (MF) and smooth muscles (SM) shown in CODEX images.
  • MF myofibroblasts
  • SM smooth muscles
  • FIGS. 4A-4C Spatiotemporal cellular dynamics of human esophageal development.
  • A Representative CODEX images with selected markers showing the epithelium and the local mesenchyme at E45 (Early), E72 (Mid) and E120 (Late). Scale bars: 100 ⁇ m. Bottom panel showing enlarged boxed regions with representative cells annotated. Scale bar: 10 ⁇ m.
  • FIGS.5A-5F Spatiotemporal candidate signal nomination.
  • A Schematic: determining local mesenchymal cellular components at different developing stages first, followed by identifying potential inductive signals from the local mesenchyme that could be received by Epi_PG/BC using CellChat.
  • C- D Heatmaps showing the CellChat predicted incoming signal strength received by Epi_PG/BC (C) and sender-receiver interaction scores (D).
  • FIGS.6A-6C Manatee screens for the optimal signal combinations to promote basal cell derivation.
  • A The Manatee workflow. The Manatee model was adapted from VAE by constraining its latent space to represent TF expression. To predict perturbation-induced expression profiles, latent variables associated with candidate TFs will be adjusted.
  • FIGS.7A-7I Establishment of esophageal mucosa manufacturing platform using the Manatee-predicted strategies.
  • A Schematic illustration of the hPSC-to-esophageal basal cell differentiation protocol. DE, definitive endoderm;
  • AFE (dorsal) anterior foregut endoderm;
  • EPC esophageal progenitor.
  • EPCs were further subjected to a 5-day perturbation.
  • C IF staining of cells after the 5-day perturbation with canonical basal cell marker KRT5.
  • EPCAM+ITGB4+ double positive candidate basal cells DP
  • EPCAM+ITGB4- single positive cells SP were sorted and expanded on collagen peptide coated plates.
  • DP cells can be cryopreserved during expansion. After 3 passages, DP and SP groups maintained 75.8% and 19.1% candidate basal cells, respectively.
  • G Immunofluorescent staining of Calcium-induced stratification assay. Upper panel showed KRT5 + KRT15 + basal cells before confluency with KRT18 absence. Lower panel showed that upon Calcium treatment, basal cells underwent esophageal squamous differentiation, marked by KRT13+KRT4+ cells with large and flat morphology.
  • FIGS.8A-8M scRNA-seq data quality control and filtering, related to FIG.s 1, 2 and 3.
  • A Summary of dataset collection for multiomic atlas of human esophageal development.
  • B Quality control of scRNA-seq datasets. For each dataset, sample-wise distributions of number of cells/spots, number of UMIs per cell and number of genes per cell were reported.
  • C Iterative clustering of epithelium and stromal scRNA-seq profiles generated using TrpLE and Collagenase, respectively.
  • FIGS. 9A-9N Cellular heterogeneity and lineage analysis of epithelium and mesenchyme, related to FIG.s 2 and 3.
  • A-B Feature plots showing the expression of selected markers depicting transition from epithelial progenitors to basal cells (A), and from SB-5 to ciliated cells (B).
  • C 2 dimensional UMAP visualization of mesenchymal cells color-coded based on cell type/state, split by developmental timepoint.
  • D, F Feature plots showing the expression of selected markers distinguishing smooth muscles from myofibroblasts (D), pericytes from fibroblasts (F).
  • E, G Monocle2 trajectory of the smooth muscle cells (SM, E) and pericytes (PERI, G), color-coded based on cell type/state.
  • H-I 3 dimensional UMAP visualization of enteric nervous system (ENS) cells color-coded based on cell type/state (H) and developmental timepoint (I).
  • J Proportions of the identified cell types at each timepoint.
  • K Dot plot showing the expression of differential marker genes in the enteric nervous system.
  • L-FM Representative CODEX images showing the presence of ENS_PG at E45 (Early, L) and Neu and Glia at E120 (Late, M). Scale bars: 10 ⁇ m.
  • N Monocle2 trajectory of the whole enteric nervous system, color-coded based on cell type.
  • FIGS 10A-10O Cellular heterogeneity of endothelial, skeletal muscle and immune compartments, related to FIG. 3.
  • A-B 3 dimensional UMAP visualization of endothelial (ENDO) cells color-coded based on cell type/state (A) and developmental timepoint (B).
  • C Proportions of the identified cell types at each timepoint.
  • D Dot plot showing the expression of differential marker genes in endothelial compartment.
  • E Dot plot showing selected markers of arterial (Art), venous (Ven), lymphatic cells (Lym), pericytes (Peri) and immune cells (Im) shown in CODEX images.
  • F-G Representative CODEX images showing the presence of lymphatic, arterial, venous cells, pericytes (F) and immune cells (G) at E120 (Late). Scale bars: 10 ⁇ m.
  • FIGS.11A-11H Integrated analysis of single cell and Visium spatial transcriptomics, related to FIG.4.
  • FIGS. 12A-12D CODEX antibody panel and whole-mount tissue staining, related to FIGS. 2-4.
  • A Dot plot showing RNA expression of CODEX marker genes in different cell types.
  • B Dendrogram workflow for cell type annotation using selected markers.
  • C Violin plots of PITX1, ⁇ SMA and POSTN expression in fibroblast 1-5, interstitial cells of Cajal, myofibroblast and smooth muscle, color-coded based on cell types.
  • D 3D reconstruction of whole-mount immunostaining of E87 human fetal esophageal tissue. Note POSTN expression in subepithelial region and adventitia. Co-expression of PITX1 and POSTN marked Fib_1 lining the basement membrane, while lack of PITX1 in adventitia suggested distribution of Fib_4 and Fib_5 there.
  • FIGS. 13A-13D Human developmental signal combination screening by Manatee, related to FIG. 6.
  • A Quality control plot of in vitro single cell collections.
  • B-C UMAP visualization of in vitro single cell collections color-coded based on cell type (B) and stage (C).
  • D Original single cell expression profiles and predictions yielded by all 81 possible strategies were visualized on the two-dimensional UMAP space. Cells were color-coded by their types, and prediction effects were noted by arrows.
  • FIGS. 13A-13D Human developmental signal combination screening by Manatee, related to FIG. 6.
  • B-C UMAP visualization of in vitro single cell collections color-coded based on cell type (B) and stage (C).
  • D Original single cell expression profiles and predictions yielded by all 81 possible strategies were visualized on the two-dimensional UMAP space. Cells were color-coded by their types, and prediction effects were noted by arrows.
  • C Representative flow cytometry quantification of differentiation efficiency, showing high efficiency in deriving definitive endoderm on D4 and esophageal progenitors on D16, reduced progenitor efficiency on D24, and low efficiency in EPCAM+ ITGB4+ esophageal basal cells on D45.
  • D IF of human esophageal transcription factors on D16 cells.
  • E IF of BC markers and squamous markers on D24, D35, and D45 cells. Note that BC keratins are gradually acquired over the time course.
  • F-H Statistical quantification of the flow cytometry analyses, to evaluate the essentiality of each candidate pathway (F), sufficiency (G), and different combinations with EGF (H).
  • FIG. S7 showed representative results generated using H9 embryonic stem cell line. Both H9 and RUES2 lines were used, and consistent results were obtained in more than 3 independent runs for each line.
  • a cell includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.
  • the publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. None herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication date, which may need to be independently confirmed. Definitions [0040]
  • the terms “treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired therapeutic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting development of a disease and/or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and/or symptom(s).
  • the terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • "Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human.
  • pluripotent progenitor cells refer to cells that are capable of differentiating into two or more different cell types and proliferating.
  • pluripotent precursor cells include but are not limited to embryonic stem cells, blastocyst derived stem cells, fetal stem cells, induced pluripotent stem cells.
  • Pluripotent progenitor cells may be acquired from public or commercial sources or may be newly derived. As described herein, in some instances, pluripotent progenitor cells of the subject disclosure are those cells capable of giving rise to eBC.
  • the term “lineage bifurcation” and “lineage segregation” are used interchangeably herein and refer to a cell-fate decision where a stem cell and/or progenitor cell has the ability to differentiate into two or more cell-types.
  • the term “population”, e.g., “cell population” or “population of cells”, as used herein means a grouping (i.e., a population) of two or more cells that are separated (i.e., isolated) from other cells and/or cell groupings.
  • a 6-well culture dish can contain 6 cell populations, each population residing in an individual well.
  • the cells of a cell population can be, but need not be, clonal derivatives of one another.
  • a cell population can be derived from one individual cell. For example, if individual cells are each placed in a single well of a 6-well culture dish and each cell divides one time, then the dish will contain 6 cell populations.
  • the cells of a cell population can be, but need not be, derived from more than one cell, i.e. non- clonal.
  • the cells from which a non-clonal cell population may be derived may be related or unrelated and include but are not limited to, e.g., cells of a particular tissue, cells of a particular sample, cells of a particular lineage, cells having a particular morphological, physical, behavioral, or other characteristic, etc.
  • a cell population can be any desired size and contain any number of cells greater than one cell.
  • a cell population can be 2 or more, 10 or more, 100 or more, 1,000 or more, 5,000 or more, 10 4 or more, 10 5 or more, 10 6 or more, 10 7 or more, 10 8 or more, 10 9 or more, 10 10 or more, 10 11 or more, 10 12 or more, 10 13 or more, 10 14 or more, 10 15 or more, 10 16 or more, 10 17 or more, 10 18 or more, 10 19 or more, or 10 20 or more cells.
  • significant amount in this context, is meant an amount of undesired or contaminating cell types that negatively impacts the use of the isolated desired cell population.
  • the actual amount of undesired or contaminating cells that defines a significant amount will vary and depend on the particular type of undesired or contaminating cells and/or the particular use of the desired cell type. For example, in a population of differentiated cells used in the treatment of a subject, a significant amount of improperly differentiated contaminating cell types will be small as such cells may a high capacity to negatively impact the use of the generated desired cell population.
  • a significant amount of contaminating progenitor cells may be relatively large as such cells may have a low capacity to negatively impact the use of the generated desired cell population.
  • a homogenous population may refer to a highly enriched population.
  • heterologous as it refers to a “heterologous sequence” or “heterologous nucleic acid”, means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.
  • the term “high cell density”, as it relates to cells, means the cell numbers within an area or volume is high. For example, cells are in close contact with one another when cultured in high cell density. In some embodiments high cell density refers to a density of at least about 1.25x10 6 cells/cm 2 .
  • Methods and compositions are provided for producing a population of human esophogeal basal cells (eBC) in defined monolayer conditions in media comprising extracellular signaling agents that guide differentiation. Aspects of the disclosure include methods for deriving eBC from pluripotent progenitor cells.
  • Pluripotent stem cells may be acquired from any convenient source, including but not limited to newly derived from a subject of interest or tissue specimen or other cellular sample, obtained from a public repository, obtained from a commercial vendor, and the like.
  • pluripotent cells of interest include human cells including but not limited to, e.g., human embryonic stem cells, human induced pluripotent stem cells, human fetal stem cells, and the like.
  • pluripotent stem cells are unmodified, such that the cells have not been genetically or otherwise modified from their natural state, prior to modification according the methods described herein. In other instances, pluripotent stem cells may be unmodified, such that the cells have been genetically or otherwise modified from their natural state, prior to modification according the methods described herein.
  • Generation of eBC from pluripotent progenitors as described herein involves one or more lineage restriction events, in which cultured pluripotent stem cells are subjected to one or more treatments causing the cultured cells or a population thereof to differentiate along specific pathways. Multiple lineage restriction events are required to achieve desired eBC.
  • lineage restriction events are performed successively such that a first cell type is achieved by a first lineage restriction event and the first cell type is subjected to a second lineage restriction event to achieve a desired second cell type; etc.
  • Lineage restriction events as described herein are induced by compositions of extracellular agents that act on specific signaling pathways in cultured cells, including those agents that activate or inhibit developmental signaling pathways that drive development. As will be clear from the instant disclosure, whether activation or inhibition of a particular signaling pathway is necessary to generate a particular cell type of interest will depend on a number of factors including but not limited to, e.g., the particular desired cell type, the timing of use of the particular agent and/or composition, the starting cell type to be induced, etc.
  • the PSCs utilized in the differentiation procedures may comprise pluripotent cells of any kind.
  • pluripotent cells include human cells, including but not limited to, e.g., human embryonic stem cells, human induced pluripotent stem cells, human fetal stem cells, and the like.
  • Exemplary pluripotent cells include H1, H7 and H9 hESCs, as known in the art.
  • Exemplary induced pluripotent stem cells include iPSCs derived from peripheral blood mononuclear cells, fibroblasts, and other somatic cell sources, as known in the art.
  • the methods of the invention may be carried out by any number of cell culture processes.
  • one or more culturing steps are performed using cells plated on a substrate.
  • exemplary substrates include recombinant proteins, e.g. recombinant human proteins, for example, vitronectin, collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glucosaminoglycan, fibronectin, laminin, collagen IV, heparan sulfate proteoglycan, entactin and nidogen.
  • basement membrane extracts for example, containing proteins such as laminin, collagen IV, entactin, and heparin sulfate proteoglycans, for example, GELTREX(TM) substrate may be used.
  • a recombinant Laminin-511 E8 Fragment matrix is utilized as the culture substrate, for example iMatrix 511 (TM) (Takara Bio).
  • one or more culturing steps for example, all culturing steps, are performed using cells in a liquid culture system.
  • Cells may be seeded on the substrate at any effective density, for example, in the range of 10-50,000 cells per cm 2 .
  • the cell culture medium may be any basal medium, supplemented with differentiation factors as set forth herein.
  • Exemplary media include E8, MCDB131, for example, prepared as described herein, SFD medium, for example, prepared as described herein, and others known in the art.
  • the various methods described herein in some embodiments, encompass the differentiation of a first cell type to a second cell type by contacting cells of the first type with one or more selected agents for a period of time sufficient to induce differentiation to the second cell type.
  • the contacting may be achieved by any exposure of the cells to the selected agents.
  • the one or more selected factors are present in a liquid medium in which the cells are cultured, e.g. dissolved, suspended, or otherwise present.
  • the contacting is achieved by admixture of the one or more agents into the substrate material on which the cells are cultured, in planar culture implementations of the invention.
  • a method of producing a population of definitive esophogeal basal cells (eBC) from hPSCs is provided.
  • the method provides a means of producing a substantially pure population of eBC in defined culture conditions, in media comprising extracellular signaling agents to guide differentiation, the method comprising: (a) differentiating human pluripotent stem cells into definitive endoderm cells; (b) differentiating definitive endoderm cells into anterior foregut endoderm cells; (c) differentiating anterior foregut endoderm cells into dorsal anterior foregut endoderm cells; (d) differentiating dorsal anterior foregut endoderm cells into early eBC; (e) differentiating early eBC into definitive eBC.
  • the scope of the invention encompasses a method of differentiating human pluripotent stem cells to definitive endoderm cells.
  • the definitive endoderm cell differentiation process is performed as a first step in the differentiation of eBC from hPSCs. Undifferentiated hPSCs are seeded for differentiation as single cells.
  • the pluripotent stem cells are induced by contacting the cells in the presence of a ROCK pathway inhibitor on a suitable matrix, including without limitation a defined matrix such as recombinant laminin 511 for a period of time sufficient to differentiate human pluripotent stem cells to definitive endoderm cells, for example a period of time from about 1 day, e.g. from about 12 hour to about 36 hours.
  • the cells may then be moved to culture in the presence of a GSK-3 pathway inhibitor and TGF ⁇ pathway agonist for a period of about 3 days, for example from about 2 to about 4 days, to generate definitive endoderm.
  • the GSK-3 pathway inhibitor is optionally removed after the first day or the second day.
  • a ROCK inhibitor is Y-27632, for example at a concentration of from about 0.5 to about 50 ⁇ M, and may be around 10 ⁇ M.
  • a GSK-3 pathway inhibitor is CHIR99032, for example at a concentration of from about 0.5 to about 50 ⁇ M, and may be around 5 ⁇ M.
  • a TGF ⁇ pathway agonist is Activin A, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml.
  • a JNK inhibitor is JNK-IN-8, for example at a concentration of from about 0.1 to about 50 ⁇ M, and may be around 1 ⁇ M.
  • Anterior foregut endoderm differentiation encompasses a method of differentiating definitive endoderm cells to anterior foregut endoderm cells. Anterior foregut endoderm differentiation may be performed as a second step in the differentiation of hPSC to eBC.
  • the definitive endoderm cells are cultured in the presence of a TGF ⁇ pathway inhibitor; and BMP pathway inhibitor for a period of time sufficient to differentiate to anterior foregut endoderm ce, for example a period of time from about 2 days, e.g.
  • a TGF ⁇ pathway inhibitor is SB431542, for example at a concentration of from about 0.5 to about 50 mM, and may be around 10 mM.
  • a BMP pathway inhibitor is noggin, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml.
  • Dorsal anterior foregut endoderm differentiation In one aspect, the scope of the invention encompasses a method of differentiating anterior foregut endoderm cells to dorsal anterior foregut endoderm cells.
  • Dorsal anterior foregut endoderm differentiation may be performed as a third step in the differentiation of hPSC to eBC.
  • the anterior foregut endoderm cells are cultured in the presence of a TGF ⁇ pathway inhibitor, BMP pathway inhibitor, EGF pathway agonist and FGF pathway agonist for a period time sufficient to differentiate to dorsal anterior foregut endoderm cells, for example a period of from about 3 days, for example from about 2-4 days to generate dorsal anterior foregut endoderm.
  • a TGF ⁇ pathway inhibitor is SB431542, for example at a concentration of from about 0.5 to about 50 mM, and may be around 10 mM.
  • a BMP pathway inhibitor is noggin, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml.
  • an EGF pathway agonist is human EGF, and may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml.
  • an FGF pathway agonist is FGF10, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 50 ng/ml.
  • the scope of the invention encompasses a method of differentiating dorsal anterior foregut endoderm cells to early eBC.
  • Early esophogeal basal cell differentiation may be performed as a fourth step in the differentiation of hPSC to eBC.
  • the dorsal anterior foregut endoderm cells are cultured in the presence of an EGF pathway agonist and FGF pathway agonist for a period of time sufficient to differentiate to eBC, for example from about 3-6 days, e.g. about 5 days.
  • the cells may be cultured in the presence of an EGF agonist for a period of from about 4 to 8 days, and may be about one week. This step may also be sufficient to generate definitive eBC, although the yield can be improved with the disclosed fifth step.
  • an EGF pathway agonist is human EGF, and may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml.
  • an FGF pathway agonist is FGF10, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 50 ng/ml.
  • Definitive esophogeal basal cell differentiation In one aspect, the scope of the invention encompasses a method of differentiating early eBC to definitive eBC. Definitive esophogeal basal cell differentiation may be performed as a fifth step in in the differentiation of hPSC to eBC.
  • the early eBC are cultured in the presence of an EGF pathway agonist, a wnt pathway inhibitor; a BMP pathway agonist; and a TGF ⁇ pathway agonist; for a period of time sufficient to differentiate definitive eBC, for example from about 3-5 days, e.g. about 5 days, to generate canonical esophogeal basal cells.
  • an EGF pathway agonist is human EGF, and may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml.
  • a Wnt pathway inhibitor is IWP-2, e.g. at a concentration of from about 0.5 to about 50 ⁇ M, and may be around 4 ⁇ M.
  • a BMP pathway agonist is BMP4, e.g. at a concentration of from about 0.5 to about 500 ng/ml, and may be around 5 ng/ml.
  • a TGF ⁇ agonist is TGF ⁇ 1, e.g. at a concentration of from about 0.5 to about 500 ng/ml, and may be around 2 ng/ml.
  • greater than 50%, greater than 75%, greater than 80% or more of the final cell population express one, two, three or more of the eBC FOXA1, FOXA2, SOX2, SOX9 and P63 with ITGB4, EPCAM, Krt5, and Krt15.
  • the eBC are exapandable and are capable of self-renewal.
  • eBC are selected for expression of Epcam and ITGB4.
  • the eBCs derived by the foregoing method may be utilized in the treatment of various esophageal conditions.
  • the eBCs may be transplanted, grafted, or otherwise administered to treat genetic or acquired defects or injuries to the esophagus, for example in the treatment of recessive dystrophic epidermolysis bullosa, dominant dystrophic epidermolysis bullosa, wounding caused by cancer resection, caustic injury, esophageal stricture, esophagitis, and other wounds, dysfunctions, or pathologies of the esophagus.
  • the eBCs of the invention may be delivered by methods as known in the art for esophageal regeneration, for example, delivered in a scaffold or matrix.
  • Exemplary scaffolds include acellular scaffolds, for example comprising polymeric material, proteinaceous materials (e.g.
  • an agent useful in a particular induction composition may include an activator (agonist) or inhibitor of the TGF-beta (transforming growth factor ⁇ (TGF- ⁇ )) pathway.
  • TGF-beta transforming growth factor ⁇
  • TGF- ⁇ Transforming growth factor-beta
  • TGF- ⁇ denotes a family of proteins, TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3, which are pleiotropic modulators of cell growth and differentiation, embryonic and bone development, extracellular matrix formation, hematopoiesis, immune and inflammatory responses (Roberts and Sporn Handbook of Experimental Pharmacology (1990) 95:419-58; Massague et al. Ann Rev Cell Biol (1990) 6:597-646).
  • TGF- ⁇ initiates intracellular signaling pathways leading ultimately to the expression of genes that regulate the cell cycle, control proliferative responses, or relate to extracellular matrix proteins that mediate outside-in cell signaling, cell adhesion, migration and intercellular communication.
  • TGF- ⁇ exerts its biological activities through a receptor system including the type I and type II single transmembrane TGF- ⁇ receptors (also referred to as receptor subunits) with intracellular serine-threonine kinase domains, that signal through the Smad family of transcriptional regulators. Binding of TGF- ⁇ to the extracellular domain of the type II receptor induces phosphorylation and activation of the type I receptor (TGF ⁇ -R1) by the type II receptor (TGF ⁇ -R2).
  • a TGF ⁇ inhibitor refers to a molecule, e.g. a decoy receptor, antibody or derivative thereof, a nonpeptide small molecule, etc.
  • Activators and inhibitors of the TGF-beta pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the TGF-beta pathway resulting in a corresponding activation or inhibition in cellular TGF-beta signaling.
  • Activators (agonists) of the TGF-beta pathway include but are not limited to, e.g., TGF- beta family ligands (e.g., TGF-beta proteins and other activators of TGF-beta receptors) and portions thereof, Activin A, TGF-beta1, TGF-beta2, TGF-beta3 , IDE1/2 (IDE1 (1-[2-[(2- Carboxyphenyl)methylene]hydrazide]heptanoic acid), IDE2 (Heptanedioic acid-1-(2- cyclopentylidenehydrazide)), Nodal, and the like.
  • TGF- beta family ligands e.g., TGF-beta proteins and other activators of TGF-beta receptors
  • Activin A Activin A
  • IDE1/2 IDE1 (1-[2-[(
  • activation of the TGF- beta pathway may be achieved through repression of the a TGF-beta pathway inhibitor, e.g., including but not limited to the use of an inhibitory nucleic acid targeting an inhibitor of the TGF-beta pathway or an antibody or small molecule directed to a TGF-beta pathway inhibitor.
  • Inhibitors of the TGF-beta pathway include but are not limited to, e.g., A-83-01 (3-(6- Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), D4476 (4-[4- (2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), LY 364947 (4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-quinoline), RepSox (2-(3-(6-Methylpyridine-2-
  • an agent useful in a particular induction composition may include an inhibitor of the Wnt pathway.
  • Activators and inhibitors of the Wnt pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the Wnt pathway resulting in a corresponding activation or inhibition in cellular Wnt signaling.
  • Inhibitors of the WNT pathway include but are not limited to, e.g., C59 (4-(2-Methyl-4- pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide), DKK1, IWP-2 (N-(6-Methyl-2- benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]- acetamide), Ant1.4Br, Ant 1.4Cl, Niclosamide, apicularen, bafilomycin, XAV939 (3,5,7,8- Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), IWR-1 (4- (1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4-one),
  • a specific WNT inhibitor may be administered in such a manner as to result in a decrease in PAX3 expression and a promotion of FOXC2 expression.
  • a Wnt activator or inhibitor useful in the methods described herein may include those described in, e.g., Dodge and Lum et al. Annu Rev Pharmacol Toxicol.2011;51:289-310; Chen et al. Am J Physiol Gastrointest Liver Physiol. 2010 Aug;299(2):G293-300; Baker and Clevers, Nat Rev Drug Discov. 2006 Dec;5(12):997-1014; Meijer et al. Trends Pharmacol Sci.
  • an agent useful in a particular induction composition may include an activator of the FGF pathway.
  • an activator or inhibitor of the FGF pathway may also include activators or inhibitors of related signal transduction pathways including but not limited to, e.g., the MAPK/ERK signal transduction pathway.
  • Activators and inhibitors of the FGF pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the FGF pathway resulting in a corresponding activation or inhibition in cellular FGF signaling.
  • Activators of the FGF pathway include but are not limited to, e.g., FGF family ligands (e.g., FGF1, FGF2, FGF- 3, FGF-4, FGF-5, FGF-6, KGF/FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-15, FGF-16, FGF-17, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, etc.), SUN 11602 (4- [[4-[[2-[(4-Amino-2,3,5,6-tetramethylphenyl)amino]acetyl]methylamino]-1- piperidinyl]methyl]benzamide), t-Butylhydroquinone, U-46619, C2 Ceramide, Lactosyl Ceramide, Angiotensin II, Baicalin, and the like.
  • FGF family ligands e.g., FGF1, FGF2, FGF-
  • a FGF activator or inhibitor useful in the methods described herein may include those described in, e.g., English and Cobb, Trends Pharmacol Sci. 2002 Jan;23(1):40-5, the disclosure of which is incorporated herein by reference in its entirety.
  • an agent useful in a particular induction composition may include an activator or inhibitor of the BMP pathway.
  • Activators and inhibitors of the BMP pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the BMP pathway resulting in a corresponding activation or inhibition in cellular BMP signaling.
  • Activators of the BMP pathway include but are not limited to, e.g., BMP family ligands (e.g., BMP2, BMP4, BMP7, etc.), Alantolactone, FK506, isoliquiritigenin, 4′-hydroxychalcone, and the like.
  • BMP family ligands e.g., BMP2, BMP4, BMP7, etc.
  • Alantolactone e.g., BMP2, BMP4, BMP7, etc.
  • FK506 isoliquiritigenin
  • 4′-hydroxychalcone e.g., activation of the BMP pathway may be achieved through repression of a BMP pathway inhibitor, e.g., including but not limited to the use of an inhibitory nucleic acid targeting an inhibitor of the BMP pathway or an antibody or small molecule directed to a BMP pathway inhibitor.
  • Inhibitors of the BMP pathway include but are not limited to, e.g., NOGGIN, CHORDIN, LDN-193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride), DMH1 (4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]- quinoline), Dorsomorphin (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5- a]pyrimidine dihydrochloride), K 02288 (3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3- pyridinyl]phenol), ML 347 (5-[6-(4-Methoxyphenyl)pyr
  • an agent useful in a particular induction composition may include an inhibitor of the GSK-3 pathway.
  • Inhibitors of the GSK-3 pathway include small molecule activators, small molecule inhibitors, peptide inhibitors, antibodies, nucleic acid inhibitors, and the like that inhibit at least one component of the GSK-3 pathway resulting in a corresponding inhibition in cellular GSK-3 signaling.
  • Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase involved in various cellular processes, including metabolism, cell proliferation, and apoptosis. In cell culture studies, inhibitors of GSK-3 are commonly used to investigate its roles and mechanisms.
  • GSK-3 inhibitors suitable for cell culture including CHIR99021, which is an ATP-competitive inhibitor of GSK-3 effective at low micromolar concentrations, for example around 1 ⁇ M to 10 ⁇ M; SB216763, which is an ATP-competitive inhibitor of GSK-3 effective at low micromolar concentrations, for example around 1 ⁇ M to 10 ⁇ M; AR-A014418, which is an ATP-competitive inhibitor of GSK-3 effective at low micromolar concentrations, for example around 1 ⁇ M to 10 ⁇ M; BIO (6-Bromoindirubin-3'-oxime) which is an ATP-competitive inhibitor of GSK-3 effective at low micromolar concentrations, for example around 1 ⁇ M to 10 ⁇ M; TWS119; SB 415286; CHIR-98014 and the like.
  • an agent useful in a particular induction composition may include an agonist of the EGF pathway.
  • Activators of the EGF pathway include small molecule activators, peptide activators, nucleic acid activators, nucleic acid inhibitors, and the like that activate at least one component of the EGF pathway.
  • Epidermal Growth Factor (EGF) is a potent mitogenic peptide that stimulates cell growth, proliferation, and differentiation by binding to its receptor, EGFR (ErbB1). In cell culture studies, EGF agonists are used to activate EGFR signaling pathways to investigate their roles in various cellular processes.
  • EGF agonists of interest include recombinant human EGF (rhEGF), for example in the range of about 1 ng/mL to 100 ng/mL; transforming growth factor-alpha (TGF- ⁇ ) binds to and activates EGFR similar to EGF, and may be used at concentrations of about 0.1 ng/mL to 100 ng/mL; amphiregulin activates EGFR, and may be used at concentrations of from about 1 ng/mL to 100 ng/mL.
  • an agent useful in a particular induction composition may include an inhibitor of the ROCK pathway.
  • Inhibitors of the ROCK pathway include small molecule activators, small molecule inhibitors, peptide inhibitors, antibodies, nucleic acid inhibitors, and the like that inhibit at least one component of the ROCK pathway resulting in a corresponding inhibition in cellular ROCK signaling.
  • Rho-associated kinases are serine/threonine kinases involved in various cellular functions, including contraction, motility, proliferation, and apoptosis. In tissue culture, ROCK inhibitors are used to modulate these cellular processes.
  • ROCK inhibitors of interest include Y-27632 ((R)-(+)-trans-4-(1-Aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride), which competes with ATP for binding to the kinase domain, and may be used at concentrations of from about 10 ⁇ M to 50 ⁇ M; Fasudil (HA-1077, 5-(1,4-Diazepan-1- ylsulfonyl)isoquinoline) which competes with ATP for binding to the kinase domain, and may be used at concentrations of from about 10 ⁇ M to 100 ⁇ M; GSK269962A (4-[(2S)-2-amino-1- hydroxyethyl]phenol; (2R)-2-(3-chlorophenyl)-N-methylpyrrolidine-1-carboxamide) which competes with ATP, and may be used at concentrations of from about 1 nM to 10 ⁇ M
  • JNK inhibitors are compounds designed to specifically inhibit the activity of JNK, a member of the mitogen-activated protein kinase (MAPK) family involved in various cellular processes such as inflammation, stress responses, apoptosis, and development.
  • MPK mitogen-activated protein kinase
  • JNK inhibitors used in tissue culture, with different mechanisms of action and specificities, including, for example, SP600125, which is a broad- spectrum JNK inhibitor that competitively inhibits ATP binding to JNK.
  • JNK-IN-8 is a highly selective and irreversible inhibitor of JNK1, JNK2, and JNK3. It covalently binds to a conserved cysteine residue in the ATP-binding pocket of JNK, providing sustained inhibition.
  • AS601245 is another selective inhibitor that blocks JNK activity by competing with ATP. It has been shown to have fewer off-target effects compared to SP600125.
  • pathway modulating agents include, e.g., those that are commercially available, e.g., from such suppliers such as Tocris Bioscience (Bristol, UK), Sigma-Aldrich (St. Louis, MO), Santa Cruz Biotechnology (Santa Cruz, CA), and the like.
  • Pluripotent stem cells and derivatives thereof may be contacted with these factors by any convenient means.
  • an agent is added to culture media, as described herein, within which cells of the instant disclosure are grown or maintained, such that the agent is present, in contact with the cells, at an effective concentration to produce the desired effect.
  • the culture media in which the cells are being grown is replaced with fresh culture media containing the particular agent present in the fresh media at an effective concentration to produce the desired effect.
  • the effective concentration of a particular agent will vary and will depend on the agent. In addition, in some instances, the effective concentration may also depend on the cells being induced, the culture condition of the cells, other agents co-present in the culture media, etc.
  • the effective concentration of agents will vary and may range from 1 ng/mL to 10 ⁇ g/mL or more, including but not limited to, e.g., 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/mL, 26 ng/mL, 27 ng/mL, 28 ng/mL, 29 ng/mL, 30 ng/mL, 31
  • the effective concentration of an agent in solution may range from 1 nM to 100 ⁇ M or more, including but not limited to, e.g., 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41
  • the effective concentration of an induction agent will be below a critical concentration such that the induction produces the desired effect essentially without undesirable effects.
  • critical concentration refers to a concentration of induction agent above which undesirable effects are produced.
  • Undesirable effects that may be the result of a concentration exceeding the critical concentration include but are not limited to, e.g., off-target effects (off-target activation of signaling, off-target inhibition of signaling), reduction or loss of function (e.g., loss of desired activator function, loss of desired inhibitor function) reduction of cell viability, increase in cell mortality, lineage restriction towards an undesired cell type, differentiation into an undesired cell type, loss of expression of a particular desired marker, etc.
  • cells of the instant disclosure may be contacted with multiple agents and/or multiple compositions in order achieve a desired cell type.
  • a particular composition will contain two or more agents such that a particular cell culture is simultaneously contacted with multiple agents.
  • a particular series of compositions may be used, one at a time, in generating a desired cell type such that a particular cell culture is successively contacted with multiple agents.
  • the duration of contact of a particular composition with a particular cell type may be referred to as the “exposure time” and exposure times may range from a day to weeks or more, including but not limited to e.g., 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, 10 days, 11 days, 12, days, 13, days, 14 days, 15, days, etc.
  • exposure times are, in some instances, referred as consisting essentially of, e.g., 24 hours, indicating that the exposure time may be longer or shorter than that specified including those exposure times that are longer or shorter but do not materially affect the basic outcome of the particular exposure.
  • a time period consisting essentially of, e.g., 24 hours will be interpreted to refer to a time period ranging from about 23 hours to about 25 hours.
  • a time period consisting essentially of, e.g., 24 hours will mean a time period ranging from about 12 hours or less to about 36 hours or more.
  • an exposure period consisting essentially of 24 hours may refer to an exposure time of 22-26 hours, 21-27 hours, 20-28 hours, 19-29 hours, 18-30 hours, etc.
  • time periods of exposure may be pre-determined such that cells are contacted with an induction composition according to a schedule set forth prior to the contacting.
  • the time period of exposure may be modulated according to some feature or characteristic of the cells and/or cell culture, including but not limited to, e.g., cell morphology, cell viability, cell appearance, cellular behaviors, cell number, culture confluence, marker expression, etc.
  • cells are grown in densities that may range from but not limited to 100 cells/cm 2 , 10 3 cells/cm 2 , 10 4 cells/cm 2 , 10 5 cells/cm 2 , 10 6 cells/cm 2 , 10 7 cells/cm 2 , 10 8 cells/cm 2 , 10 9 cells/cm 2 , 10 10 cells/cm 2 .
  • markers of interest include cell surface markers that may be detected, e.g., on live cells.
  • markers of interest include expression markers, e.g., cellular expression markers indicative of cell type.
  • canonical esophogeal basal cells express one, two, three or more of the eBC markers FOXA1, FOXA2, SOX2, SOX9 and P63 with ITGB4, EPCAM, Krt5, and Krt15.
  • a desired eBC cell population is purified for cells of interest.
  • markers may be detected or measured by any convenient means as such marker detection is well-known in the art and may make use of one or more detection reagents including but not limited to, e.g., antibodies, antibody fragments, binding partners (e.g., ligands, binding pairs, etc), hybridizable nucleic acids, aptamers, etc.
  • a marker may be a cell surface marker and detection of the marker may be performed based on the use of one or more detection reagents that specifically bind to the marker.
  • Detection reagents e.g., antibodies
  • may be detectably labeled e.g., fluorescently labeled through the attachment of a fluorescent molecule, fluorescent bead, or other fluorescent label
  • a second detectably labeled detection reagent that specifically binds to the first detection reagent (e.g., a fluorescently labeled secondary antibody).
  • a detection agent e.g., having a detectable label or having been bound by a second agent having a detectable label, can be visualized or otherwise observed or detected based on the visual characteristics of the label, including e.g., fluorescent detection, colorimetric detection, and the like.
  • Detectable labels useful in detection reagents need not be visually detectable and may, in some instances, be detected by a detection device con FIG.d to detect a non-visual detectable label including but not limited to, e.g., a magnetic label, a radioactive label, etc.
  • detectable labels may be detected through the use of one or more detection reactions, including but not limited to, e.g., enzymatic detection reactions (enzymatic reactions generating a detectable substrate, e.g., a fluorescent or colorimetric substrate), amplification reactions (PCR amplification, fluorescent signal amplification (e.g., tyramide signal amplification, etc.), etc.)
  • detection and/or selection for sorting of cells may be performed using a combination of markers. Such combinations may include but combinations of positive selection markers, combinations of negative selection markers or mixed combinations of positive and negative selection markers.
  • marker detection and/or measurement of marker level is performed using flow cytometry.
  • Fluorescent signals used in flow cytometry typically are fluorescently-tagged antibody preparations or fluorescently-tagged ligands for binding to antibodies or other antigen-, epitope- or ligand-specific agent, such as with biotin/avidin binding systems or fluorescently-labeled and optionally addressable beads (e.g. microspheres or microbeads).
  • the markers or combinations of markers detected by the optics and/or electronics of a flow cytometer vary and in some cases include but are not limited to: cell surface markers, intracellular and nuclear antigens, DNA, RNA, cell pigments, cell metabolites, protein modifications, transgenic proteins, enzymatic activity, apoptosis indicators, cell viability, cell oxidative state, etc.
  • flow cytometry is performed using a detection reagent, e.g., a fluorochrome-labeled antibody, e.g., a monoclonal antibody, with specific avidity against a cell surface maker of interest.
  • a cellular sample is contacted with a detection reagent under conditions sufficient to allow the detection reagent to bind the cell surface maker and the cells of the sample are loaded into the flow cytometer, e.g., by first harvesting the cells from a cell culture using methods known in the art or described herein and re-suspending the isolated cells in a suitable buffer, e.g., running buffer.
  • the flow cytometer detects events as the cell passes one or more detection areas of the flow cytometer. Detected events are counted or otherwise evaluated by the flow cytometer with or without input from an operator and used to determine, e.g., the total number of cells, the number or proportion of cells bound to a particular detection reagent, etc.
  • cells may be sorted, e.g., into separate containers, based on the detection or measurement of a particular marker.
  • cell sorting e.g., by FACS
  • FACS cell sorting
  • Expression markers of interest may be used to identify a particular cell type or verify that a derived cell type expresses a characteristic component of the derived cell type.
  • detection of expression markers may allow for optimization of a particular differentiation protocol, e.g., to optimize production of a desired cell type based on detection of one or more expression markers.
  • Expression markers will vary depending on the type of cell to be identified or verified and/or desired downstream uses of the cell following identification or verification with the expression marker.
  • Types of expression markers will include but are not limited to, e.g., gene expression marker, protein expression markers, expressed reporters, and the like.
  • Expression marker detection and/or measurement may be detrimental to cell viability (e.g., wherein detection requires lysing or fixing a cell of interest) or may be essentially neutral to cell viability (e.g., wherein detection does not require lysing or fixing a cell of interest and may be performed on live cells).
  • Methods of detecting and/or measuring gene expression and/or protein expression include but are not limited to, e.g., Northern blot, Western blot, ELISA, PCR, quantitative PCR, in situ hybridization, fluorescent in situ hybridization, immunohistochemistry, immunofluorescence, microarray, quantitative sequencing, RNAseq, quantitative mass spectrometry, and the like.
  • Methods of modification of cells including modification of pluripotent cells and modification of eBC are well-known in the art and include but are not limited to e.g., genetic modification (e.g., through deletion mutagenesis, through substitution mutagenesis), through insertional mutagenesis (e.g., through the introduction of heterologous nucleic acid into the pluripotent cell, etc.), non-mutagenic genetic modification (e.g., the non-mutagenic insertion of heterologous nucleic acid, etc.), epigenetic modification (e.g., through the treatment with one or more specific or general epigenetic modifying agents (e.g., methylation inhibitors, methylation activators, demethylases, etc.), other modifications (e.g., non-genetic labeling, etc.).
  • genetic modification e.g., through deletion mutagenesis, through substitution mutagenesis
  • through insertional mutagenesis e.g., through the introduction of heterologous nucleic acid into the pl
  • Modifications of cells may be transient or stable.
  • a modification of a particular pluripotent cell or progenitor cell may be stable such that the modification persists through derivation of a desired cell type from the pluripotent cell or progenitor cell as described herein.
  • stable modifications may persist through introduction of a cell type into a host.
  • stable modifications may persist through proliferation of the cell such that all progenitors of a particular modified cell also contain the subject modification.
  • a modification of a particular pluripotent cell or progenitor cell may be transient such that the modification is lost after derivation of a cell type of interest from the transiently modified pluripotent cell.
  • transient modifications may persist through one or more rounds of proliferation of the modified cell such that some but not all of the progeny of the modified cell contain the subject modification. In some instances, a transient modification will not persist during proliferation such that none of the progeny of a modified cell will contain the subject modification. In some instances, a transiently modified cell may be configured such that the modification persists through certain aspects of derivation of the cell type of interest, e.g., through derivation of a particular cell type of interest, but is lost prior to introduction of the derived cell into a host. Screening [00111] Aspects of the instant disclosure include methods of screening pharmacological agents using eBC derived according to the methods described herein.
  • a plurality of cell populations derived according to the methods as described herein are contacted with a plurality of pharmacological agents in order to screen for agents producing a cellular response of interest.
  • a cellular response of interest may be any cellular response including but not limited to, e.g., cell death, cell survival, cell self-renewal, proliferation, differentiation, expression of one or more markers, loss of expression of one or more markers, change in morphology, change in cellular physiology, cellular engraftment, change in cell motility, change in cell migration, production of a particular cellular component, cease of production of a particular cellular component, change in metabolic output, response to stress, and the like.
  • Screening pharmacological agents using cells described herein may be performed in vitro, e.g., in a tissue culture chamber, on a slide, etc., or may be performed in vivo, e.g., in an animal host, etc. Cells used in such screening assays may be genetically altered or may be unaltered. In some instances, cells generated according to the methods as described herein are used in multiplexed in vitro pharmacological screening.
  • Methods for evaluating cellular responses during in vitro screening include but are not limited to, e.g., microscopic methods (e.g., light microscopy, electron microscopy, etc.), expression assays, enzymatic assays, cytological assays (e.g., cellular staining), genomics, transcriptomics, metabolomics, and the like.
  • cells generated according to the methods as described herein are introduced into a host animal and the host animal may be administered a pharmacological agent in order to screen for a response from the introduced cells.
  • the cells of the in vivo assay may be directly evaluated, e.g., for an intrinsic response to a pharmacological agent.
  • the host animal of the in vivo assay may be evaluated as an indirect measurement of the response of the cells to the pharmacological agent.
  • the subject disclosure includes screening cells derived according to the methods described herein as a method of therapy of an animal model of disease and/or a human disease. Methods of screening cells derived according to the methods described herein as a method of therapy may be, in some instances, performed according to those methods described below regarding using such cells in therapeutic protocols.
  • the subject disclosure includes screening cells derived according to the methods described herein introduced to a host animal as a method of directly evaluating the cells or particular cellular behaviors, e.g., due to an introduced genetic modification or a naturally derived mutation.
  • genetically modified cells e.g., having at least one modified genomic locus, derived according to the methods described herein may be introduced into a host animal and the ability of the cells to differentiate into a particular tissue or cell type may be evaluated.
  • genetically modified cells derived according to the methods described herein may be introduced into a host animal and the behavior of the cells within the host animal and/or within a tissue of the host animal may be evaluated.
  • cells derived from a donor organism having a particular mutation or phenotype and lineage restricted according to the methods described herein may be introduced into a host animal and the behavior of the cells within the host animal and/or within a tissue of the host animal may be evaluated, including, e.g., the ability of the cells to differentiate into one or more tissue or cell types.
  • the cells may introduced into the host animal in a autologous graft, an allograft, or a xenograft such that the introduced cells may be derived from the host animal, a separate donor of the same species as the host animal, or a separate donor of a different species as compared to the host animal, respectively.
  • aspects of the disclosure include methods for lessening the symptoms of and/or ameliorating a dysfunction in eBC and cells derived therefrom, for example in the treatment of recessive dystrophic epidermolysis bullosa; esophageal injury; cancer, etc.
  • Any and all forms of dysfunction, whether treated or untreated, or resulting from any primary condition, whether treated or untreated, are suitable dysfunctions or disorders to be treated by the subject methods described herein.
  • the treatment methods described herein include the alleviation or reduction or prevention of one or more symptoms of dysfunction or disorder. Symptoms of dysfunction or disorder will vary, may be infrequent, occasional, frequent, or constant.
  • the methods of treatment described herein include administering a therapeutically effective amount of a population, e.g., an essentially homogenous population, of eBC to a subject in need thereof in order to treat the subject for a dysfunction or deficiency.
  • Conditions of interest for treatment include any condition in which esophogeal cells are damaged and can benefit from transplantation of healthy esophogeal cells. Examples include epidermolysis bullosa; esophageal injury; Barrett's esophagus, where the lining of the esophagus is damaged by stomach acid, leading to a change in the tissue lining.
  • Esophageal strictures are narrowing of the esophagus due to injury, inflammation, or chronic acid reflux, which can cause significant swallowing difficulties.
  • Achalasia is a disorder where the esophagus has trouble moving food down into the stomach because the lower esophageal sphincter fails to relax properly.
  • Esophageal atresia is a congenital condition where a part of the esophagus is missing.
  • Transplanting esophageal cells can be a part of reconstructive surgery to repair the esophagus in infants.
  • Gastroesophageal Reflux Disease may include damage to the esophagus.
  • the effective amount administered varies depending upon the goal of the administration, the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g., human, non-human primate, primate, etc.), the degree of resolution desired (e.g., the amount of alleviation or reduction of symptoms), the formulation of the cell composition, the treating clinician's assessment of the medical situation, and other relevant factors.
  • a "therapeutically effective dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy) or reduce, alleviate, or prevent symptoms to a desired extent as determined by the patient or the clinician.
  • a therapeutically effective dose can be administered in one or more administrations.
  • a therapeutically effective dose of cells is an amount that is sufficient, when administered to (e.g., transplanted into) the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state by, for example, inducing stabilization, repair, or regeneration.
  • a therapeutically effective dose of cells is one cell or more (e.g., 1x10 2 or more, 5x10 2 or more, 1x10 3 or more, 5x10 3 or more, 1x10 4 cells, 5x10 4 or more, 1x10 5 or more, 5x10 5 or more, 1 x 10 6 or more, 2x10 6 or more, 5x10 6 or more, 1x10 7 cells, 5x10 7 or more, 1x10 8 or more, 5x10 8 or more, 1 x 10 9 or more, 5x10 9 or more, or 1x10 10 or more).
  • a therapeutically effective dose of cells is in a range of from 1x10 3 cells to 1x10 10 cells (e.g., from 5x10 3 cells to 1x10 10 cells, from 1x10 4 cells to 1x10 10 cells, from 5x10 4 cells to 1x10 10 cells, from 1x10 5 cells to 1x10 10 cells, from 5x10 5 cells to 1x10 10 cells, from 1x10 6 cells to 1x10 10 cells, from 5x10 6 cells to 1x10 10 cells, from 1x10 7 cells to 1x10 10 cells, from 5x10 7 cells to 1x10 10 cells, from 1x10 8 cells to 1x10 10 cells, from 5x10 8 cells to 1x10 10 , from 5x10 3 cells to 5x10 9 cells, from 1x10 4 cells to 5x10 9 cells, from 5x10 4 cells to 5x10 9 cells, from 1x10 5 cells to 5x10 9 cells, from 5x10 5 cells to 5x10 9 cells, from 1x10 6 cells.
  • the concentration of cells to be administered is in a range of from 1 x 10 5 cells/ml to 1 x 10 9 cells/ml (e.g., from 1 x 10 5 cells/ml to 1 x 10 8 cells/ml, from 5 x 10 5 cells/ml to 1 x 10 8 cells/ml, from 5 x 10 5 cells/ml to 5 x 10 7 cells/ml, from 1 x 10 6 cells/ml to 1 x 10 8 cells/ml, from 1 x 10 6 cells/ml to 5 x 10 7 cells/ml, from 1 x 10 6 cells/ml to 1 x 10 7 cells/ml, from 1 x 10 6 cells/ml to 6 x 10 6 cells/ml, or from 2 x 10 6 cells/ml to 8 x 10 6 cells/ml).
  • the concentration of cells to be administered is 1 x 10 5 cells/ml or more (e.g., 1 x 10 5 cells/ml or more, 2 x 10 5 cells/ml or more, 3 x 10 5 cells/ml or more, 4 x 10 5 cells/ml or more, 5 x 10 5 cells/ml or more, 6 x 10 5 cells/ml or more, 7 x 10 5 cells/ml or more, 8 x 10 5 cells/ml or more, 9 x 10 5 cells/ml or more, 1 x 10 6 cells/ml or more, 2 x 10 6 cells/ml or more, 3 x 10 6 cells/ml or more, 4 x 10 6 cells/ml or more, 5 x 10 6 cells/ml or more, 6 x 10 6 cells/ml or more, 7 x 10 6 cells/ml or more, or 8 x 10 6 cells/ml or more).
  • 1 x 10 5 cells/ml or more e.g., 1 x 10 5
  • a therapeutically effective dose of cells may be delivered or prepared and any suitable medium, including but not limited to, e.g., those described herein.
  • suitable medium for the delivery of a therapeutically effective dose of cells will vary and may depend on, e.g., the type of pluripotent cells from which the effective dose of cells is derived or the type of derived cells of the effective dose.
  • a suitable medium may be a basal medium.
  • Cell medium as used herein are not limited to liquid media may, in some instances, include non- liquid components or combinations of liquid media and non-liquid components. Non-liquid components that may find use a delivery or preparation medium include those described herein and those known in the art.
  • non-liquid components include natural or synthetic extra cellular matric components including but not limited to, e.g., basement membrane matrix components and the like.
  • an effective dose of the cells described herein may be co- administered with one or more additional agents (e.g., prepared in a suitable medium). Additional agents useful in such co-administration include agents that improve the overall effectiveness of the effective dose of cells or decrease the dose of cells necessary to achieve an effect essentially equal to administration of an effective dose of the cells without the additional agent.
  • additional agents that may be co-administered include: conventional agents for treating diseases, pro-survival factors, pro-engraftment factors, functional mobilization agents, and the like.
  • pro-survival factors a factor or agent that may be added to the medium, culture media, delivery excipient, or storage solution that promotes the survival of a desired cell type.
  • pro-survival factors may be general pro-survival factors that generally promote the survival of most cell types or may be specific pro-survival factors that only promote the survival of certain specific cell types.
  • pro-survival factors of the subject disclosure include but are not limited to, e.g., Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g., low does, i.e., sub-immunosuppressive dose, cyclosporine), ZVAD-fmk, pro-survival cytokines (e.g., insulin-like growth factor-1 (IGF-1)), extra cellular matrix (ECM) components, hydrogels, matrigel, collagen, gelatin, agarose, alginate, poly(ethylene glycol), hyaluronic acid, etc.
  • ROCK Rho-associated kinase
  • pinacidil e.g., pinacidil, allopurinol, uricase
  • cyclosporine e.g., low does, i.e., sub-immunosuppressive dose, cyclosporine
  • pro-engraftment factors is meant a factor or agent that may be added to the administered dose or the delivery excipient or the cell storage solution that, upon delivery of the cells into a subject for treatment, increase the engraftment of the administered cells into the tissue targeted for engraftment and therapy.
  • pro-engraftment factors include factors that physically retain the administered cells at the delivery site, e.g., the injection site in the case of direct injection to the affected area, including but not limited to, e.g., gels, polymers, and highly viscous liquids that have physical properties that prevent the administered cells from freely diffusing.
  • Such gels, polymers, and highly viscous liquids include but are not limited to e.g., ECM components, hydrogels, matrigel, collagen, gelatin, agarose, alginate, poly(ethylene glycol), and the like.
  • co-administration and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits.
  • the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time.
  • the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms.
  • a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.
  • the cells may be introduced by injection, catheter, intravenous perfusion, or the like.
  • the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being capable of use upon thawing. Once thawed, the cells may be expanded by use of growth factors and/or feeder cells or in feeder-free conditions associated with progenitor cell proliferation and differentiation. In some instances, the cells may be administered fresh such that the cells are expanded and differentiated and administer without being frozen.
  • the cells of this disclosure can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient or buffer or media prepared under sufficiently sterile conditions for human administration.
  • composition may also comprise or be accompanied with one or more other ingredients that facilitate the engraftment or functional mobilization of the cells. Suitable ingredients include matrix proteins that support or promote adhesion of the cells, or complementary cell types.
  • the cells are derived from the subject that is to be treated with the cells.
  • the cells may be derived from a tissue sample obtained from the subject including but not limited to, e.g., a blood sample (e.g., a peripheral blood sample), a skin sample, a bone marrow sample, and the like.
  • the sample from which cells are derived may be a biopsy or swab, e.g., a biopsy or swab collected to diagnose, monitor, or otherwise evaluate the subject, e.g., diagnose the subject for a dysfunction or deficiency, e.g., bone disease or a muscle disease or a cartilage disease or a related condition, or for cell collection.
  • the autologous sample from which the cells are derived may be a previously collected and stored sample, e.g., a banked tissue sample, from the subject to be treated, including but not limited to e.g., banked cardiac tissue or cells, banked musculoskeletal tissue or cells, banked reproductive tissue or cells, banked skin tissue or cells, banked bone tissue or cells, banked bone marrow tissue or cells, banked vascular tissue or cells, banked umbilical cord blood tissue or cells, and the like.
  • cells of the subject methods are non-autologously derived. By non- autologously derived it is meant that the cells are not derived from the subject that is to be treated with the cells.
  • non-autologously derived cells may be xeno-derived (i.e., derived from a non-human animal) or allo-derived (i.e. derived from a human donor other than the subject to be treated).
  • Non-autologously derived cells or tissue may be derived from any convenient source of cells or tissue collected by any convenient means.
  • Whether to use autologously derived or non-autologously derived cells may be determined according to the discretion of the subject’s clinician and may depend on, e.g., the health, age, genetic predisposition or other physical state of the subject. In some instances, autologous cells may be preferred, including, e.g., to decrease the risk or immune rejection of the transplanted cells.
  • non-autologous cells may be preferred, including, e.g., when the subject has a genetic defect.
  • Pluripotent Stem cells [00135] Methods of derivation of pluripotent stem cells from an autologous or non-autologous tissue useful in the methods described herein include but are not limited to, e.g., methods of embryonic stem cell derivation and methods of induced pluripotent stem cell derivation. In some instances, methods as described herein may be performed using non-autologous pluripotent progenitor cells previously derived including, e.g., those publically or available or commercially available (e.g., from Biotime, Inc., Alameda, CA).
  • methods as described herein may be performed using newly derived non-autologous pluripotent progenitor cells or newly derived autologous pluripotent progenitor cells including but not limited to, e.g., newly derived embryonic stem cells (ESC) (including, e.g., those derived under xeno-free conditions as described in, e.g., Lei et al. (2007) Cell Research, 17:682-688) and newly derived induced pluripotent stem cells (iPS).
  • ESC embryonic stem cells
  • iPS newly derived induced pluripotent stem cells
  • pluripotent progenitor cells e.g., iPS cells
  • pluripotent progenitor cells useful in the methods described herein are derived by reprogramming and are genetically unmodified, including e.g., those derived by integration- free reprogramming methods, including but not limited to those described in Goh et al.
  • the pluripotent cells are dissociated, e.g., to generate a single-cell suspension.
  • the dissociation of the pluripotent cells is chemical, molecular (e.g., enzyme mediated), or mechanical dissociation.
  • Methods of chemical, molecular, and/or enzyme mediated dissociation will vary and in some instances may include but are not limited to the use of, e.g., trypsin, TrypLE Express TM , TrypLE Select TM , Accutase®, StemPro® (Life Technologies, Inc., Grand Island, NY), calcium and magnesium free media, low calcium and magnesium medium, and the like.
  • the dissociation media may further include pro-survival factors including but not limited to, e.g., Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g., low does, i.e., sub- immunosuppressive dose, cyclosporine), ZVAD-fmk, pro-survival cytokines (e.g., insulin-like growth factor-1 (IGF-1)), Thiazovivin, etc.
  • pro-survival factors including but not limited to, e.g., Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g., low does, i.e., sub- immunosuppressive dose, cyclosporine), ZVAD-fmk, pro-survival cytokines (e.g., insulin-like growth factor-1 (IGF-1)), Thi
  • methods of culturing pluripotent stem cells include xeno-free culture conditions wherein, e.g., human cells are not cultured with any reagents derived from non-human animals.
  • methods culturing of pluripotent stem cells include feeder-free culture conditions, wherein the pluripotent stem cells are cultured under conditions that do not require feeder cells and/or in feeder cell free medium, including e.g., commercially available feeder-free mediums, such as, e.g., those available from STEMCELL Technologies, Inc. (Vancouver, BC).
  • methods culturing of pluripotent stem cells include culture conditions that include supplemental serum, including e.g.
  • methods include culture conditions that are serum-free, meaning the culture media does not contain animal, mammal, or human derived serum. Serum-free culture conditions may be performed for only a portion of the life of the culture or may performed for the entire life of the culture. As is known in the art, in some instances, cells may be cultured in two dimensional or three dimensional formats (e.g., on non-coated or coated surfaces or within a solid or semi-solid matrix).
  • the pluripotent progenitor cells used according to the methods described herein may be genetically unmodified.
  • genetically unmodified is meant that essentially no modification of the genome of the cells transplanted into the subject has been performed.
  • transient genetic modification is performed at some point during the derivation of the cells but essentially no genetic modification persists in the cells that are eventually transplanted into the subject (i.e. the cells are essentially indistinguishable before the transient genetic modification and after the course of the transient modification).
  • genetically unmodified instances wherein the genome of the cells is not transiently or stably modified, e.g., where the cells are manipulated, e.g., pluripotent progenitors are derived or cells are transformed, without genetic modification (e.g., modification of the nucleotide sequence of the genome) of the cells.
  • the cells used according to the methods described herein may be genetically modified.
  • genetically modified is meant that at least one nucleotide is added to, changed within, or deleted from of the genome of the cell.
  • the genetic modification may be an insertion of a heterologous sequence, e.g., a sequence that encodes a tag, a label sequence, a reporter, a selectable marker, a gene encoding a protein from a species different from that of the host cell, etc.
  • the genetic modification corrects a defect or a mutation within the cell, e.g., corrects an anomalous mutation that confers a tissue dysfunction or deficiency.
  • the genetic modification deletes or renders inoperable an endogenous gene of the host cell.
  • the genetic modification enhances an endogenous gene of the host cell.
  • the genetic modification represents a change that enhances survival, control of proliferation, and the like.
  • Cells may be genetically altered by transfection or transduction with a suitable vector, homologous recombination, or other appropriate technique, so that they express a heterologous sequence or have altered expression of an endogenous gene.
  • a suitable vector homologous recombination, or other appropriate technique
  • the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, and embryology. With respect to tissue culture and stem cells, the reader may wish to refer to Teratocarcinomas and embryonic stem cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd.1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al.
  • Systems of the subject disclosure may include a cell production system, e.g., for the production of a homogenous or highly pure population of eBC from pluripotent progenitor cells.
  • the cell production system includes a cell culture chamber or cell culture vessel for the culture of desired cell types.
  • Such cell culture chambers may be con FIG.d for the expansion of pluripotent progenitor cells and for the differentiation and/or lineage restriction of such pluripotent progenitor cells into desired cell types.
  • the cell culture chamber is also con FIG.d for the expansion of eBC.
  • the cell culture chamber or cell culture vessel may be an open culture system, including but not limited to e.g., tissue culture dishes, tissue culture plates, tissue culture multi-well plates, tissue culture flasks, etc.
  • the cell culture chamber or cell culture vessel may be a closed culture system, including e.g., a bioreactor, a stacked tissue culture vessel (e.g., CellSTACK Culture Chambers available from Corning, Inc. Corning, NY).
  • culture media and or other factors or agents may be exchanged in and out of the cell culture chamber through the use of one or more pumps (e.g., syringe pumps, peristaltic pumps, etc.) or gravity flow devices.
  • the culture system may allow for the sterile exchange of culture media, e.g., through the use of sterile tubing connected, sealed, and reconnected through the use of a sterile devices, including but not limited to, e.g., a sterile tube welder and/or a sterile tube sealer.
  • the cell culture system may be con FIG.d to control certain environmental conditions, including but not limited to e.g., temperature, humidity, light exposure, air composition (e.g., oxygen levels, carbon dioxide levels, etc.) to achieve the conditions necessary for expansion and/or differentiation of desired cell types.
  • the cell culture chamber may include a cell culture vessel that includes one or more patterned cell culture substrates or one or more arrays of patterned cell culture substrates as described herein.
  • the cell culture chamber may be configured for the production of cells for clinical use, e.g., according to current good manufacturing practice (cGMP) compliant cell culture practices, including the methods and configurations described in e.g., Fekete et al. PLoS ONE (2012) 7(8): e43255; Pham et al. (2014) J Trans Med 12:56; Gastens et al. (2007) Cell Transplant 16(7):685-96; Fernandes et al.
  • cGMP current good manufacturing practice
  • the cell production system may, in some instances, be computer controlled and/or automated. Automated and/or computer controlled cell production systems may include a “memory” that is capable of storing information such that it is accessible and retrievable at a later time or date by a computer. Any convenient data storage structure may be chosen, based on the means used to access the stored information. In certain aspects, the information may be stored in a “permanent memory” (i.e. memory that is not erased by termination of the electrical supply to a computer or processor) or “non-permanent memory”.
  • a computer controlled and/or automated cell culture system may include a module or program stored in memory for production of cells according to the methods described herein. Such a module may include instructions for the administration of induction agent and/or induction compositions, e.g., at particular timing intervals or according to a particular schedule, in order to generate a desired cell type.
  • a computer module may further include additional modules for routine cell culture tasks including but not limited to, e.g., monitoring and record keeping, media changes, environmental monitoring, etc.
  • Systems of the present disclosure include components and/or devices for delivering cells produced according to the methods described herein to a subject in need thereof.
  • a system for treating a subject with a derived tissue dysfunction or deficiency includes a cell injection system for delivering cells in a carrier, with or without optional adjuvants, to a desired injection site, including diseased tissue, adjacent to diseased tissue, and/or within, on or near a dysfunctioning organ.
  • Such systems utilize known injection devices (e.g., including but not limited to needles, bent needles, cannulas, syringes, pumps, infusion devices, diffusion devices, etc.) and techniques (e.g., including but not limited to intramuscular injection, subcutaneous injection, device-guided injection, etc.).
  • a device or technique used for the delivery of a cell scaffold or other bioengineered device may be con FIG.d or adapted for use in a cell delivery system for use in delivering cells derived according to the methods described herein.
  • compositions and kits for use in the subject methods.
  • the subject compositions and kits include any combination of components for performing the subject methods.
  • a composition can include, but is not limited to and does not require, the following: cell dissociation agents and/or media, cell reprogramming agents and/or media, pluripotent progenitor cells, cell culture agents and/or media, cell differentiation agents and/or media; lineage restriction agents (e.g., induction agents) and/or media; conventional agents for treating diseases and/or dysfunctions, pro-survival factors, pro-engraftment factors, functional mobilization agents and any combination thereof.
  • lineage restriction agents e.g., induction agents
  • a kit can include, but is not limited to and does not require, the following: any of the above described composition components, a sample collection container, a sample collection device (e.g., a sample collection container that includes a sample enrichment mechanism including, e.g., a filter), a tissue collection device (e.g., a biopsy device), a tissue dissociation device, a cell culture vessel, a cell production system; and any combination thereof.
  • a kit can include, but is not limited to and does not require, a cell delivery system and/or a cell injection system con FIG.d for delivery of cells derived according to the methods described herein.
  • kits may include a cell injection system con FIG.d for injection or delivery of cells into a desired area of the subject in order to effectively treat the subject for a tissue dysfunction or deficiency, e.g., through delivery of cells to the tissue.
  • kits may include a cell delivery or injection system, as described herein, including individual components of such systems in assembled or unassembled form.
  • cells derived according to the methods described herein may be “preloaded” into a cell injection or delivery system such that the system is provided in a “ready-to-use” configuration.
  • a cell injection or delivery system may be provided in an “unloaded” configuration such that cells derived according to the methods described herein must be loaded into the system, with any desired carrier or vehicle, prior to use.
  • the subject kits may further include (in certain embodiments) instructions for practicing the subject methods.
  • These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • a computer readable medium e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded.
  • Machine Learning Embodiments are provided for the use of machine learning to develop methods of differentiating stem or progenitor cells to a cell type of interest. Such methods may utilize single cell and spatial technologies to generate a spatiotemporal multi-omics cell atlas for development of the cell or tissue of interest. Alternatively, an existing cell atlas such as that disclosed herein may be used. A machine learning algorithm is used to prioritize the combinations of candidate developmental signals for in silico method of differentiating a cell type of interest. Functional validation of the predicted developmental signals can be performed.
  • the cell atlas may utilize a program, e.g. Seurat, for single cell gene expression quantification, dimension reduction, clustering analysis and marker gene identification.
  • a tissue of interest at different stages of development is used, and can be merged separately for downstream analysis.
  • Cells expressing no more than 200 genes or more than 8000 genes may be removed.
  • Cells having more than 20% of reads mapped to mitochondria may be also removed.
  • Highly variable features are identified, and PCA is performed using variable features.
  • Cells can be clustered using multiple dimensions and visualized, e.g.using UMAP.
  • the initial iterative clustering revealed clusters for gross cellular compartments. These cellular compartments may be subset for further iterative clustering and annotation.
  • the machine learning process utilizes a model (referred to herein as the Manatee model), which is adapted from VAE by constraining its latent space to represent transcription factor (TF) expression.
  • TF transcription factor
  • latent variables associated with candidate TFs are adjusted. Such an adjusted latent space will then be decoded as final predictions.
  • the perturbation effect was quantified by computing the Pearson correlation (R) between the average basal cell and the perturbation expression profiles.
  • R Pearson correlation
  • both the encoder and decoder neural networks consist of two fully connected layers, each with the same number of nodes as the number of genes; 2) the latent space contain the same number of variables as the number of TFs; and 3) the following loss function is optimized during training: ⁇ ⁇ ⁇ ⁇ + ⁇ ⁇ + ⁇ ⁇ [00156] where LR and DKL represent reconstruction loss and Kullback–Leibler Divergence against the N(0, 1) normal distribution respectively, as the two regular VAE loss terms.
  • the additional L r term represents the TF reconstruction loss, which is the mean square error (MSE) between reparameterized latent variables (Z) and TF expression (TF): ⁇ ⁇ ⁇ ⁇ ( ⁇ , ⁇ ) [00157]
  • MSE mean square error
  • Z reparameterized latent variables
  • TF TF expression
  • TFs may be defined based on the Gene Ontology (GO) database, under terms GO:0003700, “DNA-binding transcription factor activity”, GO:0003677, “DNA binding”, GO:0140110, “transcription regulator activity” and GO:0006355, “regulation of DNA-templated transcription”.
  • Pathway gene lists from the GO database may also be included. When perturbing specific signaling pathways, the corresponding lists will be retrieved, and non-TFs will be filtered. The yielded lists are then manually annotated based on previous literature in terms of determining perturbation directions. Manatee can be trained by merging in vivo and in vitro single cell datasets to comprehensively capture regulatory logics during the cell specification and differentiation in vivo.
  • Databases can typically comprise cell atlas information, a machine learning model, databases of transcription factors and gene pathways, etc. The results and databases thereof may be provided in a variety of media to facilitate their use.
  • Media can refer to a manufacture that contains database information; and methods of analysis as described above.
  • the databases and comparative algorithms can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the information provided herein.
  • the minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • the data storage means may comprise any manufacture comprising a recording of the present information as described above, or a memory access means that can access such a manufacture.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test expression repertoire.
  • the data analysis may be implemented in hardware or software, or a combination of both.
  • a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention.
  • data may be used for a variety of purposes, such as drug discovery, analysis of interactions between cellular components, and the like.
  • the analysis is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device.
  • Program code is applied to input data to perform the functions described above and generate output information.
  • the output information is applied to one or more output devices, in known fashion.
  • the computer may be, for example, a personal computer, microcomputer, or workstation of conventional design.
  • Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language.
  • Each such computer program can be stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems.
  • One format for an output tests datasets possessing varying degrees of similarity to a trusted repertoire. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test repertoire.
  • Further provided herein is a method of storing and/or transmitting, via computer, data collected by the methods disclosed herein.
  • Data can be input into a computer by a user either directly or indirectly. Additionally, any of the devices which can be used to perform or analyze NIA can be linked to a computer, such that the data is transferred to a computer and/or computer-compatible storage device.
  • Data can be stored on a computer or suitable storage device (e.g., CD).
  • Data can also be sent from a computer to another computer or data collection point via methods well known in the art (e.g., the internet, ground mail, air mail). Thus, data collected by the methods described herein can be collected at any point or geographical location and sent to any other geographical location.
  • the analysis and database storage can be implemented in hardware or software, or a combination of both.
  • a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention.
  • Such data can be used for a variety of purposes, such as patient monitoring, initial diagnosis, clinical trial analysis, and the like.
  • the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device.
  • Program code is applied to input data to perform the functions described above and generate output information.
  • the output information is applied to one or more output devices, in known fashion.
  • the computer can be, for example, a personal computer, microcomputer, or workstation of conventional design.
  • Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language.
  • Each such computer program is preferably stored on a storage media or device readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system can also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention.
  • One format for an output means test datasets possessing varying degrees of similarity to a trusted profile. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test pattern.
  • Standard abbreviations may be used, e.g., room temperature (RT); base pairs (bp); kilobases (kb); picoliters (pl); seconds (s or sec); minutes (m or min); hours (h or hr); days (d); weeks (wk or wks); nanoliters (nl); microliters (ul); milliliters (ml); liters (L); nanograms (ng); micrograms (ug); milligrams (mg); grams ((g), in the context of mass); kilograms (kg); equivalents of the force of gravity ((g), in the context of centrifugation); nanomolar (nM); micromolar (uM), millimolar (mM); molar (M); amino acids (aa); kilobases (kb); base pairs (bp); nucleotides (nt); intramuscular (i.m.); intraperitoneal (i.p.); subcutaneous (s.c.); and the like.
  • RT room
  • Example 1 While stem cell technologies offer great promise, a broader approach to harness their potential to generate designer cell-based personalized therapeutics requires deeper understanding of tissue level interactions.
  • the human esophagus becomes lined with stratified columnar epithelium that is protected by the terminal differentiation into multi-ciliated cells. Later at 20 weeks’ gestation, squamous stratified epithelium appears to replace the ciliated epithelium as the dominant terminal differentiation trajectory. While the epithelial morphogenesis has been documented superficially, the underlying stromal composition and roles in morphogenesis remain unknown. Developmental studies in skin squamous stratified epithelium and our recent work have shown that regional mesodermal morphogenic signals wire the spatial-temporal specific stratification program in surface ectoderm progenitors, and these signals are required in our hPSC-derived skin production for optimal graftability.
  • FIG.1A In collaboration with the NIH-sponsored UW birth Defects Research Laboratory (BDRL), we ethically collected human embryonic and fetal esophagus samples from 35 individual donors for single cell and spatial profiling (FIG.1A, FIG.8A).
  • Our dataset ranged from 45 (mid first trimester) to 132 (mid second trimester) post-conception days (noted as E45 to E132), spanning several key developmental milestones: onset of epithelial morphogenesis, fetal-specific ciliogenesis and squamous stratification (FIG.1B upper panel).
  • EPI epithelium
  • MES mesenchyme
  • ENS enteric nervous system
  • ENDO endothelium
  • SKM skeletal muscle
  • IM immune cells
  • Epi_PG esophageal basal cells
  • KRT5 esophageal basal cells
  • KRT13 esophageal suprabasal cells
  • ciliated cells FOXJ1
  • PAX8 respiratory tract
  • NKX2-1 pharyngeal pouch
  • TBX1 stomach
  • CLDN18 pharyngeal pouch
  • CDX2 intestine
  • the ciliogenesis trajectory originates from basal cells, which first downregulate KRT15, maintain the expression of KRT5 while begin to express KRT13 in the transitioning suprabasal cell population (SB-1). Following that, a unique club cell-like population (SCGB1A1, UPK2, UPK1B, MSLN, named as SB-5) appears and generated multiciliated cells (FOXJ1). Our dataset also captures the complete ciliogenesis process by delineating the nascent ciliated cells (MUC1, DEP1, CDC20B, named as Cil-1) and the mature ciliated cells (TFF3, SNTN, TPPP3, CAPS, named as Cil-2) (FIGS.2E-F, FIGS.8H-J, 9C).
  • the squamous stratification trajectory transitions through multiple suprabasal descendants by gradually downregulating basal cell marker KRT5, early squamous marker KRT13, KRT6A, KRT6B and upregulating late squamous marker KRT4 (FIG.2F, FIGS.8H-J, named as SB-2, SB-3, and SB-4).
  • the ciliogenesis was predominantly observed at mid stage, marked by the peak of SB-5 before E80 (FIG.2B, FIGS.8H-J).
  • SB-5 is the only cluster that serves to replenish ciliated populations, the exhaustion of SB-5 by E101 underlies the later gradual lineage extinction.
  • FIG.9C Myofibroblasts and fibroblasts share a common developmental origin.
  • a fibroblast progenitor cluster (COL1A1, PITX1, ZEB2, SNAI2, named as Fib_PG,) that was prominent at the early stage (E47 and E53), but lacking at later stages (after E101) (FIGS. 3B-D, FIGS. 8K-M, 9C).
  • Fib_1, Fib_2, Fib_3 three major fibroblast subtypes
  • PDGFRA, ACTA2, named as MF three major fibroblast subtypes
  • All of these cells including the progenitors express FOXF1 and NKX6- 1, confirming their identity from splanchnic mesoderm (FIG. 12A).
  • the Fib_1 cells express high levels of POSTN and TNC, suggesting a potential regulator role in epithelial proliferation and differentiation through paracrine signaling (FIGS.3C, 3E, FIGS.8M, 12A-B).
  • Fib_2 cells exhibit abundant deposition of collagen (COL1A1, COL3A1, COL14A1), elastic fiber (FBLN1, FBLN5, FBN1, FBN2) and proteoglycan (DCN, LUM) genes, all of which could provide structural support for the esophagus (FIGS. 3C, 3E, FIGS. 8M, 12A-B).
  • Fib_3 cells specifically express the calcium-activated potassium channel gene KCNN3, the sodium channel gene SCN7A and the CD34 marker, suggesting their potential roles in neurotransmission and regulation of muscular motility (FIGS. 3C, 3F, FIGS. 8M, 12A-B).
  • Fib_1 Myofibroblasts share common markers with Fib_1, including PITX1, ZEB2, DPT, POSTN and TNC (FIGS. 3G-H, FIGS. 8K-M, 12A-B), suggesting potential roles in regulating epithelial regeneration.
  • Fib_4 cells express genes related to angiogenesis and vascular contractility (ANGPTL1 and PRRX1), underlying a putative perivascular fibroblast identity (FIG 8M).
  • Fib_5 cells exclusively express the PI16 gene and therefore considered to be adventitial fibroblasts.
  • Fib_5 cells also express multiple glia-related genes (DCX, PLP1, NGFR and ENTPD2), suggesting a close relationship with the enteric nervous system (FIGS. 8M).
  • Fib_4 and Fib_5 do not express splanchnic mesodermal regulator genes FOXF1 and NKX6-1 (FIGS.3C, 3F, 12A-B), bringing the questions about their developmental origin and lineage plasticity.
  • the pseudotime trajectory analysis predicts that fibroblast progenitors first differentiate into three branches, Fib_1, Fib_2, and myofibroblasts; while Fib_2 further follows two branches, one into Fib_3 and another into Fib_4 and 5 (FIG.3H).
  • Interstitial cells of Cajal and smooth muscle lineages are specified early in development.
  • the highly specialized mesenchymal cells, interstitial cells of Cajal (KIT, ETV1, ANO1, named as Fib_ICC), and smooth muscle cells (ACTA2, TAGLN, DES, SYNM, named as SM) are essential executors for the rhythmic peristalsis of esophagus.
  • KIT mesenchymal cells
  • ETV1 ETV1, ANO1, named as Fib_ICC
  • smooth muscle cells ACTA2, TAGLN, DES, SYNM, named as SM
  • FIG.3A Another key component in the mesenchymal compartment is the pericyte (PDGFRB, RGS5, NOTCH3, NDUFA4L2, named as Peri)
  • Peri Pericyte progenitors
  • the progenitor cells downregulate TUBB3 and UCHL1, and begin to exclusively express S100B as glial lineage commitment (FIG.9H-J, 9M).
  • glial lineage commitment (FIG.9H-J, 9M).
  • Glia_1 marked by BCAN, APOE, ENTPD2, Glia_2 marked by RXRG, GFRA2 and TNC, and Glia_3 marked by MAL, DHH (FIG.9K).
  • the glial cells are enriched in later stages compared to the neuronal lineage.
  • Glia_1 was first observed at E59, while Glia_2 and Glia_3 were detected only after E80 (FIG.9J).
  • capillary (Art_1, marked by GJA4, CXCR4, PGF) and large (Art_2, marked by HEY1 and GJA5) arterial cells, as well as lymphatic vessel cells (Lym_1, marked by LYV1, PROX1 and TFF3) were observed as early as E47.
  • Lymphatic valve cells (Lym_2, specifically express SCG3, FOXC2, and GATA2) emerge at E80 (FIGS. 10A-D). This suggest that the establishment of arterial and lymphatic lineages occur earlier than E47.
  • Ven_PG venous progenitors
  • Ven_1 the potential intermediate venous cells (Ven_1) are being specified.
  • Ven_3, marked by CD83, RGCC and CA4 venous cells emerge, which were first observed at E72.
  • the putative satellite cells (SC_1 and SC_2) were found heterogeneous in the expression of SOX8, PAX7 and MYF5. These four populations were observed at all developing stages, suggesting early specification during esophageal myogenesis (FIGS.10H-K).
  • the entire skeletal muscle lineage expresses pharyngeal mesoderm transcription factors (ISL1, TBX1, MSC and SIX1) and lacks PAX3 This finding is consistent with the non-somitic, cranial mesodermal origin of esophageal skeletal muscle previously revealed in mouse studies (FIGS.10K).
  • Identified populations included monocytes (Mono, CD163), dendritic cells (DC, LYZ or HLA-DRA), mast cells (Mast, KIT, TPSB2) from the myeloid lineage, as well as B cells (B, IGHM, CD79A, CD79B), natural killer cells (NK, GZMA, KLRD1) and type 2 and 3 innate lymphoid cells (ILC2/3, GATA3, RORA, RORC, KIT) from the lymphoid lineage (FIG.10O).
  • T cells (T, CCR7, CD3D, CD3G) emerge at E80, revealing the later migration from the thymus to the developing esophagus (FIGS.10N-O), and emphasizing the presence of the innate rather than adaptive immune system during esophageal development.
  • Spatiotemporal cellular dynamics establish esophageal tissue architecture. With the cell types identified, we orthogonally performed Visium and CODEX to dissect the spatiotemporal orchestration of developing esophageal epithelium with its surrounding stroma.
  • Fib_2 cells were mapped to the middle layer outside of Fib_1 and myofibroblasts since E72, while Fib_3 and interstitial cells of Cajal nicely intermingled with smooth muscle cells in the outer layer (FIGS. 11A-B, 11E-F).
  • Abundant enteric neural and glial cells were mapped at both mid and late stages (FIGS. 11C, 11G). By the late stage, neural cells were found more scattered within muscularis intestinal, while glial cells tended to aggregate in the adventitial plexuses (FIG. 11G).
  • Hierarchical clustering based cell type annotation further confirmed the spatial distribution of various mesenchymal populations at mid and late stages (FIGS.4A-B). Notably, our sampling of early-stage tissues captured the key coordinated events for basal cell specification and mesenchymal diversification (E45 to E72, FIGS. 4A-B).
  • the multilayered early esophageal epithelium is composed by the progenitors (Epi_PG, marked by NCAM1 and GATA6 and absent of KRT15 and KRT5) only.
  • Immediately surrounding the primitive epithelium are relatively homogeneous fibroblast progenitors (Fib_PG) with expression of PITX1, POSTN and DCN.
  • our multi-omics atlas catalogs the detailed spatiotemporal cellular dynamics of human esophageal development.
  • the esophageal rudiment is lined with progenitor-only epithelium which is surrounded by fibroblast progenitors and primitive muscularis basement.
  • these fibroblast progenitors differentiate into diverse lineages and establish the highly organized stromal architecture.
  • the stromal cells further mature and expand from mid to late stage when the squamous differentiation wave replaces the ciliogenesis wave in the epithelial compartment (FIG.4C).
  • Spatiotemporal candidate signal nomination inspired by the multi-omics atlas.
  • Fib_1 maintains a constant 10 ⁇ m cellular layer adjacent to the basement membrane throughout the developmental process.
  • Fib_1 layer Immediately surrounded Fib_1 layer is the myofibroblast layer that doubles its thickness only at the late stage.
  • layers of Fib_2 and muscularis basement constituted by smooth muscle, interstitial cells of Cajal and Fib_3 proliferate and thicken dramatically during development, which increases the overall distance from the basement membrane (Fib_2 is located 69.70 ⁇ 4.40 ⁇ m from the basement membrane at E58, 103.57 ⁇ 9.51 ⁇ m at E72, 223.37 ⁇ 21.26 ⁇ m at E120.
  • Muscularis basement membrane is located 104.50 ⁇ 6.74 ⁇ m from the basement membrane at E58, 219.65 ⁇ 14.68 ⁇ m at E72, 497.53 ⁇ 37.66 ⁇ m at E120) (FIG.5B).
  • Fib_1, Fib_2 and myofibroblasts emerge as key signal senders at mid and late stages.
  • EGF EGF, WNT and ncWNT were identified as inductive signals for basal cell maturation in the interrogation of cell-cell communication between basal cells and the mid-late local mesenchyme (FIG.5C).
  • Fib_2 muscularis basement no longer serves as the local mesenchyme to affect epithelial development. Instead, Fib_1 replaces interstitial cells of Cajal to provide BMP.
  • Fib_1, Fib_2 and myofibroblasts introduce WNT signal, while newly specified basal cells serve as an additional source for WNT in an autocrine manner.
  • EGF is the only signal revealed at late stage, and is provided solely by myofibroblasts (FIG.5D).
  • Manatee a deep learning framework, to screen in silico for the optimal combination of EGF, WNT, TGF ⁇ and BMP signals for esophageal basal cell specification.
  • VAE variational autoencoder
  • TF transcription factor
  • FIG.6A transcription factor
  • the adjusted latent space are further flowed through the decoder neural network.
  • FIGS.14A-E These progenitors matured into basal cells after another month (FIGS.14A-E).
  • Manatee was trained with day 4, 10, 16, 24 and 43 in vitro derived single cell transcriptomic profiles (FIGS. 13A-C), together with in vivo epithelial cell profiles (FIG.2A), to capture the complete set of regulatory logics related to the esophageal basal cell development.
  • Day 16 and 24 cells were used here as starting points for the in silico perturbation.
  • the basal cell derivation effectiveness was quantified and ranked using the Pearson correlation between the average in vivo human developing basal cell and the perturbation expression profiles (FIG. 6B).
  • #19-derived basal cells could function as their in vivo counterparts to self-renew and undergo squamous differentiation in culture and tissue organoid assays.
  • these basal cells can be cryopreserved, thus providing flexibility in the future clinical scaling up.
  • solid organoids were formed with KRT5 + basal cells in the periphery and KRT13 + suprabasal cells inside (FIGS.7H and 14I).
  • basal cells could regenerate tissue-specific squamous stratified epithelium by growing these cells on devitalized de-epidermal human dermis.
  • Stratified skin basal cells will express KRT1 and KRT10, proteins needed to maintain skin integrity.
  • the #19-derived basal cells contributed to building the COLVII + basement membrane and generated KRT13 + , KRT4 + and IVL + squamous descendants on top of KRT5 + KRT15 + ITGB4 + basal layer.
  • GATA6 plays essential roles in establishing endoderm in early development and later GATA6 expression becomes restricted posteriorly to gastric, intestinal and colonic epithelium but absent from the esophagus. Notably, re-emergence of GATA6 expression has been found in Barrett’s esophagus and esophageal adenocarcinoma, characterized by the pathological conversion of stratified epithelium to intestinal metaplastic simple epithelium. Our atlas could be leveraged in conjunction with disease and cancer databases to investigate any potential reactivation of developmental programs, and the role of the tumor microenvironment, during cancer progression.
  • fibroblast progenitors stratify into 4 layers: Fib_1 closest to the basement membrane, and myofibroblast, Fib_2, Fib_3 further away, respectively.
  • Fib_1 closest to the basement membrane
  • Fib_2, Fib_3 further away
  • the drastic expansion of the Fib_2 layer in the submucosa distances the muscularis intestinal which is composed of intertwined Fib_3, smooth muscles, and interstitial cells of Cajal, thus physically insulating morphogens secreted by muscularis basement from the epithelium.
  • DCN a matrix component abundantly secreted by Fib_2, could antagonize BMP and TGFb signaling, which are critical pathways in basal cell commitment.
  • the mesenchymal cellular dynamics alter the tissue architecture. This results in stage-dependent changes in the cellular components of the local mesenchyme, which potentially drive basal cell development. While the local mesenchyme cell types remain the same from the mid to late stage, these cells could change their secreted morphogen profiles as they mature. Specifically, myofibroblast cells begin to express GREM2, an effective BMP antagonist, from the late stage, rendering the epithelium in a SMAD inactive microenvironment to promote basal cell self-renewal (FIG.5F). [00203] Instead of relying on prior knowledge gained using model animals, we here combined our tissue atlas and machine learning to design a clinically compatible hPSC differentiation system.
  • the hPSC-to-esophageal basal cell system with our previous hPSC-to-skin basal cell differentiation systems provide a unique chance to interrogate regulatory modules controlling the tissue-specific basal cell development.
  • Our previous work revealed that during epidermal lineage commitment, the morphogen-induced expression of TFAP2C primes the chromatin landscape. Meanwhile, TFAP2C activates the canonical basal cell master regulator P63, which further effects on the primed chromatin for stratified epithelium maturation.
  • Our current work suggests a similar regulatory mechanism operates in endoderm.
  • induced key endodermal TFs such as SOX2 and GATA6 shape the endoderm-specific chromatin landscape as well as inducing P63; in turn, P63 induces stratified epithelium while repressing GATA6 to drive differentiation forward.
  • Our functional assays including seeding esophageal basal cells on skin dermis, provide strong evidence for the distinct and stable cellular specification network. Future mechanistic and evolutionary studies interrogating shared regulatory logic will help accelerate the production of other stratified epithelia such as bladder and cornea, as well as the mechanism of the master regulator P63 in specifying tissue-specific stratification programs in ectoderm and endoderm.
  • a non-autonomous feedback loop between epithelial and stromal cells might be formed upon basal cell specification since the mid stage, reinforcing the dorsal esophageal fate instead of ventral airway or posterior gastric identities.
  • the surrounding myofibroblasts acquire expression of GREM2 from mid to late stage, which inhibit BMP signaling (FIG. 5F).
  • the structural remodeling of local mesenchyme as well as stage-dependent morphogen dynamics shape the earlier differentiation-permissive and later self-renewal-enhancing microenvironment.
  • Exemplary conditions that may be treated by the cells of the invention include repair of congenital or acquired conditions, such as long-gap esophageal atresia (LGA), long-segment esophageal disruption, refractory GERD, caustic ingestions, cancer, and stricture.
  • LGA long-gap esophageal atresia
  • GERD refractory GERD
  • caustic ingestions cancer
  • stricture refractory GERD
  • a component is used at a value of X mM or X ng/ml
  • the method may be performed using a range of effective concentrations, for example about 0.01X, 0.05X, 0.10X, 0.15X, 0.20X, 0.25X, 0.30X, 0.35X, 0.40X, 0.45X, 0.50X.
  • the RUES2 line (Rockefeller University Embryonic Stem Cell Line 2, NIH approval number NIHhESC-09-0013) was originally from Rockefeller University. All experiments using hPSC lines were approved by Stanford University. The H9 line was maintained in Essential 8 media (Thermo Fisher Scientific) on culture plates coated with iMatrix-511 (Takara).
  • the RUES2 line was initially maintained on mouse embryonic fibroblasts (MEFs, irradiated CF-1 MEF, Thermo Fisher Scientific) in the maintenance medium: 400 ml of DMEM/F12 (Thermo Fisher Scientific), 100 ml of KnockOut Serum Replacement (Thermo Fisher Scientific), 5 ml of GlutaMAX (Thermo Fisher Scientific), 5 ml of MEM non-essential amino acids solution (Thermo Fisher Scientific), 3.5 ml of 2- mercaptoethanol (Sigma-Aldrich), 1 ml of primocin (Thermo Fisher Scientific), and FGF2 (R&D Systems) with a final concentration of 20 ng/ml to make a total of 500 ml of medium.
  • MEM non-essential amino acids solution Thermo Fisher Scientific
  • 2- mercaptoethanol Sigma-Aldrich
  • primocin Thermo Fisher Scientific
  • FGF2 R&D Systems
  • the RUES2 cells were thawed and plated on culture plates coated with iMatrix-511 (Takara) in StemFit Basic04 Complete medium (Amsbio).
  • hPSCs were detached with TrypLE Select (Thermo Fisher Scientific) and plated at 1M per 10 cm dish. hPSCs were maintained in an incubator with 95% humidity, 95% air and 5% CO2 at 37°C, and routinely tested for mycoplasma contamination.
  • Human Esophageal Tissue Histology Staining Human embryonic and fetal esophageal and stomach tissues were washed in cold HBSS.
  • tissues were transferred to 30% 1:1 sucrose-OCT solution at 4 °C overnight, and embedded in OCT the next day.
  • H&E staining sections were initially warmed up to room temperature for 10 minutes, rehydrated in PBS for 10 minutes, and rinsed in water for 1 minute. Then sections were stained in Hematoxylin (Millipore Sigma) for 30 seconds, rinsed in water, and stained in Bluing reagent (Dako) for 30 seconds, rinsed in water.
  • esophageal and stomach tissues were washed in cold HBSS.
  • Whole esophageal tissues were collected and cleaned up by removing stomach tissues slightly proximal from the gastroesophageal junction and other connective tissues.
  • esophageal tubes were cut open using fine scissors, and gently digested in 6 cm dishes with 25 U/ml Dispase (Corning) + 100 ⁇ g/ml DNase I (Millipore Sigma) at room temperature for 20 to 40 minutes.
  • whole esophageal tubes were digested in Dispase/DNase I solution at room temperature for 10 minutes instead.
  • Epithelium was physically peeled off from surrounding stromal tissues with fine forceps, and collected in a 15 ml Falcon tube.
  • the epithelium was further dissociated into single cells in TrypLE Express (Thermo Fisher Scientific), first triturated gently using P1000 pipette and then shaken in a Thermomixer at 37 °C at 1000 rpm for 10 to 20 minutes.
  • FACS buffer PBS supplemented with 1% BSA, 2 mM EDTA, 1X Anti-Anti, 25 mM HEPES pH 7.0
  • Stromal tissues were transferred to clean dishes and chopped into small pieces using scissors. Tissue pieces were transferred to a 15 ml Falcon tube with 10 ml collagenase solution (5 mg/ml Collagenase in DKSFM), triturated gently using P1000 pipette, and then shaken in a Thermomixer at 37°C at 1000 rpm for 10 to 30 minutes. 1 ml 0.25% Trypsin was then added to the solution for an additional 5 min dissociation. Dissociation was stopped by addition of the FACS buffer supplemented with 10% FBS. Dissociated cells were resuspended in cold 0.04% BSA in PBS and filtered through 40 ⁇ m strainers.
  • Live cells were sorted using FACSAria II (BD Biosciences) at Stanford Shared FACS Facility and collected in the FACS buffer supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 2X Anti-Anti on ice. The sorted cells were pelleted, washed with cold 0.04% BSA in PBS, and resuspended in cold 0.04% BSA in PBS for cell number counting. Cell alive rate was determined using Trypan Blue. Only samples with greater than 90% cell alive rate were further processed. Cells were then subjected to 10X Chromium scRNA-seq based on the manual.
  • Sequencing libraries were prepared using Chromium Single Cell 3’ v3 (CG000183 Rev C) and v3.1 (CG000315 Rev A) protocols according to the manufacturer’s manual. The pooled, 3’-end libraries were sequenced using Illumina NovaSeq.
  • Bioinformatic analysis of scRNA-seq data Cell Ranger version 4.0.0 was used for primary data analysis, including demultiplexing, alignment, mapping and UMI counting. Specifically, for alignment and mapping, the GRCh38 reference genome and corresponding annotation were used.
  • Seurat version 4.0.3 was used for single cell gene expression quantification, dimension reduction, clustering analysis and marker gene identification.
  • Epithelial samples and stromal samples of different developing stages were merged separately for downstream analysis. Cells expressing no more than 200 genes or more than 8000 genes were removed. Cells having more than 20% of reads mapped to mitochondria were also removed. A total of 2,000 highly variable features were identified, and PCA was performed using variable features. Cells were clustered using 10 dimensions and visualized using UMAP. The initial iterative clustering revealed clusters for gross cellular compartments: epithelium (EPI. EPCAM, PITX1, KRT5, KRT4), stomach (ST. GATA6, CLDN18, TFF1, MUC5AC), mesenchyme (MES. COL1A1, PDGFRA), enteric nervous system (ENS.
  • HAND2, CDH2, PHOX2B endothelium
  • skeletal muscle SKM. PAX7, CDH15, MYF5
  • immune cells IM. PTPRC, LAPTM5
  • ERY. HBM, HEMGN erythrocyte
  • SM_early mainly enriched in E47 to E59 samples; lack of SYNM
  • SM-mid mainly enriched in E72 to E113 samples; expressing moderate levels of SYNM
  • SM_late mainly enriched in E127 to E132 samples; expressing highest levels of SYNM
  • 13 clusters were identified as fibroblasts (PDGFRA), with one co-expressing cell cycle genes labeled as Cy.Fib (TOP2A, MKI6, TYMS).
  • Fib_1 PITX1, POSTN, OSR2, CXCL14
  • Fib_2 COL1A1, COL3A1, COL14A1, FBLN1, DCN
  • Fib_3 KCNN3, CD34, C7. All three fibroblast types exhibited two distinct developmental statuses along maturation: mid stage enriched in E59 to E82 samples while late stage enriched in E101 to E132 samples.
  • Two clusters co-expressing fibroblast markers PDGFRA, DPT
  • muscle markers ACTA2, TAGLN
  • Fib_PG One common progenitor cluster was identified (Fib_PG) enriched only in E47 to E80 samples (FIG.9C). These cells expressed low levels of DPT and high levels of mesodermal regulator genes ZEB2 and SNAI2 (FIGS.8K-M), suggesting an immature identity. Two distinct fibroblast populations (Fib_4 and Fib_5) were found only after E80 (FIG. 9C). Their absence of esophageal mesoderm regulator genes (FOXF1, NKX6-1) (FIG. 12A) indicated a potential distinct developmental origin from the above described fibroblast progenitors. A distinct cluster with unique expression of KIT, ETV1, ANO1 was annotated as interstitial cells of Cajal (Fib_ICC).
  • Pericytes were annotated based on their expression of PDGFRB, NOTCH3 and absence of PDGFRA (FIG. 9F).
  • One cluster of pericytes exhibiting expression of cycling genes (TOP2A, MKI67, TYMS) was annotated as cycling pericyte (Cy.Peri).
  • Three diverse pericyte subtypes (Peri_1, Peri_2, Peri_3) were identified based on their different expression in several key genes including RGS5, PRRX1, ACTA2, LUM (FIG.s 8K-M). All three subtypes were detected after E80.
  • pericyte cluster was found constantly throughout all the stages, and it did not show cycling features or subtype- enriched genes (FIGS 8K-M, 9C), thus annotated as pericyte progenitors (Peri_PG).
  • Peri_PG pericyte progenitors
  • MS mesothelium
  • FIG.9C mesothelium
  • the enteric nervous system was largely categorized in two major cell types with resolution at 1.3: neural (ASCL1, TUBB3, PHOX2B) and glial (00B, ERBB3, SOX10) cells, besides a cycling cluster (TOP2A, MKI67, TYMS).
  • ENS enteric nervous system
  • a cycling cluster TOP2A, MKI67, TYMS.
  • ETV1 Neu_A
  • BNC2 Neu_B
  • branch A we identified one early cluster (Neu_A_early) enriched only in E47 and E53 samples, and one later cluster afterwards. This later cluster expressed NO, VIP, NPY, SCGN and highest levels of GAL, thus representing the inhibitory motor neurons.
  • Neu_B_early enriched in E47 and E53 samples.
  • this cluster branched into two distinct subtypes (Neu_B_1, Neu_B_2), with Neu_B_2 specifically expressing CASZ1, SLC5A7, SST. Therefore we considered Neu_B_2 as excitory motor neurons with neuroendocrine features.
  • a common transitional neuroblast (NB) population was identified expressing DLL3 and higher levels of ASCL1.
  • Glia_1 was defined by the expression of BCAN, APOE and ENTPD2.
  • Glia_2 was defined by their enrichment of RXRG, TNC and GFRA2.
  • Glia_3 expressed high levels of MPZ, MAL and DHH, suggesting that they might be Schwann cell precursors. Based on differences in expression in NTRK2 and FGL2, Glia_3 was further subdivided into Glia_3_1 and Glia_3_2. All three glial cell types exhibited developmental timepoint-related maturation, thus further classified as mid and late stages (FIGS. 9H-K). One cluster was found co-expressing both neural markers (ASCL1, HAND2, TUBB3) and glial markers ( 00B, ERBB3, SOX10), and only enriched in E47, E53 and E59 timepoints (FIG.9J-K), thus annotated as progenitors (ENS_PG).
  • ENS_PG progenitors
  • the endothelium compartment was separated into 9 clusters with resolution at 0.7.
  • One with cycling features (TOP2A, MKI67, TYMS) was termed as Cy.Endo.
  • Two lymphatic clusters were identified based on their expression of LYVE1, PROX1, CCL21, TFF3 and STAB2. Lym_2 was only found after E80, and showed unique expression of lymphatic valve markers (SCG3, FOXC2, GATA2).
  • Two arterial clusters were identified based on their expression of PRND, GJA4 and CXCR4. These two arterial clusters differed from each other in the expression of PGF, LXN, CHST1 (ART_1) and HEY1, GJA5, CXCL12, GRIA2 (ART_2).
  • Ven_PG progenitor population was found abundant only in earlier stages before E82, thus named as Ven_PG (FIG.10C).
  • Ven_2 One cluster exhibiting higher expression of ACKR1, ADGRG6, SELE and SELP was termed as Ven_2, suggesting the role as large venous cells.
  • the other with higher expression of CD83, CA4 and INMT was termed as Ven_3, suggesting the identity of capillary venous cells (FIG.s S3A-D).
  • the final cluster was found across all the developmental timepoints, and showed moderate levels for both large and capillary venous markers, thus termed as Ven_1 with a transitional role (FIG. S3C).
  • the skeletal muscle compartment comprised of 8 clusters with resolution at 0.5.
  • the expression of key regulator SOX8 in one cluster led to the annotation of muscle satellite cells (SC_1).
  • SC_1 A transitioning cluster adjacent to SC_1 downregulated SOX8 and upregulated PAX7 and MYF5, thus annotated as SC_2 with a potential identity of activating/differentiating satellite cells.
  • Skeletal muscle cells were largely segregated into two major types. SkM_1 expressed PAX7, MYF5 and NOTCH3, while SkM_2 expressed high levels of MYL1, ACTA1, MYH3 and TPM1 (FIG.
  • NK cells One cluster specifically expressing GZMK, KLRD1, IFNG and GNLY was annotated as NK cells.
  • Another lymphoid cluster was classified as type 2 and 3 innate lymphoid cells (ILC2/3) due to their expression of RORC, TOX2, GATA3 and IL7R. Myeloid lineages were identified in 5 clusters.
  • a minor cluster exhibiting unique expression of KIT, TPSB2, TPSB1 and GATA2 was annotated as mast cells.
  • Monocytes were revealed with high expression of CD163 and LYVE1 (FIGS. 10L-O). Within monocytes, one early-stage subpopulation was found enriched only before E72, and the late-stage subpopulation emerged afterwards (FIG. 10N).
  • Dendric cells were annotated on the basis of expression in HLA-DRA, ITGAX, ITGAM. Dendric cells were also subdivided into two subpoulations, with one expressing high levels of LYZ, marking the immature phenotype, while others expressing high levels of HLA-DRA and HLA-DQA1. These were labeled as DC_early and DC_late, respectively (FIGS.10L-O).
  • Monocle version 2.18.0 was used for single cell trajectory analysis. In order to perform efficient monocle analysis, without losing generality, we limit the maximum number of single cells per cell type as 1000.
  • CellChat was used for single cell intercellular communication analysis.
  • CODEX profiling starts with antibody conjugation, which was performed using protocols and reagents per manufacturer instructions (Akoya; 7000009). Such a process conjugates 50 ⁇ g of immunofluorescence- validated carrier-free antibodies to specific barcodes. Specifically, antibodies were concentrated on a 50 kDa filter equilibrated with the filtration buffer. The sulfhydryl groups were activated by incubating for 30 minutes at room temperature with the reduction mix.
  • Antibodies were then washed with the conjugation buffer once. Oligonucleotide barcodes were resuspended in the conjugation buffer, added to the antibodies, and allowed to incubate for 2 hours at room temperature. The conjugated antibodies were washed 3 times, by resuspending and spinning down at 12,000 g for 8 minutes with the purification solution. Antibodies were then eluted by adding 100 ⁇ l storage buffer and spinning at 3000g for 4 minutes. The conjugated antibodies were stored at 4°C till use. [00238] Before use in multiplexed CODEX experiments, conjugated antibodies were validated with CODEX single stains on human fixed frozen fetal esophageal tissues. Staining was performed with the conjugated antibody as described below.
  • the screening buffer was prepared according to the CODEX manual provided by Akoya Biosciences. Fixed and stained tissues were incubated in the screening buffer for up to 15 minutes. Fluorescent DNA probes were prepared and added to stained tissues for 5 minutes. Tissues were washed 3 times with the screening buffer followed by 1 wash with the CODEX buffer. Tissues were then imaged using a Keyence BZ-X810 inverted fluorescent microscope for the antibody validation. [00239] All fixed frozen tissue CODEX antibody stainings were done according to Akoya Biosciences staining protocol associated with the CODEX Staining Kit with some modifications (Akoya; 7000008).
  • the blocking buffer was prepared by adding N, S, J, and G blocking solutions to the CODEX staining buffer (contained in the CODEX Staining Kit). Antibodies were added to the blocking buffer to make a total volume of 200 ⁇ l. The antibody cocktail was added to the coverslip, and staining was performed in a sealed humidity chamber at 4°C overnight. After staining, coverslips were washed twice in the hydration buffer for 4 minutes followed by fixation in the storage buffer (contained in the CODEX Staining Kit) with 1.6% paraformaldehyde for 10 minutes.
  • CODEX fixative solution (contained in CODEX Staining Kit) was prepared right before the final fixation step.20 ⁇ l of CODEX fixative reagent was added to 1000 ⁇ l 1X PBS. 200 ⁇ l fixative solution was added to the coverslip for 20 minutes followed by 3 washes in 1X PBS. Coverslips were then immediately prepped for imaging. [00240] For CODEX multicycle setup and imaging, coverslips were mounted onto Akoya’s custom-made stage between coverslip gaskets with the tissue side facing up.
  • the coverslips were cleaned from the bottom using a Kim wipe to get rid of any salts.
  • the tissue was stained with Hoechst Nuclear Stain (Thermo Fisher Scientific, cat. no.62249) at a 1:2000 dilution in the 1X CODEX buffer.
  • a 96-well plate was used to set up the multicycle experiment with different fluorescent oligonucleotides in each well.
  • a reporter stock solution was prepared to contain 1:2000 Hoechst stain and 1:12 dilution of assay reagent in the 1X CODEX buffer (contained in the CODEX Staining Kit).
  • Fluorescent oligonucleotides (Akoya Biosciences) were added to this reporter solution at a final concentration of 1:50 in a total of 250 ⁇ l per well. A blank cycle containing no fluorescent probes was performed at the start and end of the experiment to capture residual autofluorescence. Automated image acquisition was performed using the CODEX Instrument Manager (CIM, version 1.30, Akoya Biosciences). Imaging was performed using a Keyence BZ-X810 microscope, fitted with a Nikon CFI Plan Apo l 20X/0.75 objective. The BZ-X software (Keyence) multi-point option was used to define the center and the imaging area corresponding to each region.9-11 z steps were acquired with the pitch set at 1.5 in the BZ-X software.
  • CODEX Analysis Raw tiff files were processed using the CODEX Processor version 1.7.0.6 by Akoya Biosciences. CODEX Processor sequentially performs drift-compensation, deconvolution, background subtraction, tile stitching and segmentation. Such a pipeline yields spatial coordinates as well as marker protein signal intensities for all the detected cells. We then performed quality control based on the average signal intensity of all the DAPI channels. Cells with low average DAPI signals were removed, and the remaining cells were subjected to signal intensity log-transformation, hierarchical clustering and marker-guided annotation. [00242] Manatee.
  • both the encoder and decoder neural networks consist of two fully connected layers, each with the same number of nodes as the number of genes; 2) the latent space contain the same number of variables as the number of TFs; and 3) the following loss function is optimized during training: ⁇ ⁇ ⁇ ⁇ + ⁇ ⁇ + ⁇ ⁇ [00243] where LR and DKL represent reconstruction loss and Kullback–Leibler Divergence against the N(0, 1) normal distribution respectively, as the two regular VAE loss terms.
  • the additional L r term represents the TF reconstruction loss, which is the mean square error (MSE) between reparameterized latent variables (Z) and TF expression (TF): ⁇ ⁇ ⁇ ⁇ ( ⁇ , ⁇ ) [00244]
  • MSE mean square error
  • Z reparameterized latent variables
  • TF TF expression
  • TFs based on the Gene Ontology (GO) database, under terms GO:0003700, “DNA-binding transcription factor activity”, GO:0003677, “DNA binding”, GO:0140110, “transcription regulator activity” and GO:0006355, “regulation of DNA-templated transcription”.
  • H9 or RUES2 hPSCs were plated in E8 medium with 10 ⁇ M Rock inhibitor Y-27632 (Tocris) in 1 well of 12-well culture plates coated with iMatrix- 511.24 hours later, culture medium was changed to MCDB131 base medium supplemented with 5 ⁇ M CHIR99021 (Tocris) and 100 ng/ml Activin A (R&D System) for 24 hours.
  • MCDB131 base medium was prepared as following: 500 ml MCDB131 (Thermo Fisher Scientific), 5 ml of GlutaMAX (Thermo Fisher Scientific), 33 ml of 7.5% Bovine Albumin Fraction V Solution (Thermo Fisher Scientific), 10 ml of 7.5% NaHCO 3 (Thermo Fisher Scientific) and 2 ml of 45% Glucose-D (Sigma Aldrich).
  • the medium was supplemented with 0.5 ⁇ M CHIR99021 and 100 ng/ml Activin A.
  • the medium was supplemented with 100 ng/ml Activin A only.
  • Serum-Free Differentiation (SFD) medium 750 ml of reconstituted IMDM (Thermo Fisher Scientific), 250 ml of F-12 (Corning), 7.5 ml of 7.5% Bovine Albumin Fraction V Solution (Thermo Fisher Scientific), 10 ml of GlutaMAX (Thermo Fisher Scientific), 5 ml of N2 (Thermo Fisher Scientific), 10 ml of B27 (Thermo Fisher Scientific) and 10 ml of Penicillin/Streptomycin (Thermo Fisher Scientific), and adding L- Ascorbic acid (Sigma-Aldrich) and MTG (Sigma-Aldrich) on the day of use to obtain a final concentration of 50 mg/ml and 0.04 ml/ml, respectively.
  • SFD Serum-Free Differentiation
  • mice were cultured in the SFD medium supplemented with 100 ng/ml hEGF, 5 ng/ml BMP4 (R&D Systems), 2 ng/ml TGFB1 (R&D Systems) and 0.4 ⁇ M IWP2 (Selleck Chemicals).
  • negative control strategy #9 cells were cultured in the SFD medium supplemented with 100 ng/ml hEGF, 1 ⁇ M A83-01 (Tocris), 1 ⁇ M DMH-1 (Tocris) and 3 ⁇ M CHIR99021 (Tocris).
  • base control strategy #14 cells were cultured in the SFD medium supplemented with 100 ng/ml hEGF only.
  • cells were dissociated with TrypLE Select and stained with fluorophore conjugated antibodies in the FACS buffer for 20 to 30 minutes at room temperature. After washing in the FACS buffer, stained cells were resuspended in FACS buffer with SYTOX Blue Dead Cell Stain to exclude dead cells.
  • FACS buffer For intracellular staining, cells were processed using eBioscience Foxp3 /Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cells were stained with surface markers and Fixable Viability Dye eFluor 450 for 30 to 45 minutes at 4 °C.
  • UltraComp eBeads Compensation Beads were used for single color compensation control. Flow cytometric analysis was performed using LSR II (BD Biosciences) and cell sorting was performed using FACSAria II at Stanford Shared FACS Facility. Data was analyzed using FlowJo software. Cells were sorted into the FACS buffer supplemented with 10% fetal bovine serum and 2X Anti-Anti. [00253] qPCR.
  • RNA concentration and quality was measured by 2100 Bioanalyzer (Agilent Technologies). Only samples with high RIN (>8.0) were used for library preparation. Libraries were prepared using SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) and sequenced by NovaSeq (Illumina) with paired-end 100 bp reads at MedGenome. Each library was sequenced with a targeted depth of 40 million total reads.2 or 3 biological replicates were sequenced for each sample. Alignment was performed using STAR against the hg38 reference genome.
  • RPKM gene expression values were quantified using HOMER analyzeRepeats.pl.
  • raw reads were compared using DESeq2 likelihood ratio test (LRT) to assess gene expression changes along different time points.
  • LRT DESeq2 likelihood ratio test
  • Top 1000 differentially expressed genes were filtered and ranked on the basis of an adjusted P value ⁇ 0.05.
  • Stage- dependent differentially expressed genes were used for EnrichR analysis (https://maayanlab.cloud/Enrichr/) to assess enrichment in ARCHS4_TISSUES terms. [00255] Calcium Induced Stratification.
  • Cadaver skin (New York Firefighter Skin Bank) was freeze–thawed three times to devitalize cells and washed in PBS with 5X penicillin-streptomycin, 5X gentamicin and 5X fungizone.
  • the sterilized skin was stored in PBS containing 1X penicillin-streptomycin, 1X gentamicin and 1X fungizone at 37 °C for one week.
  • the epidermis was then peeled off of the dermis, which was then stored in PBS containing 1X penicillin-streptomycin at 4 °C for more than two weeks before use.
  • Devitalized dermis was cut into 1.5 cm ⁇ 1.5 cm pieces, and stored epidermis-down in 6-well dishes at 37 °C to let the dermis attach to the bottom.
  • the culture was switched to DKSFM, and 10 6 induced esophageal basal cells were seeded onto the center of DED.
  • the culture was lifted to the air–liquid interface and switched to KGM medium (DMEM: Hams F123:1, FBS 10%, 1X nonessential amino-acid, 0.18 mM adenine hydrochloride, 0.1 nM cholera toxin, 10 ng/ml EGF, 0.4 ⁇ g/ml hydrocortisone, 5 ⁇ g/ml insulin, 2 nM triiodo-l-thyronine, 5 ⁇ g/ml transferrin).
  • KGM medium DMEM: Hams F123:1, FBS 10%, 1X nonessential amino-acid, 0.18 mM adenine hydrochloride, 0.1 nM cholera toxin, 10 ng/ml EGF, 0.4 ⁇ g/ml hydrocortisone, 5 ⁇ g/ml insulin, 2 nM triiodo-l-thyronine, 5 ⁇ g/ml transferrin.
  • Prrx1 limb enhancer marks an adult subpopulation of injury-responsive dermal fibroblasts. Biology Open 8.7 (2019): bio043711. [00295] Lendahl, Urban, Lars Muhl, and Christer Betsholtz. "Identification, discrimination and heterogeneity of fibroblasts.” Nature Communications 13.1 (2022): 1-14. [00296] Buechler, Matthew B., et al. "Cross-tissue organization of the fibroblast lineage.” Nature 593.7860 (2021): 575-579. [00297] Huizinga, Jan D., and Elizabeth J. White.

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Abstract

Methods are provided for the efficient differentiation of hPSCs into eBC in defined, GMP conditions. The disclosure also provides systems, compositions, and kits for practicing the methods.

Description

METHODS OF GENERATING ESOPHAGUS BASAL CELLS FROM PLURIPOTENT STEM CELLS CROSS REFERENCE TO OTHER APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/521,601 filed June 16, 2023, the contents of which are hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT RESEARCH [0002] This invention was made with Government support under contract W81XWH-22-1- 0272 awarded by the Department of Defense. The Government has certain rights in the invention. BACKGROUND [0003] While stem cell technologies offer great promise, a broader approach to harness their potential to generate designer cell-based personalized therapeutics requires deeper understanding of tissue level interactions. [0004] The human esophagus is an endodermal-derived stratified squamous epithelium which serves as a protective barrier from abrasive food friction. The esophageal epithelium is established and maintained by basal cells (BCs), which are adult stem cells named for their proximity to the basement membrane. Underneath the epithelium lies the local mesenchyme, which emanates inductive signals guiding the specification of BCs from columnar progenitors, and wiring the downstream BC differentiation programs. Such a tissue architecture of esophagus resembles other stratified epithelial like skin, despite them being derived from different germ layers. Compared to skin, the early development of esophagus remains largely unexplored with only a handful of histological studies in the early 20th century. A holistic survey of the developing human fetal esophagus is therefore needed to close such a long-standing knowledge gap. In particular, characterizing the BC development process will shed light on the BC manufacture for clinical applications. [0005] Scalable clinical manufacturing of BCs remains an unmet need to cure severe epithelial defects, such as blistering as a result of congenital genetic mutations, and large wounds caused by caustic burn and post-cancer surgical resection. Enlightened by deep tissue knowledge in skin development, we and others have successfully generated patient- specific, hPSC-derived skin BCs for clinical trials. However, the equivalent platform for esophageal BC (eBC) production remains unavailable. Two pioneering studies leveraged key signaling mechanisms revealed in mouse studies, and differentiated hPSCs as esophageal progenitors. However, neither is compatible with clinical standards due to the use of undefined medium. Meanwhile, it remains unclear how to further induce the BC lineage commitment from those progenitors, which also hinders the clinical translation of the two studies. [0006] Pluripotent stem cell systems that differentiate into lineages of interest hold great potential for regenerative and precision medicine. Current practices for designing such systems either rely on the knowledge gained from mouse studies, or follow the trial and error paradigm. Human and mouse esophaguses are architecturally different, which might be linked to non-conserved developmental patterns. Meanwhile, trial and error methods are extremely labor-intensive and time-consuming. Therefore, the rational design of hPSC differentiation systems based on human esophageal development processes and milestones is in pressing demand. [0007] Developing methods for efficient production of eBC is therefore of great interest and is addressed herein. SUMMARY [0008] Methods are provided for a GMP-compatible, efficient differentiation of human pluripotent stem cells (hPSC) into human esophogeal basal cells (eBC) in vitro using extracellular factors to drive differentiation. The ability to efficiently and rapidly generate hPSC- derived eBC cells provides a means to produce these human cells for research and therapeutic purposes. As adult stem cells, eBCs are capable of regenerating the entire esophageal epithelium. The disclosure provides a versatile method to accelerate cell replacement therapy for lineage-specific stratified epithelial genetic defects and wounds. The derived esophogeal basal cells are useful for transplantation and tissue regeneration for various purposes, including without limitation rare genetic diseases like recessive or dominant esophagitis, dystrophic epidermolysis bullosa; esophageal injury; cancer; etc. [0009] The in vitro differentiation of eBC occurs through sequential stages. At each stage, specific factors are provided, and in some cases, specific signaling pathways are activated or inhibited, to drive differentiation and achieve purified populations of the desired cells at each stage. [0010] In an embodiment, a method of producing a population of definitive esophogeal basal cells (eBC) from hPSCs is provided. In one embodiment, the method provides a means of producing a substantially pure population of eBC in defined culture conditions, in media comprising extracellular signaling agents to guide differentiation, the method comprising: (a) differentiating human pluripotent stem cells into definitive endoderm cells; (b) differentiating definitive endoderm cells into anterior foregut endoderm cells; (c) differentiating anterior foregut endoderm cells into dorsal anterior foregut endoderm cells; (d) differentiating dorsal anterior foregut endoderm cells into early eBC; (e) differentiating early eBC into definitive eBC. [0011] The desired eBC cell population is optionally purified for cells of interest, and may be maintained on organoid culture. The eBC are expandable and are capable of self-renewal. In some embodiments greater than 50%, greater than 75%, greater than 80% or more of the final cell definitive eBC population express one, two, three or more of the eBC markers FOXA1, FOXA2, SOX2, SOX9 and P63 with ITGB4, EPCAM, Krt5, and Krt15. In some embodiments the cells express EPCAM and ITG4. As described herein, the level of purity of a particular purified population will vary depending on various factors and may be achieved through use of the cell derivation methods described herein including or excluding the use of one or more binding agents used to isolate particular cell types. [0012] In some embodiments, methods are provided for the use of eBCs in screening for cellular responses. In some embodiment methods are provided for treating a subject for a condition requiring esophogeal cells by administration of eBCs or cells derived therefrom, produced by the methods of the disclosure. In some embodiments, systems and kits for producing eBC types and/or screening for cellular responses and/or treating subjects with such eBC are provided. [0013] Aspects of the disclosure relate to screening a substantially pure population of eBC or progeny therefrom produced according to the methods described herein for a cellular response. In certain aspects, a method of screening a substantially pure population of eBC for a cellular response may include contacting a population of substantially pure population of eBC with a pharmacological agent and evaluating the population of cells for a cellular response induced by the pharmacological agent. In certain aspects, the screening may be in vitro screening and the contacting may be performed in vitro. In certain aspects, the screening may be in vivo screening and the contacting may be performed by administering the pharmacological agent to a host animal that contains the population of cells. [0014] Aspects of the disclosure relate to methods of treating a subject for a condition through the administration of a population of eBC derived or produced according to the methods described herein. In certain aspects, the method of treating a subject for a condition through administration of cells derived according to the methods as described herein may further include co-administration with at least one pro-survival or pro-engraftment factor. In certain aspects, the cells administered to a subject are genetically modified in at least one genetic locus. [0015] Aspects of the disclosure include kits for the production, derivation, purification, and use of a derived population of eBC that include one or more induction compositions and/or one or more specific binding agents and/or combinations thereof. In certain aspects, such kits may or may not include one or more cell types described herein. [0016] Aspects of the disclosure include systems for the production, derivation, purification, and use of a substantially pure population of eBC that include one or more components configured to administer one or more induction compositions and/or one or more specific inducing agents and/or one or more specific binding agents and/or combinations thereof. In certain aspects, such systems are configured to administer such compositions and/or agents at specific amounts or for specific periods of time according to the methods described herein. [0017] In an embodiment, methods are provided for the use of machine learning to develop methods of differentiating stem or progenitor cells to a cell type of interest. Such methods may utilize single cell and spatial technologies to generate a spatiotemporal multi-omics cell atlas for development of the cell or tissue of interest. Alternatively, an existing cell atlas such as that disclosed herein may be used. A machine learning algorithm is used to prioritize the combinations of candidate developmental signals for in silico method of differentiating a cell type of interest. Functional validation of the predicted developmental signals can be performed. BRIEF DESCRIPTION OF THE DRAWINGS [0018] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures. [0019] FIGS. 1A-1B. A multi-omics cell atlas of human esophageal development. (A) Workflow schematic: first integration of scRNA-seq, Visium spatial transcriptomics and CODEX multiplexed immunofluorescent cell imaging to build the multi-omics cell atlas to depict human esophageal development, followed by investigation into intercellular signal dynamics to nominate basal cell specification signals. Then the deep-learning algorithm Manatee predicts the optimal signal combination to drive basal cell specification, which is further used to build the hPSC-based esophageal mucosa manufacturing platform. (B) H&E staining of developing human esophageal sections from E45 to E130 with the timeline and summary of analyzed samples colored by assays. Scale bars: 100 μm. [0020] FIGS.2A-2F. The cellular heterogeneity and lineage trajectory of human esophageal epithelium during development. (A) Epithelial single cells were projected on the UMAP space, and color-coded based on cell type. Inset showing UMAP visualization by developmental timepoint. (B) Proportions of the identified cell types at each timepoint. (C) Dot plot showing the expression levels for selected markers of epithelial progenitors (Epi_PG), basal cells (BC), immature ciliated cells (Cil-1) and mature ciliated cells (Cil-2) for CODEX image representation. (D-E) CODEX images confirming the presence of Epi_PG, BC (D), Cil-1, Cil- 2 (E) using selected markers. Early: Representative images from E45 (Early), E120 (Late) and E72 (Mid) samples. Scale bars: 10 μm. (F) Monocle2 trajectory of the whole epithelial compartment, color-coded based on cell type. Insets: projection of epithelial cells collected at representative stages on the Monocle2 trajectory. Early: E47; Mid: E72; Late: E130. SB: suprabasal cells. [0021] FIGS. 3A-3H. Esophageal stromal diversity and mesenchymal lineages. (A) Stromal single cells were projected on the UMAP space, and color-coded based on cell types/states. Inset showing UMAP visualization by cellular compartment (top right) and developmental timepoint (bottom right). (B) Proportions of the identified cell types at each timepoint. (C) Dot plot showing the expression levels for selected markers of different fibroblasts, myofibroblasts (MF) and smooth muscles (SM) shown in CODEX images. (D-G) CODEX images confirming the presence of fibroblast progenitors (Fib_PG) (D), Fib_1/2 (E), Fib_3/4/5, interstitial cells of Cajal (Fib_ICC, F), MF/SM (G) using selected markers. Early: Representative images from E45 (Early) and E120 (Late) samples. Scale bars: 10 μm. (H) Monocle2 trajectory of the Fib- MF lineages, color-coded based on cell type. Other abbreviation: MS: mesothelium; Peri: pericytes; NB: neuroblasts; SkM: skeletal muscle; SC: satellite cells; Art: arterial cells; Ven: venous cells; Lym: lymphatic cells; Mono: monocytes: DC: dendritic cells. [0022] FIGS. 4A-4C. Spatiotemporal cellular dynamics of human esophageal development. (A) Representative CODEX images with selected markers showing the epithelium and the local mesenchyme at E45 (Early), E72 (Mid) and E120 (Late). Scale bars: 100 μm. Bottom panel showing enlarged boxed regions with representative cells annotated. Scale bar: 10 μm. (B) Cell type annotation of the representative CODEX images based on hierarchical clustering analysis of the fluorescent intensities of 42 markers. (C) Diagram of the cellular composition and tissue architecture at early, mid, late stages of the developing human esophagus. Abbreviation: Epi_PG: epithelial progenitor; BC: basal cell; SB: suprabasal cell; Cil: ciliated cell; Fib_PG: fibroblast progenitor; Fib: fibroblast; Fib_ICC: interstitial cell of Cajal; MF: myofibroblast; SM: smooth muscle; Neu: neuronal cell; Im: immune cell; Endo: endothelium; Peri: pericyte; ENS: enteric nervous system. [0023] FIGS.5A-5F. Spatiotemporal candidate signal nomination. (A) Schematic: determining local mesenchymal cellular components at different developing stages first, followed by identifying potential inductive signals from the local mesenchyme that could be received by Epi_PG/BC using CellChat. (B) Quantification of spatial distance to basement membrane (left panel) and layer thickness (right panel) of key mesenchymal cell types. Boxplots represent mean ± SEM. Each dot represents one random measurement. For each stage, n >= 30. (C- D) Heatmaps showing the CellChat predicted incoming signal strength received by Epi_PG/BC (C) and sender-receiver interaction scores (D). (E) Diagram summarizing the candidate intercellular communication model of esophageal BC development. (F) Signaling gene expression patterns of key signaling pathways summarized in a dot-plot. For each pathway, expression patterns of ligand, antagonist and receptor genes among early, mid and late stage cell populations were visualized. The expression level and percentage were coded by dot color and size, respectively. [0024] FIGS.6A-6C. Manatee screens for the optimal signal combinations to promote basal cell derivation. (A) The Manatee workflow. The Manatee model was adapted from VAE by constraining its latent space to represent TF expression. To predict perturbation-induced expression profiles, latent variables associated with candidate TFs will be adjusted. Such an adjusted latent space will then be decoded as final predictions. (B) Screening all 81 possible perturbation combinations of EGF, WNT, TGFb and BMP signaling pathways for basal cell derivation (red, up-regulate; blue, down-regulate; white, intact). The perturbation effect was quantified by computing the Pearson correlation (R) between the average basal cell and the perturbation expression profiles. Specifically, 4 combinations were highlighted, including the candidate optimal combination #19, negative control #9, base control #14 and #41 which has not been perturbed. (C) Original single cell expression profiles and predictions yielded by the above-highlighted strategies were visualized on the two-dimensional UMAP space. D4, 10, 16, 24 and 43, single cells harvested at day 4, 10, 16, 24 and 43 from the hPSC culture, respectively. D16_P and D24_P, predicted single cell profiles with D16 and D24 as starting points, respectively. Epi_PG, BC, SB-1 to 5, epithelium single cell populations as described in FIG.2. Arrows indicate perturbation effects. [0025] FIGS.7A-7I. Establishment of esophageal mucosa manufacturing platform using the Manatee-predicted strategies. (A) Schematic illustration of the hPSC-to-esophageal basal cell differentiation protocol. DE, definitive endoderm; (d)AFE, (dorsal) anterior foregut endoderm; EPC, esophageal progenitor. EPCs were further subjected to a 5-day perturbation. (B) Representative flow cytometry plots of cells after the 5-day perturbation. Percentage of candidate basal cells (EPCAM+ITGB4+) among all cells were quantified in strategy #19 (33.9%), negative control #9 (0.84%) and base control #14 (7.37%). Statistical quantification of the flow cytometry analysis. Candidate basal cell percentage was calculated against epithelial cells (EPCAM+). Boxplots represent mean ± SEM; n = 3. Student t-test, * p < 0.05, ** p < 0.01. (C) IF staining of cells after the 5-day perturbation with canonical basal cell marker KRT5. (D) qPCR analysis of canonical basal cell markers KRT5 and KRT15. Marker expression was quantified by log2 fold change against day 0. Boxplots represent mean ± SEM; n = 3. Hu-Fetal: human fetal esophageal tissues used as endogenous control, n = 2. (E) Bulk RNA-seq heatmap of day 24 cells before treatment and day 29 cells after 5- day perturbation. Expression of selected basal cell markers were quantified. The effectiveness of pathway perturbation was quantified by expression of representative WNT, BMP and TGFb downstream targets. n = 3. (F) Representative flow cytometry plots showing the self-renewing capability of cells after the 5-day perturbation. EPCAM+ITGB4+ double positive candidate basal cells (DP) and EPCAM+ITGB4- single positive cells (SP) were sorted and expanded on collagen peptide coated plates. DP cells can be cryopreserved during expansion. After 3 passages, DP and SP groups maintained 75.8% and 19.1% candidate basal cells, respectively. (G) Immunofluorescent staining of Calcium-induced stratification assay. Upper panel showed KRT5+KRT15+ basal cells before confluency with KRT18 absence. Lower panel showed that upon Calcium treatment, basal cells underwent esophageal squamous differentiation, marked by KRT13+KRT4+ cells with large and flat morphology. (H) Immunofluorescent staining of 3D organoids derived from DP cells showing KRT5+COLVII+ basal cells and KRT13+ suprabasal cells. (I) IF staining of organotypic culture sections. hPSC- derived basal cells expanded and differentiated to esophageal squamous cells on decellularized dermis. Scale bars: 10 μm. For (G-I), Merge images were shown as single channel stainings with grey DAPI staining. (B-D, F-I) Representative results generated using H9 embryonic stem cell line. Both H9 and RUES2 lines were used, and consistent results were obtained in more than 3 independent runs for each line. (E) RUES2 line was used for bulk RNA-seq. [0026] FIGS.8A-8M. scRNA-seq data quality control and filtering, related to FIG.s 1, 2 and 3. (A) Summary of dataset collection for multiomic atlas of human esophageal development. (B) Quality control of scRNA-seq datasets. For each dataset, sample-wise distributions of number of cells/spots, number of UMIs per cell and number of genes per cell were reported. (C) Iterative clustering of epithelium and stromal scRNA-seq profiles generated using TrpLE and Collagenase, respectively. (D, F) UMAP visualization of the coarse-grain initial clustering of epithelium (EPI, D) and stroma (STROMA, F), color-coded based on cell type. (H, I, K, L) UMAP visualization of the fine-grain clustering of epithelium and mesenchyme (MES), color- coded based on cell type/state (H, K) and developmental timepoint (I, L). (E, G, J, M) Dot plots showing the expression of selected differentially expressed genes for each cellular compartment or cell type/state. [0027] FIGS. 9A-9N. Cellular heterogeneity and lineage analysis of epithelium and mesenchyme, related to FIG.s 2 and 3. (A-B) Feature plots showing the expression of selected markers depicting transition from epithelial progenitors to basal cells (A), and from SB-5 to ciliated cells (B). (C) 2 dimensional UMAP visualization of mesenchymal cells color-coded based on cell type/state, split by developmental timepoint. (D, F) Feature plots showing the expression of selected markers distinguishing smooth muscles from myofibroblasts (D), pericytes from fibroblasts (F). (E, G) Monocle2 trajectory of the smooth muscle cells (SM, E) and pericytes (PERI, G), color-coded based on cell type/state. (H-I) 3 dimensional UMAP visualization of enteric nervous system (ENS) cells color-coded based on cell type/state (H) and developmental timepoint (I). (J) Proportions of the identified cell types at each timepoint. (K) Dot plot showing the expression of differential marker genes in the enteric nervous system. (L-FM) Representative CODEX images showing the presence of ENS_PG at E45 (Early, L) and Neu and Glia at E120 (Late, M). Scale bars: 10 μm. (N) Monocle2 trajectory of the whole enteric nervous system, color-coded based on cell type. [0028] FIGS 10A-10O. Cellular heterogeneity of endothelial, skeletal muscle and immune compartments, related to FIG. 3. (A-B) 3 dimensional UMAP visualization of endothelial (ENDO) cells color-coded based on cell type/state (A) and developmental timepoint (B). (C) Proportions of the identified cell types at each timepoint. (D) Dot plot showing the expression of differential marker genes in endothelial compartment. (E) Dot plot showing selected markers of arterial (Art), venous (Ven), lymphatic cells (Lym), pericytes (Peri) and immune cells (Im) shown in CODEX images. (F-G) Representative CODEX images showing the presence of lymphatic, arterial, venous cells, pericytes (F) and immune cells (G) at E120 (Late). Scale bars: 10 μm. (H-I) 3 dimensional UMAP visualization of skeletal muscle (SKM) cells colorcoded based on cell type/state (H) and developmental timepoint (I). (J) Proportions of the identified cell types at each timepoint. (K) Dot plot showing the expression of differential marker genes in skeletal muscle compartment. (L-M) 3 dimensional UMAP visualization of immune (IM) cells color-coded based on cell type/state (L) and developmental timepoint (M). (N) Proportions of the identified cell types at each timepoint. (O) Dot plot showing the expression of differential marker genes in immune compartment. [0029] FIGS.11A-11H. Integrated analysis of single cell and Visium spatial transcriptomics, related to FIG.4. (A-H) Visium spatial feature plots visualizing selected cell types in different combinations on E72 (Mid, A-D) and E120 (Late, E-H) esophageal sections, color-coded based on mapping scores. (A1-A6, B1-B2, C1-C2, D1-D5, E1-E7, F1-F2, G1-G2, H1-H5) Visium spatial feature plots visualizing single cell type on E72 (Mid, A1-D5) and E120 (Late, E1- H5) esophageal sections, color-coded based on mapping scores. [0030] FIGS. 12A-12D. CODEX antibody panel and whole-mount tissue staining, related to FIGS. 2-4. (A) Dot plot showing RNA expression of CODEX marker genes in different cell types. (B) Dendrogram workflow for cell type annotation using selected markers. (C) Violin plots of PITX1, αSMA and POSTN expression in fibroblast 1-5, interstitial cells of Cajal, myofibroblast and smooth muscle, color-coded based on cell types. (D) 3D reconstruction of whole-mount immunostaining of E87 human fetal esophageal tissue. Note POSTN expression in subepithelial region and adventitia. Co-expression of PITX1 and POSTN marked Fib_1 lining the basement membrane, while lack of PITX1 in adventitia suggested distribution of Fib_4 and Fib_5 there. Co-expression of αSMA and PITX1 marked myofibroblasts lining the Fib_1 layer, while αSMA+ only smooth muscle cells were found outside of submucosa. Scale bar: 100 μm. [0031] FIGS. 13A-13D. Human developmental signal combination screening by Manatee, related to FIG. 6. (A) Quality control plot of in vitro single cell collections. (B-C) UMAP visualization of in vitro single cell collections color-coded based on cell type (B) and stage (C). (D) Original single cell expression profiles and predictions yielded by all 81 possible strategies were visualized on the two-dimensional UMAP space. Cells were color-coded by their types, and prediction effects were noted by arrows. [0032] FIGS. 14A-14H S7. Characterization of the chemically-defined, xeno-free hPSC- toesophageal basal cell differentiation system, related to FIG. 7. (A) Principal-component analysis (PCA) of bulk RNA-seq data from hPSC-to-esophageal basal cell differentiation at different timepoints. (B) Heatmap of differential gene expression along hPSC-to-esophageal basal cell differentiation. Selected markers were labeled to the left. Right panel showing the top enriched ARCHS4_TISSUES terms by EnrichR, represented by bar graph for -log10(p). (C) Representative flow cytometry quantification of differentiation efficiency, showing high efficiency in deriving definitive endoderm on D4 and esophageal progenitors on D16, reduced progenitor efficiency on D24, and low efficiency in EPCAM+ ITGB4+ esophageal basal cells on D45. (D) IF of human esophageal transcription factors on D16 cells. (E) IF of BC markers and squamous markers on D24, D35, and D45 cells. Note that BC keratins are gradually acquired over the time course. (F-H) Statistical quantification of the flow cytometry analyses, to evaluate the essentiality of each candidate pathway (F), sufficiency (G), and different combinations with EGF (H). Candidate basal cell percentage was calculated against epithelial cells (EPCAM+). Boxplots represent mean ± SEM; n = 3. Student t-test, * p < 0.05, ** p < 0.01. *** p < 0.001. Blue dashed line marked the control: full cocktail of 4 pathways on D29 (F), D24 (G) and EGF only on D29 (H). (I) Immunofluorescent staining of 3D organoids derived from DP cells showing P63+SOX2+ basal cells. Scale bars, 10 μm. FIG. S7 showed representative results generated using H9 embryonic stem cell line. Both H9 and RUES2 lines were used, and consistent results were obtained in more than 3 independent runs for each line. DETAILED DESCRIPTION OF THE EMBODIMENTS [0033] Methods are provided for the generation of substantially purified eBC. Treatment methods making use of the generated eBC are also provided. The instant disclosure also provides systems, compositions, and kits for practicing the methods of the disclosure. [0034] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0035] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction. [0037] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. [0038] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth. [0039] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication date, which may need to be independently confirmed. Definitions [0040] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired therapeutic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting development of a disease and/or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and/or symptom(s). [0041] The terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human. [0042] The terms “pluripotent progenitor cells”, “pluripotent progenitors”, “pluripotent stem cells”, “multipotent progenitor cells” and the like, as used herein refer to cells that are capable of differentiating into two or more different cell types and proliferating. Non limiting examples of pluripotent precursor cells include but are not limited to embryonic stem cells, blastocyst derived stem cells, fetal stem cells, induced pluripotent stem cells. Pluripotent progenitor cells may be acquired from public or commercial sources or may be newly derived. As described herein, in some instances, pluripotent progenitor cells of the subject disclosure are those cells capable of giving rise to eBC. [0043] The term “lineage bifurcation” and “lineage segregation” are used interchangeably herein and refer to a cell-fate decision where a stem cell and/or progenitor cell has the ability to differentiate into two or more cell-types. [0044] The term “population”, e.g., “cell population” or “population of cells”, as used herein means a grouping (i.e., a population) of two or more cells that are separated (i.e., isolated) from other cells and/or cell groupings. For example, a 6-well culture dish can contain 6 cell populations, each population residing in an individual well. The cells of a cell population can be, but need not be, clonal derivatives of one another. A cell population can be derived from one individual cell. For example, if individual cells are each placed in a single well of a 6-well culture dish and each cell divides one time, then the dish will contain 6 cell populations. The cells of a cell population can be, but need not be, derived from more than one cell, i.e. non- clonal. The cells from which a non-clonal cell population may be derived may be related or unrelated and include but are not limited to, e.g., cells of a particular tissue, cells of a particular sample, cells of a particular lineage, cells having a particular morphological, physical, behavioral, or other characteristic, etc. A cell population can be any desired size and contain any number of cells greater than one cell. For example, a cell population can be 2 or more, 10 or more, 100 or more, 1,000 or more, 5,000 or more, 104 or more, 105 or more, 106 or more, 107 or more, 108 or more, 109 or more, 1010 or more, 1011 or more, 1012 or more, 1013 or more, 1014 or more, 1015 or more, 1016 or more, 1017 or more, 1018 or more, 1019 or more, or 1020 or more cells. [0045] The terms “homogenous population”, as it relates to cell populations, refers to a cell population that is essentially pure and does not consist of a significant amount of undesired or contaminating cell types. By significant amount, in this context, is meant an amount of undesired or contaminating cell types that negatively impacts the use of the isolated desired cell population. As such, the actual amount of undesired or contaminating cells that defines a significant amount will vary and depend on the particular type of undesired or contaminating cells and/or the particular use of the desired cell type. For example, in a population of differentiated cells used in the treatment of a subject, a significant amount of improperly differentiated contaminating cell types will be small as such cells may a high capacity to negatively impact the use of the generated desired cell population. In comparison, e.g., in a population of differentiated cells used in the treatment of a subject, a significant amount of contaminating progenitor cells may be relatively large as such cells may have a low capacity to negatively impact the use of the generated desired cell population. In some instances, a homogenous population may refer to a highly enriched population. Levels of homogeneity will vary, as described, and may, in some instances, be greater than 60% pure, including e.g., more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, more than 99.6%, more than 99.7%, more than 99.8%, and more than 99.9%. [0046] The term “heterologous”, as it refers to a “heterologous sequence” or “heterologous nucleic acid”, means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. [0047] The term “high cell density”, as it relates to cells, means the cell numbers within an area or volume is high. For example, cells are in close contact with one another when cultured in high cell density. In some embodiments high cell density refers to a density of at least about 1.25x106 cells/cm2. Methods [0048] Methods and compositions are provided for producing a population of human esophogeal basal cells (eBC) in defined monolayer conditions in media comprising extracellular signaling agents that guide differentiation. Aspects of the disclosure include methods for deriving eBC from pluripotent progenitor cells. [0049] Pluripotent stem cells may be acquired from any convenient source, including but not limited to newly derived from a subject of interest or tissue specimen or other cellular sample, obtained from a public repository, obtained from a commercial vendor, and the like. In some instances, pluripotent cells of interest include human cells including but not limited to, e.g., human embryonic stem cells, human induced pluripotent stem cells, human fetal stem cells, and the like. [0050] In some instances, pluripotent stem cells are unmodified, such that the cells have not been genetically or otherwise modified from their natural state, prior to modification according the methods described herein. In other instances, pluripotent stem cells may be unmodified, such that the cells have been genetically or otherwise modified from their natural state, prior to modification according the methods described herein. [0051] Generation of eBC from pluripotent progenitors as described herein involves one or more lineage restriction events, in which cultured pluripotent stem cells are subjected to one or more treatments causing the cultured cells or a population thereof to differentiate along specific pathways. Multiple lineage restriction events are required to achieve desired eBC. In certain instances, lineage restriction events are performed successively such that a first cell type is achieved by a first lineage restriction event and the first cell type is subjected to a second lineage restriction event to achieve a desired second cell type; etc. [0052] Lineage restriction events as described herein are induced by compositions of extracellular agents that act on specific signaling pathways in cultured cells, including those agents that activate or inhibit developmental signaling pathways that drive development. As will be clear from the instant disclosure, whether activation or inhibition of a particular signaling pathway is necessary to generate a particular cell type of interest will depend on a number of factors including but not limited to, e.g., the particular desired cell type, the timing of use of the particular agent and/or composition, the starting cell type to be induced, etc. [0053] The PSCs utilized in the differentiation procedures may comprise pluripotent cells of any kind. In some instances, pluripotent cells include human cells, including but not limited to, e.g., human embryonic stem cells, human induced pluripotent stem cells, human fetal stem cells, and the like. Exemplary pluripotent cells include H1, H7 and H9 hESCs, as known in the art. Exemplary induced pluripotent stem cells include iPSCs derived from peripheral blood mononuclear cells, fibroblasts, and other somatic cell sources, as known in the art. [0054] The methods of the invention may be carried out by any number of cell culture processes. In some embodiments, one or more culturing steps, for example, all culturing steps, are performed using cells plated on a substrate. Defined and xeno-free substrates are advantageously used. Exemplary substrates include recombinant proteins, e.g. recombinant human proteins, for example, vitronectin, collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glucosaminoglycan, fibronectin, laminin, collagen IV, heparan sulfate proteoglycan, entactin and nidogen. Commercially available basement membrane extracts, for example, containing proteins such as laminin, collagen IV, entactin, and heparin sulfate proteoglycans, for example, GELTREX(TM) substrate may be used. In some embodiments, a recombinant Laminin-511 E8 Fragment matrix is utilized as the culture substrate, for example iMatrix 511 (TM) (Takara Bio). In alternative implementations, one or more culturing steps, for example, all culturing steps, are performed using cells in a liquid culture system. [0055] Cells may be seeded on the substrate at any effective density, for example, in the range of 10-50,000 cells per cm2. For example, about 30,000, about 50,000, about 75,000, about 100,000, about 125,000, about 150,000, or about 200,000 cells may be cultured in substrate- coated wells of culture plates, for example a 3.5 cm2 well of a 12-well plate. [0056] The cell culture medium may be any basal medium, supplemented with differentiation factors as set forth herein. Exemplary media include E8, MCDB131, for example, prepared as described herein, SFD medium, for example, prepared as described herein, and others known in the art. [0057] The various methods described herein, in some embodiments, encompass the differentiation of a first cell type to a second cell type by contacting cells of the first type with one or more selected agents for a period of time sufficient to induce differentiation to the second cell type. The contacting may be achieved by any exposure of the cells to the selected agents. In a primary implementation, the one or more selected factors are present in a liquid medium in which the cells are cultured, e.g. dissolved, suspended, or otherwise present. In an alternative embodiment, the contacting is achieved by admixture of the one or more agents into the substrate material on which the cells are cultured, in planar culture implementations of the invention. [0058] The in vitro differentiation of eBC occurs through sequential stages. At each stage, specific factors are provided, and in some cases, specific signaling pathways are activated or inhibited, to achieve highly purified populations. [0059] In an embodiment, a method of producing a population of definitive esophogeal basal cells (eBC) from hPSCs is provided. In one embodiment, the method provides a means of producing a substantially pure population of eBC in defined culture conditions, in media comprising extracellular signaling agents to guide differentiation, the method comprising: (a) differentiating human pluripotent stem cells into definitive endoderm cells; (b) differentiating definitive endoderm cells into anterior foregut endoderm cells; (c) differentiating anterior foregut endoderm cells into dorsal anterior foregut endoderm cells; (d) differentiating dorsal anterior foregut endoderm cells into early eBC; (e) differentiating early eBC into definitive eBC. [0060] Methods of achieving each step of the foregoing process are described next. [0061] Definitive Endoderm Cell Differentiation. In one aspect, the scope of the invention encompasses a method of differentiating human pluripotent stem cells to definitive endoderm cells. The definitive endoderm cell differentiation process is performed as a first step in the differentiation of eBC from hPSCs. Undifferentiated hPSCs are seeded for differentiation as single cells. [0062] To induce definitive endoderm cells, the pluripotent stem cells are induced by contacting the cells in the presence of a ROCK pathway inhibitor on a suitable matrix, including without limitation a defined matrix such as recombinant laminin 511 for a period of time sufficient to differentiate human pluripotent stem cells to definitive endoderm cells, for example a period of time from about 1 day, e.g. from about 12 hour to about 36 hours. The cells may then be moved to culture in the presence of a GSK-3 pathway inhibitor and TGFβ pathway agonist for a period of about 3 days, for example from about 2 to about 4 days, to generate definitive endoderm. The GSK-3 pathway inhibitor is optionally removed after the first day or the second day. Optionally an inhibitor of JNK is included on the first day. [0063] In some embodiments a ROCK inhibitor is Y-27632, for example at a concentration of from about 0.5 to about 50 µM, and may be around 10 µM. In some embodiments a GSK-3 pathway inhibitor is CHIR99032, for example at a concentration of from about 0.5 to about 50 µM, and may be around 5 µM. In some embodiments a TGFβ pathway agonist is Activin A, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml. In some embodiments a JNK inhibitor is JNK-IN-8, for example at a concentration of from about 0.1 to about 50 µM, and may be around 1 µM. [0064] Anterior foregut endoderm differentiation. In one aspect, the scope of the invention encompasses a method of differentiating definitive endoderm cells to anterior foregut endoderm cells. Anterior foregut endoderm differentiation may be performed as a second step in the differentiation of hPSC to eBC. The definitive endoderm cells are cultured in the presence of a TGFβ pathway inhibitor; and BMP pathway inhibitor for a period of time sufficient to differentiate to anterior foregut endoderm ce, for example a period of time from about 2 days, e.g. from about 18 to about 36 hours to generate anterior foregut endoderm. [0065] In some embodiments a TGFβ pathway inhibitor is SB431542, for example at a concentration of from about 0.5 to about 50 mM, and may be around 10 mM. In some embodiments a BMP pathway inhibitor is noggin, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml. [0066] Dorsal anterior foregut endoderm differentiation. In one aspect, the scope of the invention encompasses a method of differentiating anterior foregut endoderm cells to dorsal anterior foregut endoderm cells. Dorsal anterior foregut endoderm differentiation may be performed as a third step in the differentiation of hPSC to eBC. The anterior foregut endoderm cells are cultured in the presence of a TGFβ pathway inhibitor, BMP pathway inhibitor, EGF pathway agonist and FGF pathway agonist for a period time sufficient to differentiate to dorsal anterior foregut endoderm cells, for example a period of from about 3 days, for example from about 2-4 days to generate dorsal anterior foregut endoderm. [0067] In some embodiments a TGFβ pathway inhibitor is SB431542, for example at a concentration of from about 0.5 to about 50 mM, and may be around 10 mM. In some embodiments a BMP pathway inhibitor is noggin, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml. In some embodiments an EGF pathway agonist is human EGF, and may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml. In some embodiments an FGF pathway agonist is FGF10, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 50 ng/ml. [0068] Early esophogeal basal cell differentiation. In one aspect, the scope of the invention encompasses a method of differentiating dorsal anterior foregut endoderm cells to early eBC. Early esophogeal basal cell differentiation may be performed as a fourth step in the differentiation of hPSC to eBC. The dorsal anterior foregut endoderm cells are cultured in the presence of an EGF pathway agonist and FGF pathway agonist for a period of time sufficient to differentiate to eBC, for example from about 3-6 days, e.g. about 5 days. The cells may be cultured in the presence of an EGF agonist for a period of from about 4 to 8 days, and may be about one week. This step may also be sufficient to generate definitive eBC, although the yield can be improved with the disclosed fifth step. [0069] In some embodiments an EGF pathway agonist is human EGF, and may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml. In some embodiments an FGF pathway agonist is FGF10, which may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 50 ng/ml. [0070] Definitive esophogeal basal cell differentiation. In one aspect, the scope of the invention encompasses a method of differentiating early eBC to definitive eBC. Definitive esophogeal basal cell differentiation may be performed as a fifth step in in the differentiation of hPSC to eBC. The early eBC are cultured in the presence of an EGF pathway agonist, a wnt pathway inhibitor; a BMP pathway agonist; and a TGFβ pathway agonist; for a period of time sufficient to differentiate definitive eBC, for example from about 3-5 days, e.g. about 5 days, to generate canonical esophogeal basal cells. [0071] In some embodiments an EGF pathway agonist is human EGF, and may be present at a concentration of from about 1 to about 500 ng/ml, and may be around 100 ng/ml. In some embodiments a Wnt pathway inhibitor is IWP-2, e.g. at a concentration of from about 0.5 to about 50 µM, and may be around 4 µM. In some embodiments a BMP pathway agonist is BMP4, e.g. at a concentration of from about 0.5 to about 500 ng/ml, and may be around 5 ng/ml. In some embodiments a TGFβ agonist is TGFβ1, e.g. at a concentration of from about 0.5 to about 500 ng/ml, and may be around 2 ng/ml. [0072] The final cell population is optionally purified for cells of interest, and may be maintained on organoid culture. In some embodiments greater than 50%, greater than 75%, greater than 80% or more of the final cell population express one, two, three or more of the eBC FOXA1, FOXA2, SOX2, SOX9 and P63 with ITGB4, EPCAM, Krt5, and Krt15. The eBC are exapandable and are capable of self-renewal. In some embodiments eBC are selected for expression of Epcam and ITGB4. [0073] The eBCs derived by the foregoing method may be utilized in the treatment of various esophageal conditions. The eBCs may be transplanted, grafted, or otherwise administered to treat genetic or acquired defects or injuries to the esophagus, for example in the treatment of recessive dystrophic epidermolysis bullosa, dominant dystrophic epidermolysis bullosa, wounding caused by cancer resection, caustic injury, esophageal stricture, esophagitis, and other wounds, dysfunctions, or pathologies of the esophagus. The eBCs of the invention may be delivered by methods as known in the art for esophageal regeneration, for example, delivered in a scaffold or matrix. Exemplary scaffolds include acellular scaffolds, for example comprising polymeric material, proteinaceous materials (e.g. collagen), and decellularized extracellular matrices, for example derived include urinary bladder matrix, skin, pericardium, dura, gastric matrix, esophageal acellular matrix and small intestinal submucosa. Factors [0074] In some instances, as disclosed above, an agent useful in a particular induction composition may include an activator (agonist) or inhibitor of the TGF-beta (transforming growth factor β (TGF-β)) pathway. Transforming growth factor-beta (TGF-β) denotes a family of proteins, TGF-β1, TGF-β2, and TGF-β3, which are pleiotropic modulators of cell growth and differentiation, embryonic and bone development, extracellular matrix formation, hematopoiesis, immune and inflammatory responses (Roberts and Sporn Handbook of Experimental Pharmacology (1990) 95:419-58; Massague et al. Ann Rev Cell Biol (1990) 6:597-646). TGF-β initiates intracellular signaling pathways leading ultimately to the expression of genes that regulate the cell cycle, control proliferative responses, or relate to extracellular matrix proteins that mediate outside-in cell signaling, cell adhesion, migration and intercellular communication. [0075] TGF-β exerts its biological activities through a receptor system including the type I and type II single transmembrane TGF-β receptors (also referred to as receptor subunits) with intracellular serine-threonine kinase domains, that signal through the Smad family of transcriptional regulators. Binding of TGF-β to the extracellular domain of the type II receptor induces phosphorylation and activation of the type I receptor (TGFβ-R1) by the type II receptor (TGFβ-R2). [0076] A TGFβ inhibitor refers to a molecule, e.g. a decoy receptor, antibody or derivative thereof, a nonpeptide small molecule, etc. specifically binding to a TGFβ-R1 receptor having the ability to inhibit the biological function of a native TGF-β molecule. Activators and inhibitors of the TGF-beta pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the TGF-beta pathway resulting in a corresponding activation or inhibition in cellular TGF-beta signaling. [0077] Activators (agonists) of the TGF-beta pathway include but are not limited to, e.g., TGF- beta family ligands (e.g., TGF-beta proteins and other activators of TGF-beta receptors) and portions thereof, Activin A, TGF-beta1, TGF-beta2, TGF-beta3 , IDE1/2 (IDE1 (1-[2-[(2- Carboxyphenyl)methylene]hydrazide]heptanoic acid), IDE2 (Heptanedioic acid-1-(2- cyclopentylidenehydrazide)), Nodal, and the like. In some instances, activation of the TGF- beta pathway may be achieved through repression of the a TGF-beta pathway inhibitor, e.g., including but not limited to the use of an inhibitory nucleic acid targeting an inhibitor of the TGF-beta pathway or an antibody or small molecule directed to a TGF-beta pathway inhibitor. [0078] Inhibitors of the TGF-beta pathway include but are not limited to, e.g., A-83-01 (3-(6- Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), D4476 (4-[4- (2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), LY 364947 (4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-quinoline), RepSox (2-(3-(6-Methylpyridine-2- yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2- pyridinyl)-1H-imidazol-2-yl]benzamide), SB-505124 (2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1- dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine), SB 525334 (6-[2-(1,1-Dimethylethyl)-5- (6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), SD208 (2-(5-Chloro-2-fluorophenyl)-4- [(4-pyridyl)amino]pteridine), ITD1 (4-[1,1'-Biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7- trimethyl-5-oxo-3-quinolinecarboxylic acid ethyl ester), DAN/Fc, antibodies to TGF-beta and TGF-beta receptors, TGF-beta inhibitory nucleic acids, and the like. [0079] In some instances, an agent useful in a particular induction composition may include an inhibitor of the Wnt pathway. Activators and inhibitors of the Wnt pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the Wnt pathway resulting in a corresponding activation or inhibition in cellular Wnt signaling. [0080] Inhibitors of the WNT pathway include but are not limited to, e.g., C59 (4-(2-Methyl-4- pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide), DKK1, IWP-2 (N-(6-Methyl-2- benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]- acetamide), Ant1.4Br, Ant 1.4Cl, Niclosamide, apicularen, bafilomycin, XAV939 (3,5,7,8- Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), IWR-1 (4- (1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl- Benzamide), NSC668036 (N-[(1,1-Dimethylethoxy)carbonyl]-L-alanyl-(2S)-2-hydroxy-3- methylbutanoyl-L-Alanine-(1S)-1-carboxy-2-methylpropyl ester hydrate), 2,4-diamino- quinazoline, Quercetin, ICG-001 ((6S,9aS)-Hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1- naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)- carboxamide), PKF115-584, BML-284 (2-Amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3- methoxyphenyl)pyrimidine), FH-535, iCRT-14, JW-55, JW-67, antibodies to Wnts and Wnt receptors, Wnt inhibitory nucleic acids, and the like. In some instances, a specific WNT inhibitor may be administered in such a manner as to result in a decrease in PAX3 expression and a promotion of FOXC2 expression. In some instances, a Wnt activator or inhibitor useful in the methods described herein may include those described in, e.g., Dodge and Lum et al. Annu Rev Pharmacol Toxicol.2011;51:289-310; Chen et al. Am J Physiol Gastrointest Liver Physiol. 2010 Aug;299(2):G293-300; Baker and Clevers, Nat Rev Drug Discov. 2006 Dec;5(12):997-1014; Meijer et al. Trends Pharmacol Sci. 2004 Sep;25(9):471-80; and Lepourcelet et al. Cancer Cell. 2004 Jan;5(1):91-102, the disclosures of which are incorporated herein by reference in their entirety. [0081] In some instances, an agent useful in a particular induction composition may include an activator of the FGF pathway. In some instances, an activator or inhibitor of the FGF pathway may also include activators or inhibitors of related signal transduction pathways including but not limited to, e.g., the MAPK/ERK signal transduction pathway. Activators and inhibitors of the FGF pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the FGF pathway resulting in a corresponding activation or inhibition in cellular FGF signaling. Activators of the FGF pathway include but are not limited to, e.g., FGF family ligands (e.g., FGF1, FGF2, FGF- 3, FGF-4, FGF-5, FGF-6, KGF/FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-15, FGF-16, FGF-17, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, etc.), SUN 11602 (4- [[4-[[2-[(4-Amino-2,3,5,6-tetramethylphenyl)amino]acetyl]methylamino]-1- piperidinyl]methyl]benzamide), t-Butylhydroquinone, U-46619, C2 Ceramide, Lactosyl Ceramide, Angiotensin II, Baicalin, and the like. In some instances, a FGF activator or inhibitor useful in the methods described herein may include those described in, e.g., English and Cobb, Trends Pharmacol Sci. 2002 Jan;23(1):40-5, the disclosure of which is incorporated herein by reference in its entirety. [0082] In some instances, an agent useful in a particular induction composition may include an activator or inhibitor of the BMP pathway. Activators and inhibitors of the BMP pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the BMP pathway resulting in a corresponding activation or inhibition in cellular BMP signaling. [0083] Activators of the BMP pathway include but are not limited to, e.g., BMP family ligands (e.g., BMP2, BMP4, BMP7, etc.), Alantolactone, FK506, isoliquiritigenin, 4′-hydroxychalcone, and the like. In some instances, activation of the BMP pathway may be achieved through repression of a BMP pathway inhibitor, e.g., including but not limited to the use of an inhibitory nucleic acid targeting an inhibitor of the BMP pathway or an antibody or small molecule directed to a BMP pathway inhibitor. [0084] Inhibitors of the BMP pathway include but are not limited to, e.g., NOGGIN, CHORDIN, LDN-193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride), DMH1 (4-[6-[4-(1-Methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]- quinoline), Dorsomorphin (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5- a]pyrimidine dihydrochloride), K 02288 (3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3- pyridinyl]phenol), ML 347 (5-[6-(4-Methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), DMH-1, antibodies to BMPs and BMP receptors, BMP inhibitory nucleic acids, and the like. [0085] In some instances, an agent useful in a particular induction composition may include an inhibitor of the GSK-3 pathway. Inhibitors of the GSK-3 pathway include small molecule activators, small molecule inhibitors, peptide inhibitors, antibodies, nucleic acid inhibitors, and the like that inhibit at least one component of the GSK-3 pathway resulting in a corresponding inhibition in cellular GSK-3 signaling. [0086] Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase involved in various cellular processes, including metabolism, cell proliferation, and apoptosis. In cell culture studies, inhibitors of GSK-3 are commonly used to investigate its roles and mechanisms. Here are some widely used GSK-3 inhibitors suitable for cell culture, including CHIR99021, which is an ATP-competitive inhibitor of GSK-3 effective at low micromolar concentrations, for example around 1 µM to 10 µM; SB216763, which is an ATP-competitive inhibitor of GSK-3 effective at low micromolar concentrations, for example around 1 µM to 10 µM; AR-A014418, which is an ATP-competitive inhibitor of GSK-3 effective at low micromolar concentrations, for example around 1 µM to 10 µM; BIO (6-Bromoindirubin-3'-oxime) which is an ATP-competitive inhibitor of GSK-3 effective at low micromolar concentrations, for example around 1 µM to 10 µM; TWS119; SB 415286; CHIR-98014 and the like. [0087] In some instances, an agent useful in a particular induction composition may include an agonist of the EGF pathway. Activators of the EGF pathway include small molecule activators, peptide activators, nucleic acid activators, nucleic acid inhibitors, and the like that activate at least one component of the EGF pathway. [0088] Epidermal Growth Factor (EGF) is a potent mitogenic peptide that stimulates cell growth, proliferation, and differentiation by binding to its receptor, EGFR (ErbB1). In cell culture studies, EGF agonists are used to activate EGFR signaling pathways to investigate their roles in various cellular processes. EGF agonists of interest include recombinant human EGF (rhEGF), for example in the range of about 1 ng/mL to 100 ng/mL; transforming growth factor-alpha (TGF-α) binds to and activates EGFR similar to EGF, and may be used at concentrations of about 0.1 ng/mL to 100 ng/mL; amphiregulin activates EGFR, and may be used at concentrations of from about 1 ng/mL to 100 ng/mL. [0089] In some instances, an agent useful in a particular induction composition may include an inhibitor of the ROCK pathway. Inhibitors of the ROCK pathway include small molecule activators, small molecule inhibitors, peptide inhibitors, antibodies, nucleic acid inhibitors, and the like that inhibit at least one component of the ROCK pathway resulting in a corresponding inhibition in cellular ROCK signaling. [0090] Rho-associated kinases (ROCKs) are serine/threonine kinases involved in various cellular functions, including contraction, motility, proliferation, and apoptosis. In tissue culture, ROCK inhibitors are used to modulate these cellular processes. ROCK inhibitors of interest include Y-27632 ((R)-(+)-trans-4-(1-Aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride), which competes with ATP for binding to the kinase domain, and may be used at concentrations of from about 10 µM to 50 µM; Fasudil (HA-1077, 5-(1,4-Diazepan-1- ylsulfonyl)isoquinoline) which competes with ATP for binding to the kinase domain, and may be used at concentrations of from about 10 µM to 100 µM; GSK269962A (4-[(2S)-2-amino-1- hydroxyethyl]phenol; (2R)-2-(3-chlorophenyl)-N-methylpyrrolidine-1-carboxamide) which competes with ATP, and may be used at concentrations of from about 1 nM to 10 µM; thiazovivin ((E)-N-methyl-N-(3-pyridinylmethylene)-1-(2-thiazolylsulfonyl)-1-propanamine) which inhibits by binding to the kinase domain, and may be used at concentrations of from about 0.5 µM and 2 µM; etc. [0091] c-Jun N-terminal kinase (JNK) inhibitors are compounds designed to specifically inhibit the activity of JNK, a member of the mitogen-activated protein kinase (MAPK) family involved in various cellular processes such as inflammation, stress responses, apoptosis, and development. There are several types of JNK inhibitors used in tissue culture, with different mechanisms of action and specificities, including, for example, SP600125, which is a broad- spectrum JNK inhibitor that competitively inhibits ATP binding to JNK. JNK-IN-8 is a highly selective and irreversible inhibitor of JNK1, JNK2, and JNK3. It covalently binds to a conserved cysteine residue in the ATP-binding pocket of JNK, providing sustained inhibition. AS601245 is another selective inhibitor that blocks JNK activity by competing with ATP. It has been shown to have fewer off-target effects compared to SP600125. [0092] In some instances, pathway modulating agents, as described above and including pathway activators and pathway inhibitors include, e.g., those that are commercially available, e.g., from such suppliers such as Tocris Bioscience (Bristol, UK), Sigma-Aldrich (St. Louis, MO), Santa Cruz Biotechnology (Santa Cruz, CA), and the like. [0093] Pluripotent stem cells and derivatives thereof may be contacted with these factors by any convenient means. Generally an agent is added to culture media, as described herein, within which cells of the instant disclosure are grown or maintained, such that the agent is present, in contact with the cells, at an effective concentration to produce the desired effect. In other instances, the culture media in which the cells are being grown is replaced with fresh culture media containing the particular agent present in the fresh media at an effective concentration to produce the desired effect. [0094] The effective concentration of a particular agent will vary and will depend on the agent. In addition, in some instances, the effective concentration may also depend on the cells being induced, the culture condition of the cells, other agents co-present in the culture media, etc. As such, the effective concentration of agents will vary and may range from 1 ng/mL to 10 µg/mL or more, including but not limited to, e.g., 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/mL, 26 ng/mL, 27 ng/mL, 28 ng/mL, 29 ng/mL, 30 ng/mL, 31 ng/mL, 32 ng/mL, 33 ng/mL, 34 ng/mL, 35 ng/mL, 36 ng/mL, 37 ng/mL, 38 ng/mL, 39 ng/mL, 40 ng/mL, 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46 ng/mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 1-5 ng/mL, 1-10 ng/mL, 1-20 ng/mL , 1-30 ng/mL, 1-40 ng/mL, 1-50 ng/mL, 5-10 ng/mL, 5-20 ng/mL, 10-20 ng/mL, 10-30 ng/mL, 10-40 ng/mL, 10-50 ng/mL, 20-30 ng/mL, 20-40 ng/mL, 20-50 ng/mL, 30-40 ng/mL, 30-50 ng/mL, 40-50 ng/mL, 1-100 ng/mL, 50-100 ng/mL, 60-100 ng/mL, 70-100 ng/mL, 80-100 ng/mL, 90-100 ng/mL, 10-100 ng/mL, 50-200 ng/mL, 100-200 ng/mL, 50-300 ng/mL, 100-300 ng/mL, 200-300 ng/mL, 50-400 ng/mL, 100- 400 ng/mL, 200-400 ng/mL, 300-400 ng/mL, 50-500 ng/mL, 100-500 ng/mL, 200-500 ng/mL, 300-500 ng/mL, 400 to 500 ng/mL, 0.001-1 µg/mL, 0.001-2 µg/mL, 0.001-3 µg/mL, 0.001-4 µg/mL, 0.001-5 µg/mL, 0.001-6 µg/mL, 0.001-7 µg/mL, 0.001-8 µg/mL, 0.001-9 µg/mL, 0.001- 10 µg/mL, 0.01-1 µg/mL, 0.01-2 µg/mL, 0.01-3 µg/mL, 0.01-4 µg/mL, 0.01-5 µg/mL, 0.01-6 µg/mL, 0.01-7 µg/mL, 0.01-8 µg/mL, 0.01-9 µg/mL, 0.01-10 µg/mL, 0.1-1 µg/mL, 0.1-2 µg/mL, 0.1-3 µg/mL, 0.1-4 µg/mL, 0.1-5 µg/mL, 0.1-6 µg/mL, 0.1-7 µg/mL, 0.1-8 µg/mL, 0.1-9 µg/mL, 0.1-10 µg/mL, 0.5-1 µg/mL, 0.5-2 µg/mL, 0.5-3 µg/mL, 0.5-4 µg/mL, 0.5-5 µg/mL, 0.5-6 µg/mL, 0.5-7 µg/mL, 0.5-8 µg/mL, 0.5-9 µg/mL, 0.5-10 µg/mL, and the like. [0095] In some instances, the effective concentration of an agent in solution, e.g., cell culture media, may range from 1 nM to 100 µM or more, including but not limited to, e.g., 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 1-2 nM, 1-3 nM, 1-4 nM, 1-5 nM, 1-6 nM, 1-7 nM, 1-8 nM, 1-9 nM, 1-10 nM, 1.5 nM, 1.5-2 nM, 1.5-3 nM, 1.5-4 nM, 1.5-5 nM, 1.5-6 nM, 1.5-7 nM, 1.5-8 nM, 1.5-9 nM, 1.5-10 nM, 2-3 nM, 2-4 nM, 2-5 nM, 2-6 nM, 2-7 nM, 2-8 nM, 2-9 nM, 2-10 nM, 3-4 nM, 3-5 nM, 3-6 nM, 3-7 nM, 3-8 nM, 3-9 nM, 3-10 nM, 4-5 nM, 4-6 nM, 4-7 nM, 4-8 nM, 4-9 nM, 4-10 nM, 5-6 nM, 5-7 nM, 5-8 nM, 5-9 nM, 5-10 nM, 6-7 nM, 6-8 nM, 6-9 nM, 6-10 nM, 7-8 nM, 7-9 nM, 7-10 nM, 8-9 nM, 8-10 nM, 9-10 nM, 5-15 nM, 5-20 nM, 5-25 nM, 5-30 nM, 5-35 nM, 5-40 nM, 5-45 nM, 5-50 nM, 10-15 nM, 10-20 nM, 10-25 nM, 10-30 nM, 10-35 nM, 10-40 nM, 10-50 nM, 15-20 nM, 15-25 nM, 15-30 nM, 15-35 nM, 15-40 nM, 15-45 nM, 15-50 nM, 20-25 nM, 20-30 nM, 20-35 nM, 20-40 nM, 20-45 nM, 20-50 nM, 25-30 nM, 25-35 nM, 25-40 nM, 25-45 nM, 25-50 nM, 30-35 nM, 30-40 nM, 30-45 nM, 30-50 nM, 35-40 nM, 35-45 nM, 35-50 nM, 40-45 nM, 40-50 nM, 45-50 nM, 10-100 nM, 20-100 nM, 30-100 nM, 40-100 nM, 50-100 nM, 60-100 nM, 70-100 nM, 80-100 nM, 90- 100 nM, 50-150 nM, 50-200 nM, 50-250 nM, 50-300 nM, 50-350 nM, 50-400 nM, 50-450 nM, 50-500 nM, 10-150 nM, 10-200 nM, 10-250 nM, 10-300 nM, 10-350 nM, 10-400 nM, 10-450 nM, 10-500 nM, 100-150 nM, 100-200 nM, 100-250 nM, 100-300 nM, 100-350 nM, 100-400 nM, 100-450 nM, 100-500 nM, 200-500 nM, 300-500 nM,400-500 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM,500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM,900 nM, 950 nM, 200-400 nM, 300-500 nM, 400-600 nM, 500-700 nM, 600-800 nM, 700-900 nM, 800 nM to 1µM, 0.5-1 µM, 0.5-1.5 µM, 0.5-2 µM, 0.5-2.5 µM, 0.5-3 µM, 0.5-3.5 µM, 0.5-4 µM, 0.5-4.5 µM, 0.5-5 µM, 1 µM, 2 µM, 3 µM, 4 µM, 5 µM, 6 µM, 7 µM, 8 µM, 9 µM, 10 µM, 11 µM, 12 µM, 13 µM, 14 µM, 15 µM, 16 µM, 17 µM, 18 µM, 19 µM, 20 µM, 21 µM, 22 µM, 23 µM, 24 µM, 25 µM, 26 µM, 27 µM, 28 µM, 29 µM, 30 µM, 31 µM, 32 µM, 33 µM, 34 µM, 35 µM, 36 µM, 37 µM, 38 µM, 39 µM, 40 µM, 41 µM, 42 µM, 43 µM, 44 µM, 45 µM, 46 µM, 47 µM, 48 µM, 49 µM, 50 µM, 1-2 µM, 1-3 µM, 1-4 µM, 1-5 µM, 1-6 µM, 1- 7 µM, 1-8 µM, 1-9 µM, 1-10 µM, 1.5 µM, 1.5-2 µM, 1.5-3 µM, 1.5-4 µM, 1.5-5 µM, 1.5-6 µM, 1.5-7 µM, 1.5-8 µM, 1.5-9 µM, 1.5-10 µM, 2-3 µM, 2-4 µM, 2-5 µM, 2-6 µM, 2-7 µM, 2-8 µM, 2-9 µM, 2-10 µM, 3-4 µM, 3-5 µM, 3-6 µM, 3-7 µM, 3-8 µM, 3-9 µM, 3-10 µM, 4-5 µM, 4-6 µM, 4-7 µM, 4-8 µM, 4-9 µM, 4-10 µM, 5-6 µM, 5-7 µM, 5-8 µM, 5-9 µM, 5-10 µM, 6-7 µM, 6-8 µM, 6-9 µM, 6-10 µM, 7-8 µM, 7-9 µM, 7-10 µM, 8-9 µM, 8-10 µM, 9-10 µM, 5-15 µM, 5-20 µM, 5- 25 µM, 5-30 µM, 5-35 µM, 5-40 µM, 5-45 µM, 5-50 µM, 10-15 µM, 10-20 µM, 10-25 µM, 10- 30 µM, 10-35 µM, 10-40 µM, 10-50 µM, 15-20 µM, 15-25 µM, 15-30 µM, 15-35 µM, 15-40 µM, 15-45 µM, 15-50 µM, 20-25 µM, 20-30 µM, 20-35 µM, 20-40 µM, 20-45 µM, 20-50 µM, 25-30 µM, 25-35 µM, 25-40 µM, 25-45 µM, 25-50 µM, 30-35 µM, 30-40 µM, 30-45 µM, 30-50 µM, 35-40 µM, 35-45 µM, 35-50 µM, 40-45 µM, 40-50 µM, 45-50 µM, 10-100 µM, 20-100 µM, 30- 100 µM, 40-100 µM, 50-100 µM, 60-100 µM, 70-100 µM, 80-100 µM, 90-100 µM, and the like. [0096] In some instances, the effective concentration of an induction agent will be below a critical concentration such that the induction produces the desired effect essentially without undesirable effects. As used herein, the term “critical concentration” refers to a concentration of induction agent above which undesirable effects are produced. Undesirable effects that may be the result of a concentration exceeding the critical concentration include but are not limited to, e.g., off-target effects (off-target activation of signaling, off-target inhibition of signaling), reduction or loss of function (e.g., loss of desired activator function, loss of desired inhibitor function) reduction of cell viability, increase in cell mortality, lineage restriction towards an undesired cell type, differentiation into an undesired cell type, loss of expression of a particular desired marker, etc. Whether a particular induction agent will have a critical concentration and what the critical concentrations of those agents having a critical concentration are will depend on the agent and the specific conditions in which the agent is used. [0097] In some instances, cells of the instant disclosure may be contacted with multiple agents and/or multiple compositions in order achieve a desired cell type. In some instances, a particular composition will contain two or more agents such that a particular cell culture is simultaneously contacted with multiple agents. In some instances, a particular series of compositions may be used, one at a time, in generating a desired cell type such that a particular cell culture is successively contacted with multiple agents. [0098] The duration of contact of a particular composition with a particular cell type, in some instances, may be referred to as the “exposure time” and exposure times may range from a day to weeks or more, including but not limited to e.g., 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, 10 days, 11 days, 12, days, 13, days, 14 days, 15, days, etc. As used herein, exposure times are, in some instances, referred as consisting essentially of, e.g., 24 hours, indicating that the exposure time may be longer or shorter than that specified including those exposure times that are longer or shorter but do not materially affect the basic outcome of the particular exposure. As such, in some instances where a particular exposure is more time sensitive such that under or over exposure, e.g., of more or less than 1 hour, materially affects the outcome of the exposure, a time period consisting essentially of, e.g., 24 hours, will be interpreted to refer to a time period ranging from about 23 hours to about 25 hours. In some other instances where a particular exposure is less time sensitive such that under or over exposure, e.g., of more than 12 hours, does not materially affect the outcome of the exposure, a time period consisting essentially of, e.g., 24 hours will mean a time period ranging from about 12 hours or less to about 36 hours or more. In some instances, depending on the context, an exposure period consisting essentially of 24 hours may refer to an exposure time of 22-26 hours, 21-27 hours, 20-28 hours, 19-29 hours, 18-30 hours, etc. [0099] In some instances, time periods of exposure may be pre-determined such that cells are contacted with an induction composition according to a schedule set forth prior to the contacting. In some instances, the time period of exposure, whether pre-determined or otherwise, may be modulated according to some feature or characteristic of the cells and/or cell culture, including but not limited to, e.g., cell morphology, cell viability, cell appearance, cellular behaviors, cell number, culture confluence, marker expression, etc. [00100] In some instances, cells are grown in densities that may range from but not limited to 100 cells/cm2, 103 cells/cm2, 104 cells/cm2, 105 cells/cm2, 106 cells/cm2, 107 cells/cm2, 108 cells/cm2, 109 cells/cm2, 1010 cells/cm2. Markers [00101] Aspects of the present disclosure include identifying cells based on the presence or absence or relative amount of one or more markers. In some instances, markers of interest include cell surface markers that may be detected, e.g., on live cells. In other instances, markers of interest include expression markers, e.g., cellular expression markers indicative of cell type. [00102] In an embodiment canonical esophogeal basal cells express one, two, three or more of the eBC markers FOXA1, FOXA2, SOX2, SOX9 and P63 with ITGB4, EPCAM, Krt5, and Krt15. In some embodiments a desired eBC cell population is purified for cells of interest. In some embodiments greater than 50%, greater than 75%, greater than 80% or more of the final cell population express one, two, three or more of the eBC markers. [00103] Markers may be detected or measured by any convenient means as such marker detection is well-known in the art and may make use of one or more detection reagents including but not limited to, e.g., antibodies, antibody fragments, binding partners (e.g., ligands, binding pairs, etc), hybridizable nucleic acids, aptamers, etc. In some instances, a marker may be a cell surface marker and detection of the marker may be performed based on the use of one or more detection reagents that specifically bind to the marker. Detection reagents, e.g., antibodies, may be detectably labeled (e.g., fluorescently labeled through the attachment of a fluorescent molecule, fluorescent bead, or other fluorescent label) or may be detected through the use of a second detectably labeled detection reagent that specifically binds to the first detection reagent (e.g., a fluorescently labeled secondary antibody). In some instances, a detection agent, e.g., having a detectable label or having been bound by a second agent having a detectable label, can be visualized or otherwise observed or detected based on the visual characteristics of the label, including e.g., fluorescent detection, colorimetric detection, and the like. Detectable labels useful in detection reagents need not be visually detectable and may, in some instances, be detected by a detection device con FIG.d to detect a non-visual detectable label including but not limited to, e.g., a magnetic label, a radioactive label, etc. In some instances, detectable labels may be detected through the use of one or more detection reactions, including but not limited to, e.g., enzymatic detection reactions (enzymatic reactions generating a detectable substrate, e.g., a fluorescent or colorimetric substrate), amplification reactions (PCR amplification, fluorescent signal amplification (e.g., tyramide signal amplification, etc.), etc.) [00104] In some instances, identification and/or selection for sorting of cells may be performed using a combination of markers. Such combinations may include but combinations of positive selection markers, combinations of negative selection markers or mixed combinations of positive and negative selection markers. [00105] In certain embodiments marker detection and/or measurement of marker level is performed using flow cytometry. Fluorescent signals used in flow cytometry, for instance when quantifying and/or sorting cells by any marker present on or in the cell, typically are fluorescently-tagged antibody preparations or fluorescently-tagged ligands for binding to antibodies or other antigen-, epitope- or ligand-specific agent, such as with biotin/avidin binding systems or fluorescently-labeled and optionally addressable beads (e.g. microspheres or microbeads). The markers or combinations of markers detected by the optics and/or electronics of a flow cytometer vary and in some cases include but are not limited to: cell surface markers, intracellular and nuclear antigens, DNA, RNA, cell pigments, cell metabolites, protein modifications, transgenic proteins, enzymatic activity, apoptosis indicators, cell viability, cell oxidative state, etc. [00106] In certain instances, flow cytometry is performed using a detection reagent, e.g., a fluorochrome-labeled antibody, e.g., a monoclonal antibody, with specific avidity against a cell surface maker of interest. A cellular sample is contacted with a detection reagent under conditions sufficient to allow the detection reagent to bind the cell surface maker and the cells of the sample are loaded into the flow cytometer, e.g., by first harvesting the cells from a cell culture using methods known in the art or described herein and re-suspending the isolated cells in a suitable buffer, e.g., running buffer. The flow cytometer detects events as the cell passes one or more detection areas of the flow cytometer. Detected events are counted or otherwise evaluated by the flow cytometer with or without input from an operator and used to determine, e.g., the total number of cells, the number or proportion of cells bound to a particular detection reagent, etc. In instances where FACS is utilized cells may be sorted, e.g., into separate containers, based on the detection or measurement of a particular marker. In some instances, cell sorting, e.g., by FACS, may be utilized to generate a purified population of a desired cell type. [00107] Expression markers of interest may be used to identify a particular cell type or verify that a derived cell type expresses a characteristic component of the derived cell type. In some instances, detection of expression markers may allow for optimization of a particular differentiation protocol, e.g., to optimize production of a desired cell type based on detection of one or more expression markers. Expression markers will vary depending on the type of cell to be identified or verified and/or desired downstream uses of the cell following identification or verification with the expression marker. Types of expression markers will include but are not limited to, e.g., gene expression marker, protein expression markers, expressed reporters, and the like. Expression marker detection and/or measurement may be detrimental to cell viability (e.g., wherein detection requires lysing or fixing a cell of interest) or may be essentially neutral to cell viability (e.g., wherein detection does not require lysing or fixing a cell of interest and may be performed on live cells). [00108] Methods of detecting and/or measuring gene expression and/or protein expression are well-known in the art and include but are not limited to, e.g., Northern blot, Western blot, ELISA, PCR, quantitative PCR, in situ hybridization, fluorescent in situ hybridization, immunohistochemistry, immunofluorescence, microarray, quantitative sequencing, RNAseq, quantitative mass spectrometry, and the like. Cell Modification [00109] Methods of modification of cells, including modification of pluripotent cells and modification of eBC are well-known in the art and include but are not limited to e.g., genetic modification (e.g., through deletion mutagenesis, through substitution mutagenesis), through insertional mutagenesis (e.g., through the introduction of heterologous nucleic acid into the pluripotent cell, etc.), non-mutagenic genetic modification (e.g., the non-mutagenic insertion of heterologous nucleic acid, etc.), epigenetic modification (e.g., through the treatment with one or more specific or general epigenetic modifying agents (e.g., methylation inhibitors, methylation activators, demethylases, etc.), other modifications (e.g., non-genetic labeling, etc.). [00110] Modifications of cells may be transient or stable. In some instances, a modification of a particular pluripotent cell or progenitor cell may be stable such that the modification persists through derivation of a desired cell type from the pluripotent cell or progenitor cell as described herein. In some instances, stable modifications may persist through introduction of a cell type into a host. In some instances, stable modifications may persist through proliferation of the cell such that all progenitors of a particular modified cell also contain the subject modification. In some instances, a modification of a particular pluripotent cell or progenitor cell may be transient such that the modification is lost after derivation of a cell type of interest from the transiently modified pluripotent cell. In certain instances, transient modifications may persist through one or more rounds of proliferation of the modified cell such that some but not all of the progeny of the modified cell contain the subject modification. In some instances, a transient modification will not persist during proliferation such that none of the progeny of a modified cell will contain the subject modification. In some instances, a transiently modified cell may be configured such that the modification persists through certain aspects of derivation of the cell type of interest, e.g., through derivation of a particular cell type of interest, but is lost prior to introduction of the derived cell into a host. Screening [00111] Aspects of the instant disclosure include methods of screening pharmacological agents using eBC derived according to the methods described herein. In some instances, a plurality of cell populations derived according to the methods as described herein are contacted with a plurality of pharmacological agents in order to screen for agents producing a cellular response of interest. A cellular response of interest may be any cellular response including but not limited to, e.g., cell death, cell survival, cell self-renewal, proliferation, differentiation, expression of one or more markers, loss of expression of one or more markers, change in morphology, change in cellular physiology, cellular engraftment, change in cell motility, change in cell migration, production of a particular cellular component, cease of production of a particular cellular component, change in metabolic output, response to stress, and the like. [00112] Screening pharmacological agents using cells described herein may be performed in vitro, e.g., in a tissue culture chamber, on a slide, etc., or may be performed in vivo, e.g., in an animal host, etc. Cells used in such screening assays may be genetically altered or may be unaltered. In some instances, cells generated according to the methods as described herein are used in multiplexed in vitro pharmacological screening. Methods for evaluating cellular responses during in vitro screening are well-known in the art and include but are not limited to, e.g., microscopic methods (e.g., light microscopy, electron microscopy, etc.), expression assays, enzymatic assays, cytological assays (e.g., cellular staining), genomics, transcriptomics, metabolomics, and the like. [00113] In some instances, cells generated according to the methods as described herein are introduced into a host animal and the host animal may be administered a pharmacological agent in order to screen for a response from the introduced cells. In some instances, the cells of the in vivo assay may be directly evaluated, e.g., for an intrinsic response to a pharmacological agent. In some instances, the host animal of the in vivo assay may be evaluated as an indirect measurement of the response of the cells to the pharmacological agent. [00114] In certain embodiments, the subject disclosure includes screening cells derived according to the methods described herein as a method of therapy of an animal model of disease and/or a human disease. Methods of screening cells derived according to the methods described herein as a method of therapy may be, in some instances, performed according to those methods described below regarding using such cells in therapeutic protocols. [00115] In certain embodiments, the subject disclosure includes screening cells derived according to the methods described herein introduced to a host animal as a method of directly evaluating the cells or particular cellular behaviors, e.g., due to an introduced genetic modification or a naturally derived mutation. In one embodiment, genetically modified cells, e.g., having at least one modified genomic locus, derived according to the methods described herein may be introduced into a host animal and the ability of the cells to differentiate into a particular tissue or cell type may be evaluated. In another embodiment, genetically modified cells derived according to the methods described herein may be introduced into a host animal and the behavior of the cells within the host animal and/or within a tissue of the host animal may be evaluated. In another embodiment, cells derived from a donor organism having a particular mutation or phenotype and lineage restricted according to the methods described herein may be introduced into a host animal and the behavior of the cells within the host animal and/or within a tissue of the host animal may be evaluated, including, e.g., the ability of the cells to differentiate into one or more tissue or cell types. The cells may introduced into the host animal in a autologous graft, an allograft, or a xenograft such that the introduced cells may be derived from the host animal, a separate donor of the same species as the host animal, or a separate donor of a different species as compared to the host animal, respectively. Therapy [00116] Aspects of the disclosure include methods for lessening the symptoms of and/or ameliorating a dysfunction in eBC and cells derived therefrom, for example in the treatment of recessive dystrophic epidermolysis bullosa; esophageal injury; cancer, etc. Any and all forms of dysfunction, whether treated or untreated, or resulting from any primary condition, whether treated or untreated, are suitable dysfunctions or disorders to be treated by the subject methods described herein. In some instances, the treatment methods described herein include the alleviation or reduction or prevention of one or more symptoms of dysfunction or disorder. Symptoms of dysfunction or disorder will vary, may be infrequent, occasional, frequent, or constant. [00117] The methods of treatment described herein include administering a therapeutically effective amount of a population, e.g., an essentially homogenous population, of eBC to a subject in need thereof in order to treat the subject for a dysfunction or deficiency. [00118] Conditions of interest for treatment include any condition in which esophogeal cells are damaged and can benefit from transplantation of healthy esophogeal cells. Examples include epidermolysis bullosa; esophageal injury; Barrett's esophagus, where the lining of the esophagus is damaged by stomach acid, leading to a change in the tissue lining. This condition can sometimes progress to esophageal cancer; esophogeal cancer, such as adenocarcinoma, squamous cell carcinoma, small cell carcinoma, sarcoma, lymphoma, melanoma and choriocarcinoma. Esophageal strictures are narrowing of the esophagus due to injury, inflammation, or chronic acid reflux, which can cause significant swallowing difficulties. Achalasia is a disorder where the esophagus has trouble moving food down into the stomach because the lower esophageal sphincter fails to relax properly. Esophageal atresia is a congenital condition where a part of the esophagus is missing. Transplanting esophageal cells can be a part of reconstructive surgery to repair the esophagus in infants. Gastroesophageal Reflux Disease (GERD) may include damage to the esophagus. [00119] The effective amount administered varies depending upon the goal of the administration, the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g., human, non-human primate, primate, etc.), the degree of resolution desired (e.g., the amount of alleviation or reduction of symptoms), the formulation of the cell composition, the treating clinician's assessment of the medical situation, and other relevant factors. [00120] A "therapeutically effective dose" or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy) or reduce, alleviate, or prevent symptoms to a desired extent as determined by the patient or the clinician. A therapeutically effective dose can be administered in one or more administrations. For purposes of this disclosure, a therapeutically effective dose of cells is an amount that is sufficient, when administered to (e.g., transplanted into) the individual, to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state by, for example, inducing stabilization, repair, or regeneration. [00121] In some embodiments, a therapeutically effective dose of cells is one cell or more (e.g., 1x102 or more, 5x102 or more, 1x103 or more, 5x103 or more, 1x104 cells, 5x104 or more, 1x105 or more, 5x105 or more, 1 x 106 or more, 2x106 or more, 5x106 or more, 1x107 cells, 5x107 or more, 1x108 or more, 5x108 or more, 1 x 109 or more, 5x109 or more, or 1x1010 or more). [00122] In some embodiments, a therapeutically effective dose of cells is in a range of from 1x103 cells to 1x1010 cells (e.g., from 5x103 cells to 1x1010 cells, from 1x104 cells to 1x1010 cells, from 5x104 cells to 1x1010 cells, from 1x105 cells to 1x1010 cells, from 5x105 cells to 1x1010 cells, from 1x106 cells to 1x1010 cells, from 5x106 cells to 1x1010 cells, from 1x107 cells to 1x1010 cells, from 5x107 cells to 1x1010 cells, from 1x108 cells to 1x1010 cells, from 5x108 cells to 1x1010, from 5x103 cells to 5x109 cells, from 1x104 cells to 5x109 cells, from 5x104 cells to 5x109 cells, from 1x105 cells to 5x109 cells, from 5x105 cells to 5x109 cells, from 1x106 cells to 5x109 cells, from 5x106 cells to 5x109 cells, from 1x107 cells to 5x109 cells, from 5x107 cells to 5x109 cells, from 1x108 cells to 5x109 cells, from 5x108 cells to 5x109, from 5x103 cells to 1x109 cells, from 1x104 cells to 1x109 cells, from 5x104 cells to 1x109 cells, from 1x105 cells to 1x109 cells, from 5x105 cells to 1x109 cells, from 1x106 cells to 1x109 cells, from 5x106 cells to 1x109 cells, from 1x107 cells to 1x109 cells, from 5x107 cells to 1x109 cells, from 1x108 cells to 1x109 cells, from 5x108 cells to 1x109, from 5x103 cells to 5x108 cells, from 1x104 cells to 5x108 cells, from 5x104 cells to 5x108 cells, from 1x105 cells to 5x108 cells, from 5x105 cells to 5x108 cells, from 1x106 cells to 5x108 cells, from 5x106 cells to 5x108 cells, from 1x107 cells to 5x108 cells, from 5x107 cells to 5x108 cells, or from 1x108 cells to 5x108 cells). [00123] In some embodiments, the concentration of cells to be administered is in a range of from 1 x 105 cells/ml to 1 x 109 cells/ml (e.g., from 1 x 105 cells/ml to 1 x 108 cells/ml, from 5 x 105 cells/ml to 1 x 108 cells/ml, from 5 x 105 cells/ml to 5 x 107 cells/ml, from 1 x 106 cells/ml to 1 x 108 cells/ml, from 1 x 106 cells/ml to 5 x 107 cells/ml, from 1 x 106 cells/ml to 1 x 107 cells/ml, from 1 x 106 cells/ml to 6 x 106 cells/ml, or from 2 x 106 cells/ml to 8 x 106 cells/ml). [00124] In some embodiments, the concentration of cells to be administered is 1 x 105 cells/ml or more (e.g., 1 x 105 cells/ml or more, 2 x 105 cells/ml or more, 3 x 105 cells/ml or more, 4 x 105 cells/ml or more, 5 x 105 cells/ml or more, 6 x 105 cells/ml or more, 7 x 105 cells/ml or more, 8 x 105 cells/ml or more, 9 x 105 cells/ml or more, 1 x 106 cells/ml or more, 2 x 106 cells/ml or more, 3 x 106 cells/ml or more, 4 x 106 cells/ml or more, 5 x 106 cells/ml or more, 6 x 106 cells/ml or more, 7 x 106 cells/ml or more, or 8 x 106 cells/ml or more). [00125] A therapeutically effective dose of cells may be delivered or prepared and any suitable medium, including but not limited to, e.g., those described herein. Suitable medium for the delivery of a therapeutically effective dose of cells will vary and may depend on, e.g., the type of pluripotent cells from which the effective dose of cells is derived or the type of derived cells of the effective dose. In some instances, a suitable medium may be a basal medium. “Cell medium” as used herein are not limited to liquid media may, in some instances, include non- liquid components or combinations of liquid media and non-liquid components. Non-liquid components that may find use a delivery or preparation medium include those described herein and those known in the art. In some instances, non-liquid components include natural or synthetic extra cellular matric components including but not limited to, e.g., basement membrane matrix components and the like. [00126] In some instances, an effective dose of the cells described herein may be co- administered with one or more additional agents (e.g., prepared in a suitable medium). Additional agents useful in such co-administration include agents that improve the overall effectiveness of the effective dose of cells or decrease the dose of cells necessary to achieve an effect essentially equal to administration of an effective dose of the cells without the additional agent. Non-limiting examples of additional agents that may be co-administered include: conventional agents for treating diseases, pro-survival factors, pro-engraftment factors, functional mobilization agents, and the like. [00127] By pro-survival factors is meant a factor or agent that may be added to the medium, culture media, delivery excipient, or storage solution that promotes the survival of a desired cell type. Such pro-survival factors may be general pro-survival factors that generally promote the survival of most cell types or may be specific pro-survival factors that only promote the survival of certain specific cell types. In some instances, pro-survival factors of the subject disclosure include but are not limited to, e.g., Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g., low does, i.e., sub-immunosuppressive dose, cyclosporine), ZVAD-fmk, pro-survival cytokines (e.g., insulin-like growth factor-1 (IGF-1)), extra cellular matrix (ECM) components, hydrogels, matrigel, collagen, gelatin, agarose, alginate, poly(ethylene glycol), hyaluronic acid, etc. [00128] By pro-engraftment factors is meant a factor or agent that may be added to the administered dose or the delivery excipient or the cell storage solution that, upon delivery of the cells into a subject for treatment, increase the engraftment of the administered cells into the tissue targeted for engraftment and therapy. In some instances, pro-engraftment factors include factors that physically retain the administered cells at the delivery site, e.g., the injection site in the case of direct injection to the affected area, including but not limited to, e.g., gels, polymers, and highly viscous liquids that have physical properties that prevent the administered cells from freely diffusing. Such gels, polymers, and highly viscous liquids include but are not limited to e.g., ECM components, hydrogels, matrigel, collagen, gelatin, agarose, alginate, poly(ethylene glycol), and the like. [00129] The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent. [00130] The cells may be introduced by injection, catheter, intravenous perfusion, or the like. The cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being capable of use upon thawing. Once thawed, the cells may be expanded by use of growth factors and/or feeder cells or in feeder-free conditions associated with progenitor cell proliferation and differentiation. In some instances, the cells may be administered fresh such that the cells are expanded and differentiated and administer without being frozen. [00131] The cells of this disclosure can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient or buffer or media prepared under sufficiently sterile conditions for human administration. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and eBC Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. Choice of the cellular excipient and any accompanying elements of the composition will be adapted in accordance with the route and device used for administration. The composition may also comprise or be accompanied with one or more other ingredients that facilitate the engraftment or functional mobilization of the cells. Suitable ingredients include matrix proteins that support or promote adhesion of the cells, or complementary cell types. [00132] Cells of the subject methods may be autologously derived. By autologously derived it is meant that the cells are derived from the subject that is to be treated with the cells. The cells may be derived from a tissue sample obtained from the subject including but not limited to, e.g., a blood sample (e.g., a peripheral blood sample), a skin sample, a bone marrow sample, and the like. In some instances, the sample from which cells are derived may be a biopsy or swab, e.g., a biopsy or swab collected to diagnose, monitor, or otherwise evaluate the subject, e.g., diagnose the subject for a dysfunction or deficiency, e.g., bone disease or a muscle disease or a cartilage disease or a related condition, or for cell collection. In some instances, the autologous sample from which the cells are derived may be a previously collected and stored sample, e.g., a banked tissue sample, from the subject to be treated, including but not limited to e.g., banked cardiac tissue or cells, banked musculoskeletal tissue or cells, banked reproductive tissue or cells, banked skin tissue or cells, banked bone tissue or cells, banked bone marrow tissue or cells, banked vascular tissue or cells, banked umbilical cord blood tissue or cells, and the like. [00133] In some instances, cells of the subject methods are non-autologously derived. By non- autologously derived it is meant that the cells are not derived from the subject that is to be treated with the cells. In some instances, non-autologously derived cells may be xeno-derived (i.e., derived from a non-human animal) or allo-derived (i.e. derived from a human donor other than the subject to be treated). Non-autologously derived cells or tissue may be derived from any convenient source of cells or tissue collected by any convenient means. [00134] Whether to use autologously derived or non-autologously derived cells may be determined according to the discretion of the subject’s clinician and may depend on, e.g., the health, age, genetic predisposition or other physical state of the subject. In some instances, autologous cells may be preferred, including, e.g., to decrease the risk or immune rejection of the transplanted cells. In some instances, non-autologous cells may be preferred, including, e.g., when the subject has a genetic defect. Pluripotent Stem cells [00135] Methods of derivation of pluripotent stem cells from an autologous or non-autologous tissue useful in the methods described herein include but are not limited to, e.g., methods of embryonic stem cell derivation and methods of induced pluripotent stem cell derivation. In some instances, methods as described herein may be performed using non-autologous pluripotent progenitor cells previously derived including, e.g., those publically or available or commercially available (e.g., from Biotime, Inc., Alameda, CA). In some instances, methods as described herein may be performed using newly derived non-autologous pluripotent progenitor cells or newly derived autologous pluripotent progenitor cells including but not limited to, e.g., newly derived embryonic stem cells (ESC) (including, e.g., those derived under xeno-free conditions as described in, e.g., Lei et al. (2007) Cell Research, 17:682-688) and newly derived induced pluripotent stem cells (iPS). General methods of inducing pluripotency to derive pluripotent progenitor cells are described in, e.g., Rodolfa KT, (2008) Inducing pluripotency, StemBook, ed. The Stem Cell Research Community, doi/10.3824/stembook.1.22.1 and Selvaraj et al. (2010) Trends Biotechnol, 28(4)214-23, the disclosures of which are incorporated herein by reference. In some instances, pluripotent progenitor cells, e.g., iPS cells, useful in the methods described herein are derived by reprogramming and are genetically unmodified, including e.g., those derived by integration- free reprogramming methods, including but not limited to those described in Goh et al. (2013) PLoS ONE 8(11): e81622; Awe et al (2013) Stem Cell Research & Therapy, 4:87; Varga (2014) Exp Cell Res, 322(2)335-44; Jia et al. (2010) Nat Methods, 7(3):197-9; Fusaki et al. (2009) Proc Jpn Acad Ser B Phys Biol Sci.85(8):348-62; Shao & Wu, (2010) Expert Opin Biol Ther.10(2):231-42; the disclosures of which are incorporated herein by reference. [00136] In some instances, before differentiation or lineage restriction of the pluripotent cells the pluripotent cells are dissociated, e.g., to generate a single-cell suspension. In some instances, the dissociation of the pluripotent cells is chemical, molecular (e.g., enzyme mediated), or mechanical dissociation. Methods of chemical, molecular, and/or enzyme mediated dissociation will vary and in some instances may include but are not limited to the use of, e.g., trypsin, TrypLE ExpressTM, TrypLE SelectTM, Accutase®, StemPro® (Life Technologies, Inc., Grand Island, NY), calcium and magnesium free media, low calcium and magnesium medium, and the like. In some instances the dissociation media may further include pro-survival factors including but not limited to, e.g., Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g., low does, i.e., sub- immunosuppressive dose, cyclosporine), ZVAD-fmk, pro-survival cytokines (e.g., insulin-like growth factor-1 (IGF-1)), Thiazovivin, etc. [00137] In some instances, methods of culturing pluripotent stem cells include xeno-free culture conditions wherein, e.g., human cells are not cultured with any reagents derived from non-human animals. In some instances, methods culturing of pluripotent stem cells include feeder-free culture conditions, wherein the pluripotent stem cells are cultured under conditions that do not require feeder cells and/or in feeder cell free medium, including e.g., commercially available feeder-free mediums, such as, e.g., those available from STEMCELL Technologies, Inc. (Vancouver, BC). In some instances, methods culturing of pluripotent stem cells include culture conditions that include supplemental serum, including e.g. supplement of autologously derived serum, e.g., as described in Stute et al. (2004) Exp Hematol, 32(12):1212-25. In some instances, methods include culture conditions that are serum-free, meaning the culture media does not contain animal, mammal, or human derived serum. Serum-free culture conditions may be performed for only a portion of the life of the culture or may performed for the entire life of the culture. As is known in the art, in some instances, cells may be cultured in two dimensional or three dimensional formats (e.g., on non-coated or coated surfaces or within a solid or semi-solid matrix). Instances where two dimensional or three dimensional culture is appropriate for use in the methods as described herein, e.g., to promote survival or differentiation of a desired cell type, will be readily apparent to the ordinary skilled artisan. General methods of culturing human pluripotent progenitor cells are described in, e.g., Freshney et al. (2007) Culture of human stem cells, Wiley-Interscience, Hoboken, NJ and Borowski et al. (2012) Basic pluripotent stem cell culture protocols, StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook, the disclosures of which are incorporated herein by reference. [00138] In some instances, the pluripotent progenitor cells used according to the methods described herein may be genetically unmodified. By “genetically unmodified” is meant that essentially no modification of the genome of the cells transplanted into the subject has been performed. Encompassed within the term genetically unmodified are instances wherein transient genetic modification is performed at some point during the derivation of the cells but essentially no genetic modification persists in the cells that are eventually transplanted into the subject (i.e. the cells are essentially indistinguishable before the transient genetic modification and after the course of the transient modification). Also encompassed within the term genetically unmodified are instances wherein the genome of the cells is not transiently or stably modified, e.g., where the cells are manipulated, e.g., pluripotent progenitors are derived or cells are transformed, without genetic modification (e.g., modification of the nucleotide sequence of the genome) of the cells. [00139] In some instances, the cells used according to the methods described herein may be genetically modified. By “genetically modified” is meant that at least one nucleotide is added to, changed within, or deleted from of the genome of the cell. In some instances, the genetic modification may be an insertion of a heterologous sequence, e.g., a sequence that encodes a tag, a label sequence, a reporter, a selectable marker, a gene encoding a protein from a species different from that of the host cell, etc. In some instances, the genetic modification corrects a defect or a mutation within the cell, e.g., corrects an anomalous mutation that confers a tissue dysfunction or deficiency. In some instances, the genetic modification deletes or renders inoperable an endogenous gene of the host cell. In some instances, the genetic modification enhances an endogenous gene of the host cell. In some instances, the genetic modification represents a change that enhances survival, control of proliferation, and the like. Cells may be genetically altered by transfection or transduction with a suitable vector, homologous recombination, or other appropriate technique, so that they express a heterologous sequence or have altered expression of an endogenous gene. [00140] For further elaboration of general techniques useful in the practice of this disclosure, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, and embryology. With respect to tissue culture and stem cells, the reader may wish to refer to Teratocarcinomas and embryonic stem cells: A practical approach (E. J. Robertson, ed., IRL Press Ltd.1987); Guide to Techniques in Mouse Development (P. M. Wasserman et al. eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998). Systems [00141] Also provided are systems for use in practicing the subject methods. Systems of the subject disclosure may include a cell production system, e.g., for the production of a homogenous or highly pure population of eBC from pluripotent progenitor cells. [00142] In some instances, the cell production system includes a cell culture chamber or cell culture vessel for the culture of desired cell types. Such cell culture chambers may be con FIG.d for the expansion of pluripotent progenitor cells and for the differentiation and/or lineage restriction of such pluripotent progenitor cells into desired cell types. In some instances, the cell culture chamber is also con FIG.d for the expansion of eBC. In certain embodiments, the cell culture chamber or cell culture vessel may be an open culture system, including but not limited to e.g., tissue culture dishes, tissue culture plates, tissue culture multi-well plates, tissue culture flasks, etc. In certain embodiments, the cell culture chamber or cell culture vessel may be a closed culture system, including e.g., a bioreactor, a stacked tissue culture vessel (e.g., CellSTACK Culture Chambers available from Corning, Inc. Corning, NY). In some instances, culture media and or other factors or agents may be exchanged in and out of the cell culture chamber through the use of one or more pumps (e.g., syringe pumps, peristaltic pumps, etc.) or gravity flow devices. In instances where the cells are cultured under sterile conditions the culture system may allow for the sterile exchange of culture media, e.g., through the use of sterile tubing connected, sealed, and reconnected through the use of a sterile devices, including but not limited to, e.g., a sterile tube welder and/or a sterile tube sealer. The cell culture system may be con FIG.d to control certain environmental conditions, including but not limited to e.g., temperature, humidity, light exposure, air composition (e.g., oxygen levels, carbon dioxide levels, etc.) to achieve the conditions necessary for expansion and/or differentiation of desired cell types. In some instances, the cell culture chamber may include a cell culture vessel that includes one or more patterned cell culture substrates or one or more arrays of patterned cell culture substrates as described herein. [00143] The cell culture chamber may be configured for the production of cells for clinical use, e.g., according to current good manufacturing practice (cGMP) compliant cell culture practices, including the methods and configurations described in e.g., Fekete et al. PLoS ONE (2012) 7(8): e43255; Pham et al. (2014) J Trans Med 12:56; Gastens et al. (2007) Cell Transplant 16(7):685-96; Fernandes et al. (2013) Stem Cell Bioprocessing: For Cellular Therapy, Diagnostics and Drug Development, Burlington, Oxford: Elsevier Science: Woodhead Publishing, the disclosures of which are incorporated herein by reference. [00144] The cell production system may, in some instances, be computer controlled and/or automated. Automated and/or computer controlled cell production systems may include a “memory” that is capable of storing information such that it is accessible and retrievable at a later time or date by a computer. Any convenient data storage structure may be chosen, based on the means used to access the stored information. In certain aspects, the information may be stored in a “permanent memory” (i.e. memory that is not erased by termination of the electrical supply to a computer or processor) or “non-permanent memory”. Computer hard- drive, CD-ROM, floppy disk, portable flash drive and DVD are all examples of permanent memory. Random Access Memory (RAM) is an example of non-permanent memory. A file in permanent memory may be editable and re-writable. [00145] In certain instances, a computer controlled and/or automated cell culture system may include a module or program stored in memory for production of cells according to the methods described herein. Such a module may include instructions for the administration of induction agent and/or induction compositions, e.g., at particular timing intervals or according to a particular schedule, in order to generate a desired cell type. In some instances, such a computer module may further include additional modules for routine cell culture tasks including but not limited to, e.g., monitoring and record keeping, media changes, environmental monitoring, etc. [00146] Systems of the present disclosure include components and/or devices for delivering cells produced according to the methods described herein to a subject in need thereof. For example, in some instances a system for treating a subject with a derived tissue dysfunction or deficiency includes a cell injection system for delivering cells in a carrier, with or without optional adjuvants, to a desired injection site, including diseased tissue, adjacent to diseased tissue, and/or within, on or near a dysfunctioning organ. Such systems utilize known injection devices (e.g., including but not limited to needles, bent needles, cannulas, syringes, pumps, infusion devices, diffusion devices, etc.) and techniques (e.g., including but not limited to intramuscular injection, subcutaneous injection, device-guided injection, etc.). In some instances, a device or technique used for the delivery of a cell scaffold or other bioengineered device may be con FIG.d or adapted for use in a cell delivery system for use in delivering cells derived according to the methods described herein. [00147] In addition to the above described components systems of the subject disclosure may include a number of additional components, such as data output devices, e.g., monitors and/or speakers, data input devices, e.g., interface ports, keyboards, etc., fluid handling components, power sources, controllers, etc. Compositions and Kits [00148] Also provided are compositions and kits for use in the subject methods. The subject compositions and kits include any combination of components for performing the subject methods. In some embodiments, a composition can include, but is not limited to and does not require, the following: cell dissociation agents and/or media, cell reprogramming agents and/or media, pluripotent progenitor cells, cell culture agents and/or media, cell differentiation agents and/or media; lineage restriction agents (e.g., induction agents) and/or media; conventional agents for treating diseases and/or dysfunctions, pro-survival factors, pro-engraftment factors, functional mobilization agents and any combination thereof. [00149] In some embodiments, a kit can include, but is not limited to and does not require, the following: any of the above described composition components, a sample collection container, a sample collection device (e.g., a sample collection container that includes a sample enrichment mechanism including, e.g., a filter), a tissue collection device (e.g., a biopsy device), a tissue dissociation device, a cell culture vessel, a cell production system; and any combination thereof. [00150] In some embodiments, a kit can include, but is not limited to and does not require, a cell delivery system and/or a cell injection system con FIG.d for delivery of cells derived according to the methods described herein. For example, a kit may include a cell injection system con FIG.d for injection or delivery of cells into a desired area of the subject in order to effectively treat the subject for a tissue dysfunction or deficiency, e.g., through delivery of cells to the tissue. Such kits may include a cell delivery or injection system, as described herein, including individual components of such systems in assembled or unassembled form. In some instances, cells derived according to the methods described herein may be “preloaded” into a cell injection or delivery system such that the system is provided in a “ready-to-use” configuration. In other instances, a cell injection or delivery system may be provided in an “unloaded” configuration such that cells derived according to the methods described herein must be loaded into the system, with any desired carrier or vehicle, prior to use. [00151] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is electronic, e.g., a website address which may be used via the internet to access the information at a removed site. Machine Learning Embodiments [00152] In an embodiment, methods are provided for the use of machine learning to develop methods of differentiating stem or progenitor cells to a cell type of interest. Such methods may utilize single cell and spatial technologies to generate a spatiotemporal multi-omics cell atlas for development of the cell or tissue of interest. Alternatively, an existing cell atlas such as that disclosed herein may be used. A machine learning algorithm is used to prioritize the combinations of candidate developmental signals for in silico method of differentiating a cell type of interest. Functional validation of the predicted developmental signals can be performed. [00153] The cell atlas may utilize a program, e.g. Seurat, for single cell gene expression quantification, dimension reduction, clustering analysis and marker gene identification. A tissue of interest at different stages of development is used, and can be merged separately for downstream analysis. Cells expressing no more than 200 genes or more than 8000 genes may be removed. Cells having more than 20% of reads mapped to mitochondria may be also removed. Highly variable features are identified, and PCA is performed using variable features. Cells can be clustered using multiple dimensions and visualized, e.g.using UMAP. The initial iterative clustering revealed clusters for gross cellular compartments. These cellular compartments may be subset for further iterative clustering and annotation. [00154] In some embodiments the machine learning process utilizes a model (referred to herein as the Manatee model), which is adapted from VAE by constraining its latent space to represent transcription factor (TF) expression. To predict perturbation-induced expression profiles, latent variables associated with candidate TFs are adjusted. Such an adjusted latent space will then be decoded as final predictions. The perturbation effect was quantified by computing the Pearson correlation (R) between the average basal cell and the perturbation expression profiles. [00155] The Manatee deep learning framework models the generative process from TF expression to the corresponding gene expression. In order to do so, 1) both the encoder and decoder neural networks consist of two fully connected layers, each with the same number of nodes as the number of genes; 2) the latent space contain the same number of variables as the number of TFs; and 3) the following loss function is optimized during training: ^ ∼ ^^ +^^^ + ^^ [00156] where LR and DKL represent reconstruction loss and Kullback–Leibler Divergence against the N(0, 1) normal distribution respectively, as the two regular VAE loss terms. The additional Lr term represents the TF reconstruction loss, which is the mean square error (MSE) between reparameterized latent variables (Z) and TF expression (TF): ^^ ≡ ^^^(^, ^^) [00157] Manatee analysis starts with the TF expression matrix (D16 and D24 in our case), and perturbations are made by adjusting values of corresponding latent variables. The adjusted TF expression profiles are then passed through the decoder to predict expression profiles after perturbations. [00158] TFs may be defined based on the Gene Ontology (GO) database, under terms GO:0003700, “DNA-binding transcription factor activity”, GO:0003677, “DNA binding”, GO:0140110, “transcription regulator activity” and GO:0006355, “regulation of DNA-templated transcription”. Pathway gene lists from the GO database may also be included. When perturbing specific signaling pathways, the corresponding lists will be retrieved, and non-TFs will be filtered. The yielded lists are then manually annotated based on previous literature in terms of determining perturbation directions. Manatee can be trained by merging in vivo and in vitro single cell datasets to comprehensively capture regulatory logics during the cell specification and differentiation in vivo. [00159] Also provided are databases and computer systems for analysis. Databases can typically comprise cell atlas information, a machine learning model, databases of transcription factors and gene pathways, etc. The results and databases thereof may be provided in a variety of media to facilitate their use. [00160] "Media" can refer to a manufacture that contains database information; and methods of analysis as described above. The databases and comparative algorithms can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present database information. "Recorded" refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc. [00161] As used herein, "a computer-based system" refers to the hardware means, software means, and data storage means used to analyze the information provided herein. The minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in analysis. The data storage means may comprise any manufacture comprising a recording of the present information as described above, or a memory access means that can access such a manufacture. [00162] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test expression repertoire. [00163] The data analysis may be implemented in hardware or software, or a combination of both. In one embodiment, a machine-readable storage medium is provided, the medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention. Such data may be used for a variety of purposes, such as drug discovery, analysis of interactions between cellular components, and the like. In some embodiments, the analysis is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer may be, for example, a personal computer, microcomputer, or workstation of conventional design. [00164] Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language. Each such computer program can be stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein. [00165] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems. One format for an output tests datasets possessing varying degrees of similarity to a trusted repertoire. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test repertoire. [00166] Further provided herein is a method of storing and/or transmitting, via computer, data collected by the methods disclosed herein. Any computer or computer accessory including, but not limited to software and storage devices, can be utilized to practice the present invention. Data can be input into a computer by a user either directly or indirectly. Additionally, any of the devices which can be used to perform or analyze NIA can be linked to a computer, such that the data is transferred to a computer and/or computer-compatible storage device. Data can be stored on a computer or suitable storage device (e.g., CD). Data can also be sent from a computer to another computer or data collection point via methods well known in the art (e.g., the internet, ground mail, air mail). Thus, data collected by the methods described herein can be collected at any point or geographical location and sent to any other geographical location. [00167] The analysis and database storage can be implemented in hardware or software, or a combination of both. In one embodiment of the invention, a machine-readable storage medium is provided, the medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention. Such data can be used for a variety of purposes, such as patient monitoring, initial diagnosis, clinical trial analysis, and the like. Preferably, the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer can be, for example, a personal computer, microcomputer, or workstation of conventional design. [00168] Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language. Each such computer program is preferably stored on a storage media or device readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The system can also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein. [00169] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. One format for an output means test datasets possessing varying degrees of similarity to a trusted profile. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test pattern. EXAMPLES [00170] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., room temperature (RT); base pairs (bp); kilobases (kb); picoliters (pl); seconds (s or sec); minutes (m or min); hours (h or hr); days (d); weeks (wk or wks); nanoliters (nl); microliters (ul); milliliters (ml); liters (L); nanograms (ng); micrograms (ug); milligrams (mg); grams ((g), in the context of mass); kilograms (kg); equivalents of the force of gravity ((g), in the context of centrifugation); nanomolar (nM); micromolar (uM), millimolar (mM); molar (M); amino acids (aa); kilobases (kb); base pairs (bp); nucleotides (nt); intramuscular (i.m.); intraperitoneal (i.p.); subcutaneous (s.c.); and the like. Example 1 [00171] While stem cell technologies offer great promise, a broader approach to harness their potential to generate designer cell-based personalized therapeutics requires deeper understanding of tissue level interactions. We previously developed a surface ectoderm- derived stratified epithelial method for skin, but induction of the related anterior foregut-derived human fetal esophageal stratified epithelium mucosa for tissue replacement remains a hurdle. Here we use single cell and spatial technologies applied to human embryonic tissue during establishment of esophageal tissue architecture to describe the unique cellular heterogeneity, lineage transitions and intercellular signals of human embryonic esophageal epithelium and stroma. We use the recently developed variational autoencoder Manatee algorithm to prioritize candidate stromal signals for effective and efficient signaling pathway perturbations that guide in vitro eBC differentiation. Functional validation of tissue organoids leads to efficient development of a chemically-defined good manufacturing practice compatible protocol for functional esophageal mucosa. Our approach creates a versatile methodology that accelerates cell replacement therapy for lineage-specific stratified epithelial genetic defects and wounds. [00172] Classic histology studies show that the human esophagus is specified in the dorsal anterior foregut and separated from the ventral trachea at around 4-6 weeks of human gestation. At 8 weeks, the human esophagus becomes lined with stratified columnar epithelium that is protected by the terminal differentiation into multi-ciliated cells. Later at 20 weeks’ gestation, squamous stratified epithelium appears to replace the ciliated epithelium as the dominant terminal differentiation trajectory. While the epithelial morphogenesis has been documented superficially, the underlying stromal composition and roles in morphogenesis remain unknown. Developmental studies in skin squamous stratified epithelium and our recent work have shown that regional mesodermal morphogenic signals wire the spatial-temporal specific stratification program in surface ectoderm progenitors, and these signals are required in our hPSC-derived skin production for optimal graftability. Therefore, to accelerate esophageal mucosa manufacturing, a holistic molecular characterization of the coordinated development of epithelium and stroma is needed. To overcome these hurdles, we have now built an integrated tissue informatics platform and present a spatiotemporal single-cell multi-omics atlas for human esophageal development. With such an atlas, we comprehensively catalog the cellular diversity, lineage transition, anatomic architecture and intercellular communication of the esophageal epithelium and stroma. We then use the novel deep learning algorithm Manatee to prioritize signaling pathway combinations for the in vitro specification of esophageal basal cells. We identify activating EGF, BMP, TGFβ while inhibiting WNT as the optimal strategy for esophageal basal cell derivation, and functionally validate the approach to establish a clinical standard hPSC-to- esophageal basal cell differentiation system that will accelerate translational applications. RESULTS [00173] A spatiotemporal multi-omics cell atlas for human esophageal development. Aiming to manufacture esophageal mucosa using human tissue developmental signaling, we comprehensively characterized the cellular dynamics during human esophageal development and extracted the intercellular signals associated with morphogenesis for in silico screening (FIG. 1A). In collaboration with the NIH-sponsored UW Birth Defects Research Laboratory (BDRL), we ethically collected human embryonic and fetal esophagus samples from 35 individual donors for single cell and spatial profiling (FIG.1A, FIG.8A). Our dataset ranged from 45 (mid first trimester) to 132 (mid second trimester) post-conception days (noted as E45 to E132), spanning several key developmental milestones: onset of epithelial morphogenesis, fetal-specific ciliogenesis and squamous stratification (FIG.1B upper panel). In addition to the epithelium, our dataset also catalogs the drastic growth, increased tissue complexity, and organization of the surrounding stroma (FIG.1B bottom panel), thus providing the opportunity to explore tissue interactive signals during the coordinated development of epithelium and stroma. [00174] By densely sampling human esophageal development, we produced high quality single cell transcriptomic data for both epithelium and stroma using distinct cell dissociation methods (FIG. 8B-C). After iterative clustering, removal of low-quality and contaminant clusters, and removal of the erythrocyte cluster, we present 97,212 epithelial cells from 14 timepoints and 91,891 stromal cells from 11 timepoints (FIG. 8A, 8C-J). Using canonical markers, we grossly annotated 6 major cellular compartments: epithelium (EPI), mesenchyme (MES), enteric nervous system (ENS), endothelium (ENDO), skeletal muscle (SKM), immune cells (IM). Fine-grained clustering of each compartment further discovered 10 cell types/states in the epithelium (FIG.2A, FIGS.8B, 8H-J) and 66 in the stroma (FIG.3A, FIG.8B, 8K-M). [00175] Classic tissue recombination experiments demonstrate that the signal interactions between epithelium and local mesenchyme are essential for tissue morphogenesis and cell fate determination. We and others have demonstrated the significance of integrating supporting cellular signals into human tissue engineering, reinforcing the importance of detailing the spatial architecture of developing human esophagus. To systematically map the cell types revealed in single cell atlas, we orthogonally performed unbiased Visium spatial transcriptomics (16 sections from 5 timepoints, 5 donors) and high-resolution CODEX (co- detection by indexing) proteomic profiling (48 sections/905,062 cells from 13 timepoints, 15 donors) (FIG.1A, FIG.8A). We integrated single cell transcriptomics with the spatial profiles to delineate the dynamic cellular architecture for human esophageal development. The full dataset can be explored online through the EsoDev data portal. [00176] Developmental origin of esophageal basal cells identified at the early stage. We first cataloged human esophageal epithelial morphogenesis to build the molecular roadmap for esophageal mucosa manufacturing, focusing on the major tissue morphogenic transitions. Our scRNA-seq dataset of human esophageal epithelial cells showed a relatively constant proportion of basal cells (KRT5, KRT15) across all the samples, suggesting the establishment of the self-renewing basal stem cell pool in early development (FIGS.2A-B, FIGS.8H-J, 9A). To identify basal cell precursors, we focused on a small cluster of cells (Epi_PG) mainly detected in the earliest E47 scRNA-seq sample (FIGS.2A-B, FIGS.8H-I). These cells did not express definitive markers for any known differentiated cell types, including esophageal basal cells (KRT5, KRT15), esophageal suprabasal cells (KRT13, KRT4), ciliated cells (FOXJ1), thyroid (PAX8), respiratory tract (NKX2-1), pharyngeal pouch (TBX1), stomach (CLDN18, GATA4), or intestine (CDX2) (FIG.8J). The high expression of SHH and GATA6 revealed their uncommitted foregut progenitor identity (FIG. 8J). Interestingly, several epithelial- mesenchymal transition (EMT) genes, NCAM1, VIM, VCAN, were expressed in these cells and largely lost while progressing to basal cells (FIG.9A), consistent with the active epithelial morphogenesis at this early stage. Their presence was validated by immunofluorescence for NCAM1 and GATA6 in early-stage esophageal sections before the appearance of basal cells (FIGS.2C-D). By H&E, we found that these cells presented a primitive-appearing morphology in the multilayered early esophagus (FIG. 1B, E45 and E57). Furthermore, the Monocle pseudotime analysis predicted that these progenitors first give rise to basal cells, while basal cells later generate other differentiated populations (FIG.2F, FIG.9B). Therefore, for the first time we reveal the molecular features and lineage commitment of the stratified columnar epithelium documented in classic histology literature. [00177] Two differentiation waves of esophageal epithelial morphogenesis. After the basal cell specification, we observed two distinct differentiation programs that are sequentially activated: fetal-specific ciliated differentiation and canonical squamous stratified differentiation (FIGS. 2F, 4C). The ciliogenesis trajectory originates from basal cells, which first downregulate KRT15, maintain the expression of KRT5 while begin to express KRT13 in the transitioning suprabasal cell population (SB-1). Following that, a unique club cell-like population (SCGB1A1, UPK2, UPK1B, MSLN, named as SB-5) appears and generated multiciliated cells (FOXJ1). Our dataset also captures the complete ciliogenesis process by delineating the nascent ciliated cells (MUC1, DEP1, CDC20B, named as Cil-1) and the mature ciliated cells (TFF3, SNTN, TPPP3, CAPS, named as Cil-2) (FIGS.2E-F, FIGS.8H-J, 9C). In contrast, the squamous stratification trajectory transitions through multiple suprabasal descendants by gradually downregulating basal cell marker KRT5, early squamous marker KRT13, KRT6A, KRT6B and upregulating late squamous marker KRT4 (FIG.2F, FIGS.8H-J, named as SB-2, SB-3, and SB-4). Interestingly, the ciliogenesis was predominantly observed at mid stage, marked by the peak of SB-5 before E80 (FIG.2B, FIGS.8H-J). As SB-5 is the only cluster that serves to replenish ciliated populations, the exhaustion of SB-5 by E101 underlies the later gradual lineage extinction. The alternative canonical squamous stratification starts from E80, marked by the appearance of SB-3 and SB-4. The rapid increase in the proportions of SB-3/4 after E101 represents the replacement of ciliated differentiation ultimately by squamous stratification (FIGS. 2B, 4C). Thus, we depict two conversion events in epithelial morphogenesis: the primitive multilayered epithelium at the early stage first differentiating to ciliated columnar epithelium and marked by specification of basal cells and ciliated differentiation; later during the mid stage, the squamous stratified epithelium program replaces the transitional ciliated columnar epithelium and further matures the mucosa at later stages. [00178] Myofibroblasts and fibroblasts share a common developmental origin. To interrogate the accompanying stromal dynamics with these two epithelial differentiation waves, we subclustered the mesenchymal compartment and revealed 23 cell types/states (FIG.9C). Like the overlying epithelium we detect a fibroblast progenitor cluster (COL1A1, PITX1, ZEB2, SNAI2, named as Fib_PG,) that was prominent at the early stage (E47 and E53), but lacking at later stages (after E101) (FIGS. 3B-D, FIGS. 8K-M, 9C). As the progenitor abundance decreases from E59, we observe the emergence of three major fibroblast subtypes (COL1A1, PDGFRA, named as Fib_1, Fib_2, Fib_3) and one myofibroblast population (PDGFRA, ACTA2, named as MF). All of these cells including the progenitors express FOXF1 and NKX6- 1, confirming their identity from splanchnic mesoderm (FIG. 12A). The Fib_1 cells express high levels of POSTN and TNC, suggesting a potential regulator role in epithelial proliferation and differentiation through paracrine signaling (FIGS.3C, 3E, FIGS.8M, 12A-B). The Fib_2 cells exhibit abundant deposition of collagen (COL1A1, COL3A1, COL14A1), elastic fiber (FBLN1, FBLN5, FBN1, FBN2) and proteoglycan (DCN, LUM) genes, all of which could provide structural support for the esophagus (FIGS. 3C, 3E, FIGS. 8M, 12A-B). Fib_3 cells specifically express the calcium-activated potassium channel gene KCNN3, the sodium channel gene SCN7A and the CD34 marker, suggesting their potential roles in neurotransmission and regulation of muscular motility (FIGS. 3C, 3F, FIGS. 8M, 12A-B). Myofibroblasts share common markers with Fib_1, including PITX1, ZEB2, DPT, POSTN and TNC (FIGS. 3G-H, FIGS. 8K-M, 12A-B), suggesting potential roles in regulating epithelial regeneration. Two additional fibroblast subtypes, Fib_4 and Fib_5, were found only after E80 (FIGS.3B, 9C), suggesting a developmental delay in their specification, and possibly a later role in tissue function. Indeed, Fib_4 cells express genes related to angiogenesis and vascular contractility (ANGPTL1 and PRRX1), underlying a putative perivascular fibroblast identity (FIG 8M). Fib_5 cells exclusively express the PI16 gene and therefore considered to be adventitial fibroblasts. Interestingly, Fib_5 cells also express multiple glia-related genes (DCX, PLP1, NGFR and ENTPD2), suggesting a close relationship with the enteric nervous system (FIGS. 8M). Interestingly, Fib_4 and Fib_5 do not express splanchnic mesodermal regulator genes FOXF1 and NKX6-1 (FIGS.3C, 3F, 12A-B), bringing the questions about their developmental origin and lineage plasticity. The pseudotime trajectory analysis predicts that fibroblast progenitors first differentiate into three branches, Fib_1, Fib_2, and myofibroblasts; while Fib_2 further follows two branches, one into Fib_3 and another into Fib_4 and 5 (FIG.3H). [00179] Interstitial cells of Cajal and smooth muscle lineages are specified early in development. The highly specialized mesenchymal cells, interstitial cells of Cajal (KIT, ETV1, ANO1, named as Fib_ICC), and smooth muscle cells (ACTA2, TAGLN, DES, SYNM, named as SM) are essential executors for the rhythmic peristalsis of esophagus. These two cell types were constantly found in all the samples across the developmental stages (FIGS.3B-C, 3F- G, FIGS. 8K-M, 9C). Consistent with the signaling coordinator role between enteric nerves and muscles, interstitial cells of Cajal were found intermingled with smooth muscle cells (FIGS. 4B-C). Both cell types followed their isolated maturation trajectories after specification before E47, thus revealing an early establishment of the contractile muscular components (FIGS.8K- L, 9C-E). We did not observe common progenitors for interstitial cells of Cajal and smooth muscle cells, or any potential transdifferentiation between these two lineages. Even though subpopulations of these two lineages have been described based on their anatomical locations, they were both found as relatively homogenous populations that mature over time (FIGS.8K-L, 9C-E). [00180] Pericyte diversity is established with a developmental lag. Another key component in the mesenchymal compartment is the pericyte (PDGFRB, RGS5, NOTCH3, NDUFA4L2, named as Peri) (FIG.3A, FIGS.8K-M, 9C, 9F). Using co-staining of αSMA and NG2, pericytes were visualized surrounding vasculature (FIGS.10E-F). In contrast to the isolated lineages of interstitial cells of Cajal and smooth muscle, pericytes exhibit much more diversity and delayed development (FIGS.3A-B, FIG.9C, 9F-G). Pericyte progenitors (Peri_PG) were observed as early as E47. This population maintained a relatively stable proportion throughout the subsequent stages, thus potentially serving as a self-maintaining progenitor pool (FIGS.3A- B, FIG. 9C, 9F-G). From E80, we detected three other pericyte populations, differentiating (Peri_1, THB ), contractile (Peri_2, ACTA2, TAGLN, MYL9, MYH11) and angiogenic (Peri_3, PRRX1, PROCR) pericytes (FIGS. 3A-B, FIGS. 8K-M, 9C, 9F-G). Notably, the angiogenic pericytes and Fib_4 were partially overlapped on UMAP, reflecting their common features in supporting vasculature and potential common developmental origin (FIGS.8K-M). However, we could not find evidence supporting lineage transformation between pericytes and myofibroblasts, which has been suggested in some fibrosis studies. [00181] Neurogenesis precedes gliogenesis to shape the enteric nervous system. Enteric nervous system is the second largest compartment in the esophageal stroma, including 16 distinct clusters (FIG.8F-G). Enteric neural crest cells first enter the esophagus and migrate caudally to colonize the entire gut tube. Consistent with previous literature, we observed an abundant progenitor population (named as ENS_PG, SOX10, SOX2, 00B, TUBB3) in E47 and E53 samples (FIG.3A, FIGS.9H-K). These progenitors quickly shut down 00B and upregulate TUBB3 and UCHL1, and track the neuronal differentiation path via neuroblasts (named as NB, ASCL1, DLL3). Neuroblasts maintain a progenitor pool while continuously branching into two neuronal populations, Neu_A marked by ETV1 and Neu_B marked by BNC2 (FIGS.9K, 9N). The presence of both neuron types at E47 suggests an early neurogenesis (FIG.9H-J, 9L). On the other hand, the progenitor cells downregulate TUBB3 and UCHL1, and begin to exclusively express S100B as glial lineage commitment (FIG.9H-J, 9M). We identified 3 glial cell types: Glia_1 marked by BCAN, APOE, ENTPD2, Glia_2 marked by RXRG, GFRA2 and TNC, and Glia_3 marked by MAL, DHH (FIG.9K). The glial cells are enriched in later stages compared to the neuronal lineage. Glia_1 was first observed at E59, while Glia_2 and Glia_3 were detected only after E80 (FIG.9J). Interestingly, the ENS differentiation pattern described here in human esophageal development matches well with the one revealed in human intestinal development, suggesting that the gastrointestinal enteric nervous system undergoes a coordinated differentiation program despite differences in progenitor types, colonization time and migratory paths. [00182] Esophageal endothelial cells, skeletal muscles and immune cells establish cellular heterogeneity in early development. We also discovered esophageal endothelial, skeletal muscle and immune populations, which together comprise ~10% of our stromal cell collection (FIG. 3A, FIG. 8F-G). We highlighted an unexpected early establishment of cellular heterogeneity in these compartments. For the endothelial compartment, capillary (Art_1, marked by GJA4, CXCR4, PGF) and large (Art_2, marked by HEY1 and GJA5) arterial cells, as well as lymphatic vessel cells (Lym_1, marked by LYV1, PROX1 and TFF3) were observed as early as E47. Lymphatic valve cells (Lym_2, specifically express SCG3, FOXC2, and GATA2) emerge at E80 (FIGS. 10A-D). This suggest that the establishment of arterial and lymphatic lineages occur earlier than E47. Notably, while the venous progenitors (Ven_PG) were also observed at E47, their fraction peaks at E53, and becomes marginal at E80. Simultaneously, the potential intermediate venous cells (Ven_1) are being specified. Subsequently, large (Ven_2, marked by ACKR1 and ADGRG6) and capillary (Ven_3, marked by CD83, RGCC and CA4) venous cells emerge, which were first observed at E72. These observations delineate the time window of venous lineage commitment during human esophageal development (FIGS.10A-D). [00183] For the compartment of skeletal muscles, we identified two types of skeletal muscle cells: SkM_1 marked by PAX7 and MYF5, and SkM_2 marked by MYOG and MYF6. The putative satellite cells (SC_1 and SC_2) were found heterogeneous in the expression of SOX8, PAX7 and MYF5. These four populations were observed at all developing stages, suggesting early specification during esophageal myogenesis (FIGS.10H-K). In addition, the entire skeletal muscle lineage expresses pharyngeal mesoderm transcription factors (ISL1, TBX1, MSC and SIX1) and lacks PAX3 This finding is consistent with the non-somitic, cranial mesodermal origin of esophageal skeletal muscle previously revealed in mouse studies (FIGS.10K). [00184] Finally, we found the presence of six immune populations as early as E47, a surprising finding given the lack of environmental antigen exposure (FIGS.10L-N). Immune cells marked by CD45 were found scattered among epithelium and stroma (FIGS. 10E, 10G). Identified populations included monocytes (Mono, CD163), dendritic cells (DC, LYZ or HLA-DRA), mast cells (Mast, KIT, TPSB2) from the myeloid lineage, as well as B cells (B, IGHM, CD79A, CD79B), natural killer cells (NK, GZMA, KLRD1) and type 2 and 3 innate lymphoid cells (ILC2/3, GATA3, RORA, RORC, KIT) from the lymphoid lineage (FIG.10O). T cells (T, CCR7, CD3D, CD3G) emerge at E80, revealing the later migration from the thymus to the developing esophagus (FIGS.10N-O), and emphasizing the presence of the innate rather than adaptive immune system during esophageal development. [00185] Spatiotemporal cellular dynamics establish esophageal tissue architecture. With the cell types identified, we orthogonally performed Visium and CODEX to dissect the spatiotemporal orchestration of developing esophageal epithelium with its surrounding stroma. By integrating scRNA-seq and Visium profiles using cell2location, (see METHODS), we first performed systematic spatial mapping of all the major cell types at mid and late stages to depict the gross tissue architecture in esophageal development. From E72, we observed distinct spatially organized stromal layers surrounding the developing basal epithelium (FIGS. 11A-B). Fib_1 and myofibroblasts largely overlapped with the distribution of basal cells at E72, while at later stage (E120) myofibroblasts formed a clear thin layer lining the epithelium (FIGS. 11A-B, 11E-F). Fib_2 cells were mapped to the middle layer outside of Fib_1 and myofibroblasts since E72, while Fib_3 and interstitial cells of Cajal nicely intermingled with smooth muscle cells in the outer layer (FIGS. 11A-B, 11E-F). Abundant enteric neural and glial cells were mapped at both mid and late stages (FIGS. 11C, 11G). By the late stage, neural cells were found more scattered within muscularis propria, while glial cells tended to aggregate in the adventitial plexuses (FIG. 11G). The vasculature components, pericytes, arterial, venous and lymphatic cells, as well as immune cells, showed diffused distribution in the stroma with no noticeable spatial aggregation (FIGS.11D, 11H). [00186] To refine the spatial depiction of cellular dynamics during esophageal development at single cell resolution, we performed CODEX multiplexed immunofluorescent imaging on esophageal sections from E45 to E130. Based on the scRNA-seq expression pattern, we specifically designed a CODEX panel targeting 42 markers (FIG. 12A). These markers, independently and in combinations, uniquely identify most of the discovered cell populations, with a major focus on epithelium, fibroblasts, myofibroblasts and smooth muscle cells (FIGS. 11A-B). Hierarchical clustering based cell type annotation further confirmed the spatial distribution of various mesenchymal populations at mid and late stages (FIGS.4A-B). Notably, our sampling of early-stage tissues captured the key coordinated events for basal cell specification and mesenchymal diversification (E45 to E72, FIGS. 4A-B). The multilayered early esophageal epithelium is composed by the progenitors (Epi_PG, marked by NCAM1 and GATA6 and absent of KRT15 and KRT5) only. Immediately surrounding the primitive epithelium are relatively homogeneous fibroblast progenitors (Fib_PG) with expression of PITX1, POSTN and DCN. With the specification of basal cells marked by acquisition of KRT5 and KRT15 while loss of NCAM1 and GATA6, the surrounding mesenchyme stratifies into the three concentric layers formed by inner Fib_1 and myofibroblasts, middle Fib_2, and outer Fib_3/4/5 together with smooth muscles and interstitial cells of Cajal (FIGS.4A-B). Notably, whole-mount immunofluorescence of fetal esophagus clearly revealed inner distribution of PITX1+ POSTN+ Fib_1 and outer distribution of PITX1- POSTN+ Fib_4 and Fib_5 (FIGS.12C- D). [00187] In summary, our multi-omics atlas catalogs the detailed spatiotemporal cellular dynamics of human esophageal development. At early stages, the esophageal rudiment is lined with progenitor-only epithelium which is surrounded by fibroblast progenitors and primitive muscularis propria. Along with basal cell specification, these fibroblast progenitors differentiate into diverse lineages and establish the highly organized stromal architecture. The stromal cells further mature and expand from mid to late stage when the squamous differentiation wave replaces the ciliogenesis wave in the epithelial compartment (FIG.4C). [00188] Spatiotemporal candidate signal nomination inspired by the multi-omics atlas. After delineating the cellular dynamics of human esophageal development, we focused on local epithelium-mesenchyme signaling interactions driving basal cell development, which could be leveraged to manufacture esophageal tissue from hPSCs (FIG. 5A). Based on spatial distribution in Visium and CODEX analyses, we noted that the fibroblast progenitors, smooth muscles and interstitial cells of Cajal are the local mesenchyme for early epithelial progenitors, and Fib_1, Fib_2, myofibroblasts for basal cells at mid and late stages (FIG.4, and FIG.11). Importantly, Fib_1 maintains a constant 10 µm cellular layer adjacent to the basement membrane throughout the developmental process. Immediately surrounded Fib_1 layer is the myofibroblast layer that doubles its thickness only at the late stage. By contrast, layers of Fib_2 and muscularis propria (constituted by smooth muscle, interstitial cells of Cajal and Fib_3) proliferate and thicken dramatically during development, which increases the overall distance from the basement membrane (Fib_2 is located 69.70 ± 4.40 µm from the basement membrane at E58, 103.57 ± 9.51 µm at E72, 223.37 ± 21.26 µm at E120. Muscularis propria is located 104.50 ± 6.74 µm from the basement membrane at E58, 219.65 ± 14.68 µm at E72, 497.53 ± 37.66 µm at E120) (FIG.5B). Considering the effective range of common morphogen gradients, we conclude that signals emanated from the muscularis propria are less likely to strongly affect esophageal epithelial development after the early stage. Fib_1, Fib_2 and myofibroblasts, on the other hand, emerge as key signal senders at mid and late stages. [00189] Next, we used the CellChat algorithm to predict stage-dependent signaling interactions between the local mesenchyme and epithelial progenitors or basal cells in an unbiased manner. Since basal cell specification is a continuous developmental process, we reasoned that both early and mid stage local mesenchymal components would be sources for inductive signals. Therefore, we first characterized signals that are received by epithelial progenitors and basal cells at early and mid stages. NRG, GDF, BMP, WNT and ncWNT were identified ranking as top candidates (FIG. 5C). Specifically, smooth muscle is the only sender for the NRG signal. Interstitial cells of Cajal serve as the major cellular sources for BMP signal. The epithelial progenitors secret GDF through autocrine signaling (FIG. 5D). In contrast, EGF, WNT and ncWNT were identified as inductive signals for basal cell maturation in the interrogation of cell-cell communication between basal cells and the mid-late local mesenchyme (FIG.5C). From the mid stage, with the expansion of Fib_2, muscularis propria no longer serves as the local mesenchyme to affect epithelial development. Instead, Fib_1 replaces interstitial cells of Cajal to provide BMP. Fib_1, Fib_2 and myofibroblasts, introduce WNT signal, while newly specified basal cells serve as an additional source for WNT in an autocrine manner. EGF, on the other hand, is the only signal revealed at late stage, and is provided solely by myofibroblasts (FIG.5D). [00190] To summarize, by combining spatial analysis and CellChat, we revealed a dynamic signal communication pattern alongside the basal cell developmental trajectory (FIG.5E). We further confirmed cellular signal sender/receiver identities with single cell expression patterns of known ligands, agonists and antagonists (FIG.5F). Taken together, EGF superfamily (EGF and NRG), WNT pathway (WNT and ncWNT), TGFb superfamily member GDF and BMP pathway might serve as key inductive signals to drive BC development. [00191] Manatee facilitates design of hPSC differentiation strategies. Manufacturing esophageal tissue in vitro for clinical applications remains challenging as the latest protocols produce progenitors rather than lineage-specified, functional basal cells, and are also not chemically-defined. While our atlas reveals the dynamic local mesenchymal signals could potentially drive basal cell development, the presence of both ligands, agonists and antagonists obscures whether these signaling pathways are activated or inhibited, as well as how these pathways cooperate with each other. The machine-readable nature of the integrated atlas data, in combination with the rapid development of more complex computational models allowed us to take an unbiased approach in prioritizing signaling combinations. We thus developed the Manatee, a deep learning framework, to screen in silico for the optimal combination of EGF, WNT, TGFβ and BMP signals for esophageal basal cell specification. [00192] Manatee, adapted from the variational autoencoder (VAE) model, predicts gene expression alterations caused by transcription factor (TF) perturbations (FIG.6A). Specifically, we constrained the VAE latent space to represent TF expression, by adding an additional TF reconstruction loss during training. As for the prediction phase, we adjusted latent variable values associated with candidate TFs to be perturbed. The adjusted latent space are further flowed through the decoder neural network. The outputs is considered as the altered expression profiles, which is ensured by the generative capacity of VAEs (see METHODS). [00193] We used Manatee to screen all 81 combinations of EGF, WNT, TGFb and BMP (FIG. 6B), and evaluated the similarity between virtually perturbed cellular statuses and in vivo human developing basal cells. We first derived esophageal progenitors from hPSCs using a new chemically-defined, xeno-free protocol (FIG. 7A). The bulk RNA-seq analysis of hPSC differentiation confirmed endoderm/foregut commitment from day 4 to day 10, whereas esophageal progenitors were specified by day 16. These progenitors matured into basal cells after another month (FIGS.14A-E). Manatee was trained with day 4, 10, 16, 24 and 43 in vitro derived single cell transcriptomic profiles (FIGS. 13A-C), together with in vivo epithelial cell profiles (FIG.2A), to capture the complete set of regulatory logics related to the esophageal basal cell development. Day 16 and 24 cells were used here as starting points for the in silico perturbation. The basal cell derivation effectiveness was quantified and ranked using the Pearson correlation between the average in vivo human developing basal cell and the perturbation expression profiles (FIG. 6B). UMAP dimension reduction showed that the top ranked strategies, including #19, could drive the in vitro day 16 and 24 cells to the transcriptomic statuses resembling the in vivo esophageal basal cells. The bottom ranked strategies, including #9 (reversed signal combination compared to #19), interestingly pushed day 16 and 24 cells backwards along the in vitro cell culture trajectory, suggesting that strategy #9 repressed basal cell development. The middle ranked strategies, including #14, did not alter the transcriptomic profiles globally, suggesting minimal perturbation effects (FIGS.6B-C, FIG.13D). [00194] The Manatee-prioritized combination of local mesenchyme signals efficiently promotes basal cell derivation. With the nominated in vivo signals as input, Manatee predicted that activation of EGF, BMP and TGFb along with inhibition of WNT (strategy #19) might enhance basal cell derivation from hPSCs in vitro. In our modified hPSC differentiation system, although definitive endoderm (DE) and esophageal progenitors (EPC) could be efficiently derived from H9 embryonic stem cell line, basal cell efficiency dropped drastically at day 43 (> 90% CXCR4+ CKIT+ definitive endoderm at day 4, > 60% EPCAM+ P63+ esophageal progenitors at day 16, in contrast to ~4% EPCAM+ ITGB4+ basal cells at day 43, FIG.14C). This drop in basal cell efficiency hinders manufacturing of esophageal mucosa at the clinical scale, mainly due to inefficient basal cell specification from the progenitors and fast expansion of non-epithelial cells over prolonged culture. [00195] To test whether strategy #19 could effectively and efficiently produce basal cells, we compared the day 24 H9-derived culture treated with EGF, BMP and TGFb ligands BMP4, TGFB1, and WNT antagonist IWP2 for another 5 days to those supplemented with EGF only (strategy #14 as the base control). We also tested strategy #9 as the negative control, using EGF, BMP and TGFb inhibitors A83-01, DMH-1 and WNT agonist CHIR99021 (FIG.7A). Flow cytometry quantification confirmed that the Manatee-predicted strategy #19 indeed increased the proportion of ITGB4+ EPCAM+ basal cells in the EPCAM+ epithelial population compared to the base control #14 (63.17% ± 6.06% vs.43.43% ± 2.55%). Notably, the negative control #9 drastically suppressed basal cell formation (6.66% ± 0.53%. FIG.s 7B). Immunostaining showed only few cells expressing weak KRT5 by day 29 (D24+5) in the #14 base control. In contrast, large quantities of strongly-staining KRT5+ cells were observed in the #19 treated day 29 culture (FIG.7C), supporting the Manatee prioritization. No KRT5+ cells could be found in the #9 treated culture, further suggesting that activation of BMP and TGFb signaling and repression of WNT are required for the basal cell commitment from the progenitors. Consistently, #19 treated cells increased expression for the canonical basal cell markers KRT5 and KRT15 while #9 treated cells showed drastic decrease (FIG.7D). We also confirmed the results using a different human embryonic stem cell line RUES2. To comprehensively characterize the perturbation effects, we performed bulk RNA-seq for RUES2-derived day 24 culture, as well as the day 29 cultures with 5-day treatment of #14, #9 and #19. Assessment of the corresponding signaling target transcripts confirmed effective activation of BMP and TGFb and repression of WNT by #19 and the reversed effect by #9. The expression of multiple BC marker genes, including TP63, ITGB4, ITGA6, COL7A1, COL17A1, KRT15 and KRT17, were all strongly increased in #19 treated cells, and largely repressed in #9 treated ones (FIG. 7E). Therefore, as predicted by the Manatee, activation of EGF, BMP, TGFb while inhibition of WNT signaling simultaneously drive esophageal basal cell commitment from the progenitor cells. [00196] To further investigate how these pathways mechanistically determine basal cell specification, we assessed the essentiality and sufficiency of each pathway in #19 strategy. Withdrawal of EGF severely diminished the derivation of ITGB4+ EPCAM+ basal cells (6.43% ± 0.38% vs.20.8% ± 0.67% with full cocktail), and withdrawal of WNT inhibitor IWP2 modestly reduced basal cell efficiency (16.87% ± 2.27% vs. 20.8% ± 0.67% with full cocktail). In contrast, withdrawal of BMP4 (21.23% ± 1.25%) or TGFB1 (19.4% ± 1.19%) alone did not alter the basal cell efficiency (FIG. 14F). Consistently, only EGF could boost basal cell derivation when individually supplied from day 24 (FIG.14G). Although the Manatee predicted EGF as a dispensable signal here (FIG. 6B), our experimental validation underpinned the importance of EGF in basal cell differentiation, which was consistent with the previous protocols that only used EGF for long-term culture. This discrepancy might be due to our current limited knowledge of EGF pathway related gene sets, which we relied on for the in silico perturbation. We further interrogated the cooperative mechanisms among BMP, TGFb and WNT pathways here. Comparing to the culture treated with only EGF, additional treatment with IWP2 significantly increased the basal cell derivation efficiency (50.97% ± 1.71% for EGF+IWP2 vs.43.43% ± 2.55% for EGF only). While BMP4 or TGFB1 did not affect the yield individually, simultaneous treatment of BMP4 and TGFB1 increased the efficiency to 60.3% ± 2.76% (FIG. S7H). Inhibition of BMP and TGFb pathways by treating with A83-01 and DMH- 1 modestly repressed basal cell specification (34.4% ± 1.15%), while activation of WNT signaling using CHIR993021 drastically inhibited this process (7.27% ± 1.08% for EGF+CHIR993021, and 6.66% ± 0.53 for EGF+A83-01+DMH-1+CHIR993021). Addition of IWP2 could partially derepressed the effect of A83-01 and DMH-1 (55.53% ± 6.59% for EGF+A83-01+DMH-1+IWP2), while addition of CHIR993021 partially suppressed the effect from BMP4 and TGFB1 (24.23% ± 4.53% for EGF+BMP4+TGFB1+CHIR993021) (FIG. S7H). Therefore, besides EGF as the essential driver, WNT signaling is the major effector determining basal cell specification, while BMP and TGFb cooperatively promote this lineage progression. [00197] Next, we tested whether #19-derived basal cells could function as their in vivo counterparts to self-renew and undergo squamous differentiation in culture and tissue organoid assays. We sorted the ITGB4+ EPCAM+ population (DP) from day 29 culture and expanded the cells on collagen peptide coated plates for several passages. These expanded cells could maintain high expression of ITGB4, while the ITGB4- EPCAM+ population (SP) remained largely absence of ITGB4 (FIG.7H), confirming the self-renewing capacity of #19- derived basal cells in vitro. Notably, these basal cells can be cryopreserved, thus providing flexibility in the future clinical scaling up. During in vitro expansion, these #19-derived basal cells retained expression for KRT5 and KRT15 while negative for simple columnar marker KRT18, underlying their fully matured identity (FIG. 7G upper panel). After Ca2+ treatment, large flattened KRT4+ KRT13+ suprabasal cells could be observed with typical squamous cell morphology (FIG. 7G bottom panel). Hence, the #19-derived basal cells could respond to Calcium influx and undergo esophageal specific squamous stratification. [00198] To test whether hPSC-derived basal cells could generate 3D tissue organoids, we embedded the cells in Matrigel and cultured for 3 weeks. Remarkably, solid organoids were formed with KRT5+ basal cells in the periphery and KRT13+ suprabasal cells inside (FIGS.7H and 14I). In addition, we determined whether the basal cells could regenerate tissue-specific squamous stratified epithelium by growing these cells on devitalized de-epidermal human dermis. Stratified skin basal cells will express KRT1 and KRT10, proteins needed to maintain skin integrity. Indeed, the #19-derived basal cells contributed to building the COLVII+ basement membrane and generated KRT13+, KRT4+ and IVL+ squamous descendants on top of KRT5+ KRT15+ ITGB4+ basal layer. All of these cells were positive for human specific nuclei staining (huNuclei) as well as esophageal specific transcription factor PITX1, confirming their maintenance of tissue specificity even with the support of skin dermis (FIG. 7I). Taken together, we successfully translated the in vivo human tissue interactive signals revealed from our atlas to the in vitro hPSC differentiation system using Manatee. The Manatee-prioritized strategy, activation of EGF, BMP, TGFβ while inhibition of WNT, accelerated the derivation of functional esophageal basal cells and enhanced the efficiency, thus providing for the manufacturing of esophageal mucosa at the clinical scale. [00199] Taking advantage of single cell and spatial technologies, we have created a robust multi-omics atlas of human tissue morphogenesis. Through integration with machine learning approaches, we have built an innovative framework to design actionable hPSC-directed differentiation strategies. Our multi-omics atlas systematically elucidates the developmental origin of esophageal basal cells and reveals two differentiation waves of epithelial morphogenesis accompanied by dynamic stromal architecture. By integrating spatial dynamics, we nominate local mesenchymal inductive signals driving basal cell development as input for Manatee screening. Leveraging this framework, we establish the first human developmental signal-inspired tissue manufacturing system for esophageal mucosa. This promises the esophageal cell replacement therapy for RDEB as well as severe epithelial wounds caused by cancer resection or caustic injury. Our framework establishes a paradigm shift in the flexible design of human tissue manufacturing for clinical use. [00200] Our atlas uncovers three unexpectedly complex yet orchestrated cellular dynamics during human esophageal development. Given that our dataset has sampled developmental timepoints as early as the mid 1st trimester, we clarified the molecular characteristics of the esophageal epithelial progenitors before the appearance of any differentiated cell types, including basal cells. Particularly, we reveal GATA6 as a progenitor specific transcription factor. GATA6 plays essential roles in establishing endoderm in early development and later GATA6 expression becomes restricted posteriorly to gastric, intestinal and colonic epithelium but absent from the esophagus. Notably, re-emergence of GATA6 expression has been found in Barrett’s esophagus and esophageal adenocarcinoma, characterized by the pathological conversion of stratified epithelium to intestinal metaplastic simple epithelium. Our atlas could be leveraged in conjunction with disease and cancer databases to investigate any potential reactivation of developmental programs, and the role of the tumor microenvironment, during cancer progression. [00201] We reveal two differentiation waves in human esophageal epithelial morphogenesis, with ciliogenesis waning towards the end of 1st trimester, and squamous stratification taking over afterwards. Ciliated cells may help the fetus to swallow amniotic fluid, which is critical for gastrointestinal development. Meanwhile, squamous cells become indispensable as the protective barrier for the esophagus. [00202] Accompanying epithelial morphogenesis, we systematically described the coordinated mesenchymal dynamics. Accompanying basal cell specification from the epithelial progenitors, fibroblast progenitors stratify into 4 layers: Fib_1 closest to the basement membrane, and myofibroblast, Fib_2, Fib_3 further away, respectively. Notably, the drastic expansion of the Fib_2 layer in the submucosa distances the muscularis propria which is composed of intertwined Fib_3, smooth muscles, and interstitial cells of Cajal, thus physically insulating morphogens secreted by muscularis propria from the epithelium. Additionally, it has been reported that DCN, a matrix component abundantly secreted by Fib_2, could antagonize BMP and TGFb signaling, which are critical pathways in basal cell commitment. Hence, the mesenchymal cellular dynamics alter the tissue architecture. This results in stage-dependent changes in the cellular components of the local mesenchyme, which potentially drive basal cell development. While the local mesenchyme cell types remain the same from the mid to late stage, these cells could change their secreted morphogen profiles as they mature. Specifically, myofibroblast cells begin to express GREM2, an effective BMP antagonist, from the late stage, rendering the epithelium in a SMAD inactive microenvironment to promote basal cell self-renewal (FIG.5F). [00203] Instead of relying on prior knowledge gained using model animals, we here combined our tissue atlas and machine learning to design a clinically compatible hPSC differentiation system. The hPSC-to-esophageal basal cell system with our previous hPSC-to-skin basal cell differentiation systems provide a unique chance to interrogate regulatory modules controlling the tissue-specific basal cell development. Our previous work revealed that during epidermal lineage commitment, the morphogen-induced expression of TFAP2C primes the chromatin landscape. Meanwhile, TFAP2C activates the canonical basal cell master regulator P63, which further effects on the primed chromatin for stratified epithelium maturation. Our current work suggests a similar regulatory mechanism operates in endoderm. Specifically, induced key endodermal TFs such as SOX2 and GATA6 shape the endoderm-specific chromatin landscape as well as inducing P63; in turn, P63 induces stratified epithelium while repressing GATA6 to drive differentiation forward. Our functional assays, including seeding esophageal basal cells on skin dermis, provide strong evidence for the distinct and stable cellular specification network. Future mechanistic and evolutionary studies interrogating shared regulatory logic will help accelerate the production of other stratified epithelia such as bladder and cornea, as well as the mechanism of the master regulator P63 in specifying tissue-specific stratification programs in ectoderm and endoderm. [00204] Screened by Manatee, we identified and experimentally validated that activating EGF, BMP, TGFβ while inhibiting WNT simultaneously enhanced esophageal basal cell derivation in vitro. Interestingly, although activation of BMP and TGFβ pathways have been extensively studied to promote the squamous differentiation from basal cells, our work revealed an unexpected earlier requirement of this dual SMAD activation in basal cell specification from the progenitors. Besides, WNT activation has been found to be beneficial for the generation of mouse and human esophageal organoids. The organoid forming efficiency is determined by the self-renewal capacity of progenitors. [00205] Our results are consistent with WNT inhibition promoting the lineage progression from highly proliferative progenitors to relatively quiescent basal cells in culture. We consider the basal cell specification event from progenitors as a subtle and transient differentiation process that is required as a brief step on the brake. Following specification of P63+ ITGB4lo/- esophageal progenitors, WNT inhibition enhanced commitment from these progenitors to P63+ ITGB4+ basal cells, and WNT activation strongly repressed this process. SOX2 in the developing esophageal epithelium was found to be important to induce the expression of WNT antagonist SFRP2. Interestingly, SFRP2 was found abundantly expressed in Fib_1 (FIG.5F). Therefore, a non-autonomous feedback loop between epithelial and stromal cells might be formed upon basal cell specification since the mid stage, reinforcing the dorsal esophageal fate instead of ventral airway or posterior gastric identities. Notably, after basal cell specification, the surrounding myofibroblasts acquire expression of GREM2 from mid to late stage, which inhibit BMP signaling (FIG. 5F). Thus, the structural remodeling of local mesenchyme as well as stage-dependent morphogen dynamics shape the earlier differentiation-permissive and later self-renewal-enhancing microenvironment. [00206] Our work highlights the broad applicability of the Manatee to prioritize identified intercellular signals for tissue induction between tissue states. Besides screening for stem cell directed differentiation strategies, it could also be used to prioritize therapeutics to reverse diseased cellular states, or infer side effects of drugs. While the Manatee successfully prioritizes the signaling combinations to facilitate our benchwork, the magnitude of importance may not reflect the similar ranking. Next generations of the Manatee will incorporate gene regulatory network topology in the design of VAE architecture, similar to what has been done in the VEGA model. [00207] In summary, our multi-omics deepens our understanding of the development of the human upper gastrointestinal system. The comprehensive characterization of cellular diversity and dynamics provides a rich resource of in vivo landmarks for in vitro human cell engineering towards lineages across germ layers, including enteric neurons and glia cells, esophageal vascular components and fibroblasts. Instead of using ligands and small molecules, we could co-derive or assemble the supporting cell types to further enhance manufacturing efficiency of esophageal mucosa. Moreover, our benchmarked atlas-spatial analysis-Manatee framework serves as a new paradigm in leveraging the human developmental cell atlas for the rational design of hPSC differentiation. [00208] A summary of the protocol is found in the table below. The protocol is useful in the production of esophageal basal cells from pluripotent cells. The protocol may be applied to any pluripotent cell type. The cells produced by the methods disclosed herein may be used in esophageal reconstruction procedures for any animal subject, particularly human subjects. Exemplary conditions that may be treated by the cells of the invention include repair of congenital or acquired conditions, such as long-gap esophageal atresia (LGA), long-segment esophageal disruption, refractory GERD, caustic ingestions, cancer, and stricture. [00209] With regards to enumerated media and added factors, it will be understood that functional substitutions of the various media compositions and added factors may be used in place of the enumerated compositions. With regards to the amounts of added factors utilized in the protocol, it will be understood that the enumerated values are exemplary, and the method may be performed higher or lower concentrations of the composition. For example, where it is indicated that a component is used at a value of X mM or X ng/ml, it will be understood that the method may be performed using a range of effective concentrations, for example about 0.01X, 0.05X, 0.10X, 0.15X, 0.20X, 0.25X, 0.30X, 0.35X, 0.40X, 0.45X, 0.50X. 0.55X, 0.60X, 0.65X, 0.70X, 0.75X, 0.80X, 0.9, X, 1.1X, 1.2X, 1.3X, 1.4X 1.5X, 1.6X, 1.7X, 1.8X, 1.9X, 2X, 3x, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 20X, 30X, 40X, 50X, 100X etc.
Day Medium Growth Factors Time Endpoint Notes 0 E8 10 μM Rock inhibitor Y- 1 Day defined Pluripotent 27632 in iMatrix511 GMP- e or e.
Figure imgf000061_0001
METHODS [00210] Human Samples. Normal, de-identified human embryonic and fetal tissues (esophagus with stomach) were obtained from the NIH-funded University of Washington Birth Defect Research Laboratory (BDRL). All procedures were approved by the University of Washington and Stanford University Institutional Review Board (IRB#46721; SCRO#801). Tissue samples were shipped overnight in HBSS with ice packs. [00211] Human Pluripotent Stem Cells. Human pluripotent stem cell (hPSC) line H9 was obtained from Stanford Stem Cell Bank. The RUES2 line (Rockefeller University Embryonic Stem Cell Line 2, NIH approval number NIHhESC-09-0013) was originally from Rockefeller University. All experiments using hPSC lines were approved by Stanford University. The H9 line was maintained in Essential 8 media (Thermo Fisher Scientific) on culture plates coated with iMatrix-511 (Takara). The RUES2 line was initially maintained on mouse embryonic fibroblasts (MEFs, irradiated CF-1 MEF, Thermo Fisher Scientific) in the maintenance medium: 400 ml of DMEM/F12 (Thermo Fisher Scientific), 100 ml of KnockOut Serum Replacement (Thermo Fisher Scientific), 5 ml of GlutaMAX (Thermo Fisher Scientific), 5 ml of MEM non-essential amino acids solution (Thermo Fisher Scientific), 3.5 ml of 2- mercaptoethanol (Sigma-Aldrich), 1 ml of primocin (Thermo Fisher Scientific), and FGF2 (R&D Systems) with a final concentration of 20 ng/ml to make a total of 500 ml of medium. For the feeder-free chemical-defined condition, the RUES2 cells were thawed and plated on culture plates coated with iMatrix-511 (Takara) in StemFit Basic04 Complete medium (Amsbio). For passaging, hPSCs were detached with TrypLE Select (Thermo Fisher Scientific) and plated at 1M per 10 cm dish. hPSCs were maintained in an incubator with 95% humidity, 95% air and 5% CO2 at 37°C, and routinely tested for mycoplasma contamination. [00212] Human Esophageal Tissue Histology Staining. Human embryonic and fetal esophageal and stomach tissues were washed in cold HBSS. Whole esophageal tubes were cut into ~3-5 mm length pieces from proximal to distal. Such processed tissues were subjected to both paraffin and OCT embedding, followed by H&E staining and imaging. [00213] For paraffin embedding, tissues were fixed in 4% paraformaldehyde at 4 °C rotating overnight. Then tissues were washed in cold PBS for 3 times, transferred to 70% ethanol and sent to Human Pathology/Histology Service Core at Stanford for embedding, sectioning and H&E staining. [00214] For OCT embedding, tissues were transferred to 30% sucrose in PBS at 4 °C rotating overnight till tissue sinking. Then tissues were transferred to 30% 1:1 sucrose-OCT solution at 4 °C overnight, and embedded in OCT the next day. During H&E staining, sections were initially warmed up to room temperature for 10 minutes, rehydrated in PBS for 10 minutes, and rinsed in water for 1 minute. Then sections were stained in Hematoxylin (Millipore Sigma) for 30 seconds, rinsed in water, and stained in Bluing reagent (Dako) for 30 seconds, rinsed in water. Following that, sections were soaked in 50% ethanol for 1 minute, 70% ethanol for 1 minute, 95% ethanol for 1 minute, Eosin (Millipore Sigma) for 1 minute, 95% ethanol for 1 minute twice, 100% ethanol for 1 minute twice, Histo-Clear (Fisher Scientific) for 5 minutes, and fresh Histo-Clear overnight. On the following day, slides were mounted using Permount mounting medium (Fisher Scientific). [00215] H&E images were acquired using AxioImager (Zeiss, Neuroscience Microscopy Service at Stanford). [00216] Immunofluorescence Staining and Microscopy Imaging. For immunofluorescence of coverslip cell cultures, cells were fixed in 4% paraformaldehyde at room temperature for 20 minutes and washed with PBS for 3 times. Cells were permeabilized and blocked with 0.3% Triton X-100 (Sigma-Aldrich) plus 10% normal horse serum (Jackson ImmunoResearch) in PBS for 1 hour. Primary antibodies were added into 0.3% Triton X-100 and 1% bovine serum albumin (Sigma-Aldrich) in PBS and incubated at 4 °C overnight. The next day, cells were washed with PBS and incubated with secondary antibodies and NucBlue Live ReadyProbes Reagent (Hoechst 33342, Thermo Fisher Scientific) at room temperature for 1 hour. After washing, cells on coverslips were mounted onto microscope slides using ProLong Gold Antifade Mountant. [00217] For immunofluorescence of fixed frozen tissue sections, 7 μm frozen sections were warmed up to room temperature for 10 minutes and rehydrated in PBS for 10 minutes. Following that, sections were blocked and stained as described above. [00218] Fluorescent images were taken using Leica Sp8 (Leica Microsystems, Cell Sciences Imaging Facility at Stanford) and LSM710 (Zeiss, Neuroscience Microscopy Service at Stanford) confocal laser scanning microscope. [00219] scRNA-seq sample and library preparation. We performed 10X Genomics scRNA-seq on both primary samples and hPSC cell cultures. Specifically, human embryonic and fetal esophageal and stomach tissues were washed in cold HBSS. Whole esophageal tissues were collected and cleaned up by removing stomach tissues slightly proximal from the gastroesophageal junction and other connective tissues. For samples older than 8 post- conception weeks, esophageal tubes were cut open using fine scissors, and gently digested in 6 cm dishes with 25 U/ml Dispase (Corning) + 100 μg/ml DNase I (Millipore Sigma) at room temperature for 20 to 40 minutes. For samples younger than 8 post-conception weeks, whole esophageal tubes were digested in Dispase/DNase I solution at room temperature for 10 minutes instead. Digestion was stopped by transferring esophagus to cold HBSS. Epithelium was physically peeled off from surrounding stromal tissues with fine forceps, and collected in a 15 ml Falcon tube. The epithelium was further dissociated into single cells in TrypLE Express (Thermo Fisher Scientific), first triturated gently using P1000 pipette and then shaken in a Thermomixer at 37 °C at 1000 rpm for 10 to 20 minutes. Dissociation was stopped by addition of the FACS buffer (PBS supplemented with 1% BSA, 2 mM EDTA, 1X Anti-Anti, 25 mM HEPES pH 7.0) supplemented with 10% FBS. Stromal tissues were transferred to clean dishes and chopped into small pieces using scissors. Tissue pieces were transferred to a 15 ml Falcon tube with 10 ml collagenase solution (5 mg/ml Collagenase in DKSFM), triturated gently using P1000 pipette, and then shaken in a Thermomixer at 37°C at 1000 rpm for 10 to 30 minutes. 1 ml 0.25% Trypsin was then added to the solution for an additional 5 min dissociation. Dissociation was stopped by addition of the FACS buffer supplemented with 10% FBS. Dissociated cells were resuspended in cold 0.04% BSA in PBS and filtered through 40 μm strainers. Cells were transferred to 1.5 ml Eppendorf tubes and pelleted down at 300 g at 4 °C for 5 minutes. Then cells were resuspended in cold 0.04% BSA in PBS for cell number counting. Cell alive rate was determined using Trypan Blue. Only samples with greater than 80% cell alive rate were further processed. Cells were then subjected to 10X Chromium scRNA-seq based on the manual. [00220] As for hPSC cell cultures, cells were dissociated using TrypLE Select, and resuspended in FACS buffer with SYTOX Blue Dead Cell Stain (Thermo Fisher Scientific). Live cells were sorted using FACSAria II (BD Biosciences) at Stanford Shared FACS Facility and collected in the FACS buffer supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 2X Anti-Anti on ice. The sorted cells were pelleted, washed with cold 0.04% BSA in PBS, and resuspended in cold 0.04% BSA in PBS for cell number counting. Cell alive rate was determined using Trypan Blue. Only samples with greater than 90% cell alive rate were further processed. Cells were then subjected to 10X Chromium scRNA-seq based on the manual. [00221] Sequencing libraries were prepared using Chromium Single Cell 3’ v3 (CG000183 Rev C) and v3.1 (CG000315 Rev A) protocols according to the manufacturer’s manual. The pooled, 3’-end libraries were sequenced using Illumina NovaSeq. [00222] Bioinformatic analysis of scRNA-seq data. Cell Ranger version 4.0.0 was used for primary data analysis, including demultiplexing, alignment, mapping and UMI counting. Specifically, for alignment and mapping, the GRCh38 reference genome and corresponding annotation were used. [00223] Seurat version 4.0.3 was used for single cell gene expression quantification, dimension reduction, clustering analysis and marker gene identification. Epithelial samples and stromal samples of different developing stages were merged separately for downstream analysis. Cells expressing no more than 200 genes or more than 8000 genes were removed. Cells having more than 20% of reads mapped to mitochondria were also removed. A total of 2,000 highly variable features were identified, and PCA was performed using variable features. Cells were clustered using 10 dimensions and visualized using UMAP. The initial iterative clustering revealed clusters for gross cellular compartments: epithelium (EPI. EPCAM, PITX1, KRT5, KRT4), stomach (ST. GATA6, CLDN18, TFF1, MUC5AC), mesenchyme (MES. COL1A1, PDGFRA), enteric nervous system (ENS. HAND2, CDH2, PHOX2B), endothelium (ENDO. CLDN5, PECAM1, CDH5, ECSCR, EGFL7), skeletal muscle (SKM. PAX7, CDH15, MYF5), immune cells (IM. PTPRC, LAPTM5), erythrocyte (ERY. HBM, HEMGN) (FIGS.8D-E). These cellular compartments were subset for further iterative clustering and annotation. Cluster- specific markers were identified using Seurat FindAllMarkers with min.pt = 0.25 and logfc.threshold = 0.25. [00224] For esophageal epithelial characterization, only the epithelium cluster from epithelial samples digested using TrypLE was subset and reclustered.10 major clusters were identified with resolution at 0.65, and annotated based on their gene expression. One cluster expressed high levels of cell cycle-related genes (CENPF, TOP2A, MKI67, PCLAF, TYMS, GINS2), thus was annotated as cycling epithelium (Cy). Two clusters marked by ciliated cells (FOXJ1) represented a transition from immature phenotype (CDC20B, DEUP1) to mature status (TPPP3, CAPS). One basal cluster and five suprabasal clusters were annotated based on expression of keratins. One suprabasal cluster expressed both secretory markers (SCGB1A1, UPK2) and early ciliogenesis gene (CCNO), thus annotated as SB-5 as progenitors for ciliated cells. One small cluster closely associated with basal cells in UMAP, did not express canonical basal cell markers but expressed highest levels of early endodermal markers (SHH, NCAM1, GATA6). These cells were not contaminants from alternative gut lineages, as they lacked expression of other key markers (PAX8, NKX2-1, TBX1, CLDN18, GATA4, CDX2). They were only found in the earliest datasets, thus annotated as epithelial progenitors (Epi_PG) (FIGS. 8H-J, 9A-B). [00225] For esophageal stromal characterization, the cellular compartments of mesenchyme, enteric nervous system, endothelium, skeletal muscle and immune cells annotated from the initial clustering with resolution at 0.6 were subset and reclustered separately (FIGS.8F-G). [00226] The mesenchymal compartment (MES) was computationally separated into 23 clusters with resolution at 1.85. Four clusters demonstrating high expression of muscle genes (ACTA2, TAGLN, CAV1, DES) without fibroblast markers (PDGFRA, DPT) were annotated as smooth muscle cells (FIG.8D). One showing cycling features (TOP2A, MKI67, TYMS) was identified as cycling smooth muscle cells (Cy.SM). The other three represented a spectrum of maturation statuses correlating well with the developmental timepoints, thus annotated as SM_early (mainly enriched in E47 to E59 samples; lack of SYNM), SM-mid (mainly enriched in E72 to E113 samples; expressing moderate levels of SYNM), SM_late (mainly enriched in E127 to E132 samples; expressing highest levels of SYNM). 13 clusters were identified as fibroblasts (PDGFRA), with one co-expressing cell cycle genes labeled as Cy.Fib (TOP2A, MKI6, TYMS). Three major types of fibroblasts were identified based on markers from previous studies in other gut organs: Fib_1 (PITX1, POSTN, OSR2, CXCL14); Fib_2 (COL1A1, COL3A1, COL14A1, FBLN1, DCN); Fib_3 (KCNN3, CD34, C7). All three fibroblast types exhibited two distinct developmental statuses along maturation: mid stage enriched in E59 to E82 samples while late stage enriched in E101 to E132 samples. Two clusters co-expressing fibroblast markers (PDGFRA, DPT) and muscle markers (ACTA2, TAGLN) were labeled as myofibroblasts (FIGS. 8K-M). One common progenitor cluster was identified (Fib_PG) enriched only in E47 to E80 samples (FIG.9C). These cells expressed low levels of DPT and high levels of mesodermal regulator genes ZEB2 and SNAI2 (FIGS.8K-M), suggesting an immature identity. Two distinct fibroblast populations (Fib_4 and Fib_5) were found only after E80 (FIG. 9C). Their absence of esophageal mesoderm regulator genes (FOXF1, NKX6-1) (FIG. 12A) indicated a potential distinct developmental origin from the above described fibroblast progenitors. A distinct cluster with unique expression of KIT, ETV1, ANO1 was annotated as interstitial cells of Cajal (Fib_ICC). Pericytes were annotated based on their expression of PDGFRB, NOTCH3 and absence of PDGFRA (FIG. 9F). One cluster of pericytes exhibiting expression of cycling genes (TOP2A, MKI67, TYMS) was annotated as cycling pericyte (Cy.Peri). Three diverse pericyte subtypes (Peri_1, Peri_2, Peri_3) were identified based on their different expression in several key genes including RGS5, PRRX1, ACTA2, LUM (FIG.s 8K-M). All three subtypes were detected after E80. One pericyte cluster was found constantly throughout all the stages, and it did not show cycling features or subtype- enriched genes (FIGS 8K-M, 9C), thus annotated as pericyte progenitors (Peri_PG). One minor cluster uniquely expressing WT1 was annotated as mesothelium (MS) (FIGS. 8K-M). As the mesothelium cluster was only found in E47 and E53 samples (FIG.9C), we considered this as contaminants from dissecting early tiny tissues. [00227] The enteric nervous system (ENS) was largely categorized in two major cell types with resolution at 1.3: neural (ASCL1, TUBB3, PHOX2B) and glial (00B, ERBB3, SOX10) cells, besides a cycling cluster (TOP2A, MKI67, TYMS). Using definition of enteric nervous cell characterization in the intestinal tract, we annotated two branches of the neuronal cells as Neu_A (ETV1) and Neu_B (BNC2). Within branch A, we identified one early cluster (Neu_A_early) enriched only in E47 and E53 samples, and one later cluster afterwards. This later cluster expressed NO, VIP, NPY, SCGN and highest levels of GAL, thus representing the inhibitory motor neurons. Within branch B, we also revealed one early cluster (Neu_B_early) enriched in E47 and E53 samples. Afterwards, this cluster branched into two distinct subtypes (Neu_B_1, Neu_B_2), with Neu_B_2 specifically expressing CASZ1, SLC5A7, SST. Therefore we considered Neu_B_2 as excitory motor neurons with neuroendocrine features. A common transitional neuroblast (NB) population was identified expressing DLL3 and higher levels of ASCL1. For glial cells, Glia_1 was defined by the expression of BCAN, APOE and ENTPD2. Glia_2 was defined by their enrichment of RXRG, TNC and GFRA2. Glia_3 expressed high levels of MPZ, MAL and DHH, suggesting that they might be Schwann cell precursors. Based on differences in expression in NTRK2 and FGL2, Glia_3 was further subdivided into Glia_3_1 and Glia_3_2. All three glial cell types exhibited developmental timepoint-related maturation, thus further classified as mid and late stages (FIGS. 9H-K). One cluster was found co-expressing both neural markers (ASCL1, HAND2, TUBB3) and glial markers ( 00B, ERBB3, SOX10), and only enriched in E47, E53 and E59 timepoints (FIG.9J-K), thus annotated as progenitors (ENS_PG). [00228] The endothelium compartment was separated into 9 clusters with resolution at 0.7. One with cycling features (TOP2A, MKI67, TYMS) was termed as Cy.Endo. Two lymphatic clusters were identified based on their expression of LYVE1, PROX1, CCL21, TFF3 and STAB2. Lym_2 was only found after E80, and showed unique expression of lymphatic valve markers (SCG3, FOXC2, GATA2). Two arterial clusters were identified based on their expression of PRND, GJA4 and CXCR4. These two arterial clusters differed from each other in the expression of PGF, LXN, CHST1 (ART_1) and HEY1, GJA5, CXCL12, GRIA2 (ART_2). Four venous clusters showed comparable expression levels of APLNR, PLVAP (FIGS.10A- D). One progenitor population was found abundant only in earlier stages before E82, thus named as Ven_PG (FIG.10C). One cluster exhibiting higher expression of ACKR1, ADGRG6, SELE and SELP was termed as Ven_2, suggesting the role as large venous cells. The other with higher expression of CD83, CA4 and INMT was termed as Ven_3, suggesting the identity of capillary venous cells (FIG.s S3A-D). The final cluster was found across all the developmental timepoints, and showed moderate levels for both large and capillary venous markers, thus termed as Ven_1 with a transitional role (FIG. S3C). [00229] The skeletal muscle compartment comprised of 8 clusters with resolution at 0.5. One was identified as cycling cluster based on expression of cell cycle genes (TOP2A, MKI67, TYMS). The expression of key regulator SOX8 in one cluster led to the annotation of muscle satellite cells (SC_1). A transitioning cluster adjacent to SC_1 downregulated SOX8 and upregulated PAX7 and MYF5, thus annotated as SC_2 with a potential identity of activating/differentiating satellite cells. Skeletal muscle cells were largely segregated into two major types. SkM_1 expressed PAX7, MYF5 and NOTCH3, while SkM_2 expressed high levels of MYL1, ACTA1, MYH3 and TPM1 (FIG. S3H-K), suggesting potential distinct developmental origins from somites and head, respectively. Distinct developing statuses in both skeletal muscle cell types were identified based on timepoint enrichment as well as different expression pattern of key myogenetic regulators (Table S2). [00230] Within the immune compartment marked by PTPRC and LAPTM5, 10 clusters were identified with resolution at 0.3. One cluster with cycling features (TOP2A, MKI67, TYMS) was annotated as Cy.Im. B cells were classified on the basis of expression of IGHM, CD79A, CD79B, PAX5 and CD22. T cells were identified by the expression of BCL11B, TRAC, CD3G, CD3D. One cluster specifically expressing GZMK, KLRD1, IFNG and GNLY was annotated as NK cells. Another lymphoid cluster was classified as type 2 and 3 innate lymphoid cells (ILC2/3) due to their expression of RORC, TOX2, GATA3 and IL7R. Myeloid lineages were identified in 5 clusters. A minor cluster exhibiting unique expression of KIT, TPSB2, TPSB1 and GATA2 was annotated as mast cells. Monocytes were revealed with high expression of CD163 and LYVE1 (FIGS. 10L-O). Within monocytes, one early-stage subpopulation was found enriched only before E72, and the late-stage subpopulation emerged afterwards (FIG. 10N). Dendric cells were annotated on the basis of expression in HLA-DRA, ITGAX, ITGAM. Dendric cells were also subdivided into two subpoulations, with one expressing high levels of LYZ, marking the immature phenotype, while others expressing high levels of HLA-DRA and HLA-DQA1. These were labeled as DC_early and DC_late, respectively (FIGS.10L-O). [00231] Monocle version 2.18.0 was used for single cell trajectory analysis. In order to perform efficient monocle analysis, without losing generality, we limit the maximum number of single cells per cell type as 1000. [00232] CellChat was used for single cell intercellular communication analysis. Same as the monocle analysis, we limit the maximum number of single cells per cell type to 1000. [00233] Seurat, monocle and CellChat analyses were performed under R version 4.0.2. [00234] Visium Spatial Transcriptomic Profiling. Human embryonic and fetal esophageal and stomach tissues were washed in cold HBSS. Whole esophageal tubes were cut into ~3-5 mm length pieces from proximal to distal. Fresh tissues were directly embedded in OCT on dry ice. 10 μm fresh frozen sections of OCT-embedded tissues were used for Visium profiling. Such sections were collected using cryostat, sealed in airtight 50 ml Falcon tubes, stored at - 80 °C for downstream profiling. These sections were processed according to the 10X Genomics Visium protocol (CG000239_Rev F), using an optimized permeabilization time of 18 min. The pooled, 3’-end libraries were sequenced using Illumina NovaSeq. [00235] Space Ranger version 1.3.1 was used for primary data analysis, including demultiplexing, alignment, mapping and UMI counting. Specifically, for alignment and mapping, the GRCh38 reference genome and corresponding annotation were used. [00236] Cell2location Spatial Mapping Analysis. The cell2location algorithm maps all identified single cell populations onto Visium spatial transcriptomic profiles. In order to make accurate spatial mapping, we 1) merged several groups of closely-related cell types, and 2) analyzed early/mid stage and late stage profiles separately. In order to make efficient spatial mapping, without losing generality, we limit the maximum number of mapped single cells per cell type as 500. The cell2location analysis was performed under Python version 3.9.12. [00237] CODEX Multiplexed Immunofluorescence Staining. CODEX profiling starts with antibody conjugation, which was performed using protocols and reagents per manufacturer instructions (Akoya; 7000009). Such a process conjugates 50 μg of immunofluorescence- validated carrier-free antibodies to specific barcodes. Specifically, antibodies were concentrated on a 50 kDa filter equilibrated with the filtration buffer. The sulfhydryl groups were activated by incubating for 30 minutes at room temperature with the reduction mix. Antibodies were then washed with the conjugation buffer once. Oligonucleotide barcodes were resuspended in the conjugation buffer, added to the antibodies, and allowed to incubate for 2 hours at room temperature. The conjugated antibodies were washed 3 times, by resuspending and spinning down at 12,000 g for 8 minutes with the purification solution. Antibodies were then eluted by adding 100 μl storage buffer and spinning at 3000g for 4 minutes. The conjugated antibodies were stored at 4°C till use. [00238] Before use in multiplexed CODEX experiments, conjugated antibodies were validated with CODEX single stains on human fixed frozen fetal esophageal tissues. Staining was performed with the conjugated antibody as described below. The screening buffer was prepared according to the CODEX manual provided by Akoya Biosciences. Fixed and stained tissues were incubated in the screening buffer for up to 15 minutes. Fluorescent DNA probes were prepared and added to stained tissues for 5 minutes. Tissues were washed 3 times with the screening buffer followed by 1 wash with the CODEX buffer. Tissues were then imaged using a Keyence BZ-X810 inverted fluorescent microscope for the antibody validation. [00239] All fixed frozen tissue CODEX antibody stainings were done according to Akoya Biosciences staining protocol associated with the CODEX Staining Kit with some modifications (Akoya; 7000008). After tissue hydration, autofluorescence was bleached using a previously published protocol by allowing the tissue to sit in a solution (made of sodium hydroxide and hydrogen peroxide) sandwiched between a LED light panel for 45 minutes at room temperature. The tissue was then washed twice in ddH2O for 10 minutes, followed by a wash in the CODEX Hydration Buffer (contained in the CODEX Staining Kit) twice, 2 minutes each. The coverslip was allowed to equilibrate in the CODEX staining Buffer (contained in CODEX Staining Kit) for 20 minutes at room temperature. The blocking buffer was prepared by adding N, S, J, and G blocking solutions to the CODEX staining buffer (contained in the CODEX Staining Kit). Antibodies were added to the blocking buffer to make a total volume of 200 μl. The antibody cocktail was added to the coverslip, and staining was performed in a sealed humidity chamber at 4°C overnight. After staining, coverslips were washed twice in the hydration buffer for 4 minutes followed by fixation in the storage buffer (contained in the CODEX Staining Kit) with 1.6% paraformaldehyde for 10 minutes. Coverslips were then washed thrice in PBS, followed by a 5 minute incubation in ice-cold methanol on ice for 5 minutes followed by another 31X PBS washes. CODEX fixative solution (contained in CODEX Staining Kit) was prepared right before the final fixation step.20 μl of CODEX fixative reagent was added to 1000 μl 1X PBS. 200 μl fixative solution was added to the coverslip for 20 minutes followed by 3 washes in 1X PBS. Coverslips were then immediately prepped for imaging. [00240] For CODEX multicycle setup and imaging, coverslips were mounted onto Akoya’s custom-made stage between coverslip gaskets with the tissue side facing up. The coverslips were cleaned from the bottom using a Kim wipe to get rid of any salts. The tissue was stained with Hoechst Nuclear Stain (Thermo Fisher Scientific, cat. no.62249) at a 1:2000 dilution in the 1X CODEX buffer. A 96-well plate was used to set up the multicycle experiment with different fluorescent oligonucleotides in each well. A reporter stock solution was prepared to contain 1:2000 Hoechst stain and 1:12 dilution of assay reagent in the 1X CODEX buffer (contained in the CODEX Staining Kit). Fluorescent oligonucleotides (Akoya Biosciences) were added to this reporter solution at a final concentration of 1:50 in a total of 250 μl per well. A blank cycle containing no fluorescent probes was performed at the start and end of the experiment to capture residual autofluorescence. Automated image acquisition was performed using the CODEX Instrument Manager (CIM, version 1.30, Akoya Biosciences). Imaging was performed using a Keyence BZ-X810 microscope, fitted with a Nikon CFI Plan Apo l 20X/0.75 objective. The BZ-X software (Keyence) multi-point option was used to define the center and the imaging area corresponding to each region.9-11 z steps were acquired with the pitch set at 1.5 in the BZ-X software. Processed images were outputted in tiff format for downstream analysis. [00241] CODEX Analysis. Raw tiff files were processed using the CODEX Processor version 1.7.0.6 by Akoya Biosciences. CODEX Processor sequentially performs drift-compensation, deconvolution, background subtraction, tile stitching and segmentation. Such a pipeline yields spatial coordinates as well as marker protein signal intensities for all the detected cells. We then performed quality control based on the average signal intensity of all the DAPI channels. Cells with low average DAPI signals were removed, and the remaining cells were subjected to signal intensity log-transformation, hierarchical clustering and marker-guided annotation. [00242] Manatee. The Manatee deep learning framework was adapted from VAE and designed to model the generative process from TF expression to the corresponding gene expression. In order to do so, 1) both the encoder and decoder neural networks consist of two fully connected layers, each with the same number of nodes as the number of genes; 2) the latent space contain the same number of variables as the number of TFs; and 3) the following loss function is optimized during training: ^ ∼ ^^ +^^^ + ^^ [00243] where LR and DKL represent reconstruction loss and Kullback–Leibler Divergence against the N(0, 1) normal distribution respectively, as the two regular VAE loss terms. The additional Lr term represents the TF reconstruction loss, which is the mean square error (MSE) between reparameterized latent variables (Z) and TF expression (TF): ^^ ≡ ^^^(^, ^^) [00244] Manatee analysis starts with the TF expression matrix (D16 and D24 in our case), and perturbations are made by adjusting values of corresponding latent variables. The adjusted TF expression profiles are then passed through the decoder to predict expression profiles after perturbations. In order to assign biologically legitimate values to candidate latent variables, values will be sampled from the reference matrix, which tracks the TF expression pattern of the target phenotype (BC in our case). We performed a TF-wise sampling of the reference matrix, which gets values from only the corresponding TF expression vector. We also specified the perturbation direction and the sampling quantile for each TF. For up-regulated TFs, expression values will be sampled from the top quantile, and vice versa for down-regulated TFs. [00245] We defined TFs based on the Gene Ontology (GO) database, under terms GO:0003700, “DNA-binding transcription factor activity”, GO:0003677, “DNA binding”, GO:0140110, “transcription regulator activity” and GO:0006355, “regulation of DNA-templated transcription”. [00246] We also downloaded pathway gene lists from the GO database, including terms GO:0007173, “epidermal growth factor receptor signaling pathway”, GO:0007179, “transforming growth factor beta receptor signaling pathway”, GO:0016055, “Wnt signaling pathway” and GO:0030509, “BMP signaling pathway”. When perturbing specific signaling pathways, the corresponding lists are retrieved, and non-TFs are filtered. The yielded lists are then manually annotated based on previous literature in terms of determining perturbation directions. [00247] We trained Manatee by merging in vivo (D4, 10, 16, 24 and 43) and in vitro (Epi_PG, BC and SB-1 to 5) single cell datasets. We designed such a training to comprehensively capture regulatory logics during the BC specification and differentiation in vivo, as well as among the hPSC culture milestones in vitro. For each cell population, we downsampled the expression matrix to a maximum 1,000 cells. We performed 5 independent trainings and the one with the minimum loss value was used for downstream predictions. [00248] In vitro Differentiation of hPSCs into Esophageal Basal Cells. We adapted three previous protocols to differentiate hPSCs into esophageal progenitor cells in feeder-free chemically-defined conditions.0.1 million H9 or RUES2 hPSCs were plated in E8 medium with 10 μM Rock inhibitor Y-27632 (Tocris) in 1 well of 12-well culture plates coated with iMatrix- 511.24 hours later, culture medium was changed to MCDB131 base medium supplemented with 5 μM CHIR99021 (Tocris) and 100 ng/ml Activin A (R&D System) for 24 hours. MCDB131 base medium was prepared as following: 500 ml MCDB131 (Thermo Fisher Scientific), 5 ml of GlutaMAX (Thermo Fisher Scientific), 33 ml of 7.5% Bovine Albumin Fraction V Solution (Thermo Fisher Scientific), 10 ml of 7.5% NaHCO3 (Thermo Fisher Scientific) and 2 ml of 45% Glucose-D (Sigma Aldrich). On day 2, the medium was supplemented with 0.5 μM CHIR99021 and 100 ng/ml Activin A. On day 3, the medium was supplemented with 100 ng/ml Activin A only. On day 4, base medium was changed to Serum-Free Differentiation (SFD) medium: 750 ml of reconstituted IMDM (Thermo Fisher Scientific), 250 ml of F-12 (Corning), 7.5 ml of 7.5% Bovine Albumin Fraction V Solution (Thermo Fisher Scientific), 10 ml of GlutaMAX (Thermo Fisher Scientific), 5 ml of N2 (Thermo Fisher Scientific), 10 ml of B27 (Thermo Fisher Scientific) and 10 ml of Penicillin/Streptomycin (Thermo Fisher Scientific), and adding L- Ascorbic acid (Sigma-Aldrich) and MTG (Sigma-Aldrich) on the day of use to obtain a final concentration of 50 mg/ml and 0.04 ml/ml, respectively. Additional 1X GlutaMAX was also added. On day 4 and 5, cells were induced for anterior foregut endoderm differentiation in the SFD medium plus 10 mM SB431542 (Tocris) and 100 ng/ml Noggin (R&D Systems). From day 6 to day 9, anterior foregut endoderm was induced to dorsalize in the SFD medium supplemented with 10 mM SB431542, 100 ng/ml Noggin, 100 ng/ml hEGF (R&D Systems) and 50 ng/ml FGF10 (R&D Systems). From day 10 to day 15, esophageal progenitor cells were induced in the SFD medium supplemented with 100 ng/ml hEGF and 50 ng/ml FGF10. From day 16 onward, cells were cultured in the SFD medium supplemented with 100 ng/ml hEGF only. [00249] To test pathway perturbation strategies predicted by Manatee, different combinations of growth factors and chemicals were added to the SFD medium with 100 ng/ml hEGF. Cells were treated in the perturbation conditions for 5 additional days from day 16 or 24. In experimental strategy #19, cells were cultured in the SFD medium supplemented with 100 ng/ml hEGF, 5 ng/ml BMP4 (R&D Systems), 2 ng/ml TGFB1 (R&D Systems) and 0.4 μM IWP2 (Selleck Chemicals). In negative control strategy #9, cells were cultured in the SFD medium supplemented with 100 ng/ml hEGF, 1 μM A83-01 (Tocris), 1 μM DMH-1 (Tocris) and 3 μM CHIR99021 (Tocris). In base control strategy #14, cells were cultured in the SFD medium supplemented with 100 ng/ml hEGF only. [00250] Flow Cytometry and Cell Sorting. For cell surface marker staining, cells were dissociated with TrypLE Select and stained with fluorophore conjugated antibodies in the FACS buffer for 20 to 30 minutes at room temperature. After washing in the FACS buffer, stained cells were resuspended in FACS buffer with SYTOX Blue Dead Cell Stain to exclude dead cells. [00251] For intracellular staining, cells were processed using eBioscience Foxp3 /Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cells were stained with surface markers and Fixable Viability Dye eFluor 450 for 30 to 45 minutes at 4 °C. Fixation and permeabilization was performed at room temperature for 1 hour, followed by primary antibody incubation overnight at 4 °C, and secondary antibody incubation at room temperature for 1 hour. [00252] UltraComp eBeads Compensation Beads were used for single color compensation control. Flow cytometric analysis was performed using LSR II (BD Biosciences) and cell sorting was performed using FACSAria II at Stanford Shared FACS Facility. Data was analyzed using FlowJo software. Cells were sorted into the FACS buffer supplemented with 10% fetal bovine serum and 2X Anti-Anti. [00253] qPCR. Cells were lysed with TRIzol Reagent (Thermo Fisher Scientific) and RNA was extracted using Direct-zol RNA Miniprep Plus kit (Zymo Research). Quantitative PCR was performed using the TaqPath 1-Step Multiplex Master Mix kit (Thermo Fisher Scientific) using a LightCycler 480 instrument (Roche). Gene expression was normalized to the internal control GAPDH-VIC (Hs99999905_m1). All qPCR experiments were performed with at least triplicates. The following Taqman probes were used: KRT5-FAM (Hs00361185_m1), KRT15- FAM (Hs00267035_m1). [00254] Bulk RNA-seq. Cells were lysed with TRIzol Reagent (Thermo Fisher Scientific) and RNA was extracted using Direct-zol RNA Miniprep Plus kit (Zymo Research). RNA concentration and quality was measured by 2100 Bioanalyzer (Agilent Technologies). Only samples with high RIN (>8.0) were used for library preparation. Libraries were prepared using SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) and sequenced by NovaSeq (Illumina) with paired-end 100 bp reads at MedGenome. Each library was sequenced with a targeted depth of 40 million total reads.2 or 3 biological replicates were sequenced for each sample. Alignment was performed using STAR against the hg38 reference genome. RPKM gene expression values were quantified using HOMER analyzeRepeats.pl. For differential gene expression analysis, raw reads were compared using DESeq2 likelihood ratio test (LRT) to assess gene expression changes along different time points. Top 1000 differentially expressed genes were filtered and ranked on the basis of an adjusted P value < 0.05. Stage- dependent differentially expressed genes were used for EnrichR analysis (https://maayanlab.cloud/Enrichr/) to assess enrichment in ARCHS4_TISSUES terms. [00255] Calcium Induced Stratification. Sorted EPCAM+ ITGB4HI double positive cells were expanded in Defined Keratinocyte Serum-Free Medium (DKSFM, Thermo Fisher Scientific) supplemented with 10 μM Y-27632, 1 μM A83-01 (Tocris) and 1 μM DMH-1 (Tocris) on 6-well ECM collagen-I-coated peptide plates (Corning) or culture plates coated with rat tail collagen- I solution (Cell Biologics). After confluency, cells were treated with 2 mM CaCl2 for 8 days. [00256] 3D Organotypic Culture. Devitalized de-epidermal human dermis (DED) was prepared as follows. Cadaver skin (New York Firefighter Skin Bank) was freeze–thawed three times to devitalize cells and washed in PBS with 5X penicillin-streptomycin, 5X gentamicin and 5X fungizone. The sterilized skin was stored in PBS containing 1X penicillin-streptomycin, 1X gentamicin and 1X fungizone at 37 °C for one week. The epidermis was then peeled off of the dermis, which was then stored in PBS containing 1X penicillin-streptomycin at 4 °C for more than two weeks before use. Devitalized dermis was cut into 1.5 cm × 1.5 cm pieces, and stored epidermis-down in 6-well dishes at 37 °C to let the dermis attach to the bottom. The culture was switched to DKSFM, and 106 induced esophageal basal cells were seeded onto the center of DED. After 3 days, the culture was lifted to the air–liquid interface and switched to KGM medium (DMEM: Hams F123:1, FBS 10%, 1X nonessential amino-acid, 0.18 mM adenine hydrochloride, 0.1 nM cholera toxin, 10 ng/ml EGF, 0.4 μg/ml hydrocortisone, 5 μg/ml insulin, 2 nM triiodo-l-thyronine, 5 μg/ml transferrin). After two to four weeks, the organotypic co- cultures were collected and the pieces were fixed in 4% PFA at room temperature for 4 hours or 4 °C overnight, washed with PBS, and processed for OCT and paraffin embedding for downstream analysis. [00257] 3D Organoid Culture. 60,000 EPCAM+ ITGB4HI sorted double positive cells were resuspended in 60 μl SFD medium supplemented with 100 ng/ml hEGF, 20 ng/ml FGF2, 10 μM Y-27632, 1 μM A83-01 (Tocris) and 1 μM DMH-1 (Tocris) and mixed with 90 μl Growth Factor Reduced Matrigel (Corning). The mixture was added in 24-well cell culture inserts (Falcon), and incubated at 37 °C incubator for 30 minutes to solidify. 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Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

THAT WHICH IS CLAIMED IS: 1. A method of producing a population of esophogeal basal cells (eBC) in defined monolayer conditions in media comprising extracellular signaling agents to guide differentiation, the method comprising: (a) differentiating human pluripotent stem cells (hPSC) to definitive endoderm cells; (b) differentiating definitive endoderm cells to anterior foregut endoderm cells; (c) differentiating anterior foregut endoderm cells to dorsal anterior foregut endoderm cells; (d) differentiating dorsal anterior foregut endoderm cells to early esophogeal basal cells; (e) differentiating the early esophogeal basal cells to produce a population of definitive esophogeal basal cells.
2. The method of claim 1, wherein greater than greater than 50% of the cells in the eBC cell population express eBC surface markers.
3. The method of any of claims 1-2, wherein (a) a population of human pluripotent cells is cultured in media comprising an effective amount of a ROCK inhibitor for a period of about 1 day; (b) the population of cells from step (a) is next cultured in a medium comprising an effective amount of a glycogen synthase kinase 3 inhibitor, and a TGFβ pathway agonist, for a period of about two days; and (c) the population of cells from step (b) is cultured in a medium comprising an effective amount of TGFβ pathway agonist for a period of about one day; optionally providing a JNK inhibitor for a first say of step (b); to generate a population of definitive endoderm cells.
4. The method of Claim 3, wherein the ROCK inhibitor Y-27632.
5. The method of Claim 4, wherein the Y-27632 is present at a concentration between 1-50 μM, or about 10 μM.
6. The method of Claim 3, wherein the glycogen synthase kinase 3 inhibitor comprises CHIR99021.
7. The method of Claim 6, wherein the CHIR99021 is present at a concentration between 0.5 to 50 μM, or about 5 μM.
8. The method of Claim 3, wherein the TGFβ pathway agonist comprises Activin A
9. The method of Claim 8, wherein Activin A is present at a concentration between 1- 1,000 ng/ml, or a concentration of about 100 ng/ml.
10. The method of any of claims 1-9, wherein definitive endoderm cells are cultured in media comprising a TGFβ pathway inhibitor and BMP pathway inhibitor for a period of about 2 days to generate anterior foregut endoderm cells.
11. The method of Claim 10, wherein the TGFβ pathway inhibitor comprises SB431542.
12. The method of Claim 11, wherein the SB431542 is present at a concentration between 1-100 mM, or a concentration of about 10 mM.
13. The method of Claim 10, wherein the BMP pathway inhibitor comprises Noggin.
14. The method of Claim 13, wherein Noggin is present at a concentration between 1- 1,000 ng/ml, or a concentration of about 100 ng/m.
15. The method of any of claims 1-14, wherein anterior foregut endoderm cells are cultured in the presence of a TGFβ pathway inhibitor, BMP pathway inhibitor, EGF pathway agonist and FGF pathway agonist for a period of about 2-4 days to generate dorsal anterior foregut endoderm.
16. The method of Claim 15, wherein the TGFβ pathway inhibitor comprises SB431542.
17. The method of Claim 16, wherein the SB431542 is present at a concentration between 1-100 mM, or a concentration of about 10 mM.
18. The method of Claim 15, wherein the BMP pathway inhibitor comprises Noggin.
19. The method of Claim 13, wherein Noggin is present at a concentration between 1- 1,000 ng/ml, or a concentration of about 100 ng/m.
20. The method of Claim 15, wherein the EGF pathway agonist comprises human epidermal growth factor.
21. The method of Claim 20, wherein the human EFG is present in a concentration between 1-1,000 ng/ml, or a concentration of about 100 ng/ml.
22. The method of Claim 15, wherein the FGF pathway agonist comprises fibroblast growth factor 10.
23. The method of Claim 22, wherein the FGF10 is present in a concentration between 1-500 ng/ml, or about 50 ng/ml.
24. The method of any of claims 1-23, wherein dorsal anterior foregut endoderm cells are cultured in the presence of an EGF pathway agonist and FGF pathway agonist for a period of from about 3-6 days, then only the EGF pathway agonist for a period of from about 6-10 days to generate to early esophogeal basal cells.
25. The method of Claim 24, wherein the EGF pathway agonist comprises human epidermal growth factor.
26. The method of Claim 25, wherein the human EFG is present in a concentration between 1-1,000 ng/ml, or a concentration of about 100 ng/ml.
27. The method of Claim 25, wherein the FGF pathway agonist comprises fibroblast growth factor 10.
28. The method of Claim 27, wherein the FGF10 is present in a concentration between 1-500 ng/ml, or about 50 ng/ml.
29. The method of any of claims 24-28, wherein a population of definitive esophogeal cells are differentiated.
30. The method of any of claims 1-28, wherein early esophogeal basal cells are cultured in the presence of an EGF pathway agonist, a wnt pathway inhibitor, a BMP pathway agonist and a TGFβ pathway agonist for a period of from about 3-5 days to generate definitive esophogeal basal cells.
31. The method of Claim 30, wherein the EGF pathway agonist comprises human epidermal growth factor.
32. The method of Claim 31, wherein the human EFG is present in a concentration between 1-1,000 ng/ml, or a concentration of about 100 ng/ml.
33. The method of Claim 32, wherein the wnt pathway inhibitor comprises IWP-2.
34. The method of Claim 33, wherein the IWP-2 is present at a concentration between 0.1 to 5.0 micromolar or, a concentration of about 0.4 micromolar.
35. The method of Claim 34, wherein the BMP pathway agonist comprises BMP4.
36. The method of Claim 35, wherein the BMP4 is present at a concentration between 0.1 to 50 ng/ml, or a concentration of about 5 ng/ml.
37. The method of Claim 30, wherein the TGFβ pathway agonist comprises TGF-β.
38. The method of Claim 37, wherein the TGF-β is present at a concentration between 0.1 to 20 ng/ml, or a concentration of about 2 ng/ml.
39. The method of claim 3, wherein the JNK inhibitor is JNK-IN-8.
40. The method of claim 39, wherein JNK-IN-8 is present at a concentration between 0.1 to 50 µM, or a concentration of about 1 µM.
41. The method of any of Claims 1-40, wherein the population of cells is cultured on a chemically defined substrate.
42. The method of Claim 41, wherein the chemically defined substrate comprises iMatrix511.
43. The method of any of Claims 1-39, wherein the population of cells is cultured in chemically defined medium.
44. The method of any of Claims 3-9, wherein in (a) the population of cells is cultured in E8 medium; and in (b) and (c) the population of cells is cultured in MCDB131 medium.
45. A method of isolating eBC’s produced by the method of any of Claims 1-44, comprising sorting the population of cells for ITGB4+/EPCAM+ cells.
46. The method of Claim 45, wherein the cells are sorted by FACS.
47. A substantially pure population of eBC produced by the method according to any of claims 1-46.
48. A method of treatment, comprising administering to an individual the population of cells according to Claim 47.
49. The method of treatment of claim 48, wherein the method is applied in the treatment of esophageal injury, esophageal defect, recessive dystrophic epidermolysis bullosa, dominant dystrophic epidermolysis bullosa, wounding caused by cancer resection, caustic injury, esophageal stricture, or esophagitis.
50. An organotypic or organoid cell culture comprising cells of Claim 47.
51. A method of screening a substantially pure population of eBC for a cellular response, comprising contacting a population of substantially pure population of eBC of claim 44 with a pharmacological agent and evaluating the population of cells for a cellular response induced by the pharmacological agent.
52. A kit or system for use in the methods of any of claims 1-47.
53. A method of determining factors for differentiating stem or progenitor cells to a cell type of interest, comprising applying machine learning to a spatiotemporal multi-omics cell atlas to determine combinations of candidate developmental signals for in silico differentiation to the cell type of interest.
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CN120536355A (en) * 2025-07-28 2025-08-26 海南大学三亚研究院 Construction method and application of an in vitro model of the Wuzhishan pig mitral valve

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EP3694603A4 (en) * 2017-10-10 2021-07-14 Children's Hospital Medical Center COMPOSITIONS OF ESOPHAGIAN TISSUES AND / OR ORGANOIDS AND THEIR MANUFACTURING PROCESSES
US20220180975A1 (en) * 2019-01-28 2022-06-09 The Broad Institute, Inc. Methods and systems for determining gene expression profiles and cell identities from multi-omic imaging data

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