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WO2025029826A1 - Cellules thérapeutiques modifiées et méthodes associées - Google Patents

Cellules thérapeutiques modifiées et méthodes associées Download PDF

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WO2025029826A1
WO2025029826A1 PCT/US2024/040216 US2024040216W WO2025029826A1 WO 2025029826 A1 WO2025029826 A1 WO 2025029826A1 US 2024040216 W US2024040216 W US 2024040216W WO 2025029826 A1 WO2025029826 A1 WO 2025029826A1
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stem cell
cell
cells
expression
reprogrammed
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William L. Rust
Jeremy RATIU
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Seraxis Inc
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Seraxis Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/117—Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure relates generally to the field of cell biology, stem cells, cellular differentiation, and cell-based therapies.
  • Diabetes mellitus is a disease in which the body’s ability to produce or respond to the hormone insulin is impaired, resulting in abnormal metabolism of carbohydrates and elevated levels of glucose in the blood and urine.
  • the disease is subdivided into several sub-types, described alternatively as Type 1 diabetes mellitus, insulin-dependent diabetes mellitus (IDDM), mature onset diabetes of the young (MODY), latent adult diabetes (LADA), brittle diabetes, lean diabetes, Type 1.5, Type 2, Type 3, obesity-related diabetes, gestational diabetes, and other nomenclature accepted by the field.
  • Insulin-dependent diabetes patients can potentially be cured by transplant of new hormone-secreting islet cells, but this approach has been limited to date because these cells are hard to obtain in sufficient quantity, quality, and purity.
  • a cell product must be generated that meets the criteria of: (1) being able to establish a functional graft of therapeutically relevant hormone-secreting islet cells in a patient, and (2) not harming the patient through unregulated hormone secretion, neoplasms, or other adverse effects. To date, cells that meet these criteria have not been demonstrated clinically.
  • the vast majority of established protocols for generating hormone-secreting islet cells from human stem cells focus on generating the largest population of cells that express markers of mature insulin-secreting beta cells: Pdxl, Nkx6.1, and C-peptide (a surrogate marker of insulin secretion) See e.g.
  • Described herein are cells and cellular compositions that produce pancreatic hormones and may be used to treat diabetes, as well as methods of making and identifying the same.
  • the present disclosure is based on the unexpected discovery that genetically modifying stem cells to have reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) results in improved differentiation of the stem cells towards a SRE cell, and the SRE with reduced or abolished CIITA expression has downregulated expression of somatostatin, downregulated expression of glucagon, and increased secretion of insulin comparted to an unmodified SRE.
  • MHC major histocompatibility complex
  • CIITA major histocompatibility complex
  • the present disclosure relates to a modified stem cell that can differentiate into a pancreatic endocrine cell and has reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA).
  • MHC major histocompatibility complex
  • CIITA major histocompatibility complex
  • the reduced expression of CIITA is sufficient to prevent expression of MCH class II.
  • the stem cell has increased capacity for maturing into a pancreatic endocrine cell compared to a stem cell having CIITA expression sufficient to induce expression of MHC class II.
  • the present disclosure relates to an isolated synthetic replacement endocrine (SRE) cell obtained by differentiation of the modified stem cell that has reduced or abolished expression of major histocompatibility complex (MHC) class II transactivator (CIITA).
  • SRE synthetic replacement endocrine
  • MHC major histocompatibility complex
  • CIITA major histocompatibility complex
  • the isolated SRE cell obtained by differentiation of the modified stem cell that has reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) expresses somatostatin at lower levels than an SRE cells obtained from a stem cell that expresses CIITA sufficient to induce MHCII expression or a wild type stem cell.
  • the isolated SRE cell obtained by differentiation of the modified stem cell that has reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) secretes insulin at higher levels than an SRE cells obtained from a stem cell that expresses CIITA sufficient to induce MHCII expression or a wild type stem cell.
  • MHC major histocompatibility complex
  • CIITA major histocompatibility complex
  • the isolated SRE cell obtained by differentiation of the modified stem cell that has reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) expresses glucagon at lower levels than an SRE cells obtained from a stem cell that expresses CIITA sufficient to induce MHCII expression or a wild type stem cell.
  • MHC major histocompatibility complex
  • CIITA major histocompatibility complex
  • the present disclosure relates to method of preparing a modified stem cell with increased capacity for maturing into a pancreatic endocrine cell, comprising reducing or silencing expression of major histocompatibility complex (MHC) class II trans- activator (CIITA) in an undifferentiated stem cell, wherein the expression of CIITA is reduced to a level so that MCH class II is not induced compared to a stem cell expressing native levels of CIITA or a wild type stem cell.
  • MHC major histocompatibility complex
  • CIITA trans- activator
  • the present disclosure provides an isolated synthetic replacement endocrine (SRE) cell, that expresses transcription factors Pdxl and ISL1, and does not express the transcription factor Nkx6.1.
  • the isolated SRE cell is differentiated from a stem cell.
  • the stem cell is selected from an embryonic stem cell, an induced pluripotent stem cell, and a multipotent reprogrammed stem cell.
  • the stem cell is derived from a cell line.
  • the stem cell is a multipotent reprogrammed stem cell.
  • the multipotent reprogrammed stem cell was obtained by reprogramming a pancreatic cell.
  • the multipotent reprogrammed stem cell was reprogrammed with an expression plasmid encoding (i) Oct4, Sox2, Klf4, and L-Myc; (ii) Oct4, Sox2, Klf4, and C-Myc; (iii) LIN28, Oct4, Sox2, and Nanog; or (iv) Gilsl, Oct3/4, Sox2, and Klf4.
  • the stem cell is genetically modified to avoid targeting by lymphocytes.
  • the stem cell is genetically modified to lack expression of MHCI and MHCII.
  • the stem cell is genetically modified to avoid targeting by natural killer cells.
  • the multipotent reprogrammed stem cell can differentiate into endoderm or ectoderm cell types, but not mesoderm cell types. In some embodiments, the multipotent reprogrammed stem cell does not comprise any reprogramming genes incorporated into its genome.
  • the isolated SRE cell expresses C-peptide.
  • C-peptide is expressed at levels equivalent to mature, native pancreatic islet cells.
  • C-peptide is expressed at levels higher than an SRE cell that expresses Nkx6.1.
  • C-peptide expression is responsive to fluctuations in glucose concentration.
  • the isolated SRE cell secretes insulin. In some embodiments, the isolated SRE cell secretes glucagon.
  • the isolated SRE cell is human.
  • compositions comprising at least one isolated SRE cell disclosed herein and a therapeutically acceptable carrier.
  • the present disclosure provides a method of treating diabetes, comprising administering to a subject with diabetes at least one isolated SRE cell disclosed herein (e.g., a cell of any one of the foregoing aspects or embodiments) or the pharmaceutical composition comprising the same.
  • the diabetes is type 1.
  • Nkx6.1 is expressed after the cell or pharmaceutical composition is administered to the subject.
  • administration comprises implanting the isolated cell or pharmaceutical composition in the subject.
  • the subject is human.
  • the present disclosure provides a method of preparing an isolated SRE cell, comprising: contacting an undifferentiated stem cell with a combination of growth factors to drive differentiation of the stem cell to an endocrine lineage, wherein the combination of growth factors comprises retinoic acid, a Hedgehog antagonist, and a bone morphogenetic protein (BMP) signaling inhibitor.
  • the combination of growth factors comprises retinoic acid, a Hedgehog antagonist, and a bone morphogenetic protein (BMP) signaling inhibitor.
  • BMP bone morphogenetic protein
  • the Hedgehog antagonist is selected from SANT1 and cyclopamine.
  • the BMP signaling inhibitor is LDN193189.
  • the stem cell is not exposed to tri-iodothyronine (T3) or analog thereof during the differentiation process.
  • T3 tri-iodothyronine
  • the stem cell is not exposed to a protein kinase C activator during the differentiation process.
  • the stem cell is not exposed to nicotinamide, a form of vitamin B3 during the differentiation process.
  • the stem cell is not exposed to transforming growth factor beta 1 (TGFbetal) protein.
  • TGFbetal transforming growth factor beta 1
  • the stem cell is not exposed to insulin-like growth factor 1 (IGF- 1).
  • IGF- 1 insulin-like growth factor 1
  • the stem cell is derived from a cell line. [0035] In some embodiments, the stem cell is selected from an embryonic stem cell, an induced pluripotent stem cell, and a multipotent reprogrammed stem cell.
  • the stem cell is a multipotent reprogrammed stem cell.
  • the multipotent reprogrammed stem cell was obtained by reprogramming a pancreatic cell.
  • the multipotent reprogrammed stem cell was reprogrammed with an expression plasmid encoding (i) Oct4, Sox2, Klf4, and L-Myc; (ii) Oct4, Sox2, Klf4, and C-Myc; (iii) LIN28, Oct4, Sox2, and Nanog; or (iv) Gils 1 , Oct3/4, Sox2, and Klf4.
  • the multipotent reprogrammed stem cell can differentiate into endoderm or ectoderm cell types, but not mesoderm cell types. In some embodiments, the multipotent reprogrammed stem cell does not comprise any reprogramming genes incorporated into its genome. In some embodiments, the stem cell is genetically modified to avoid targeting by lymphocytes. In some embodiments, the stem cell is genetically modified to lack expression of MHCI and MHCII. In some embodiments, the stem cell is genetically modified to avoid targeting by natural killer cells. In some embodiments, the stem cell is genetically modified to express CD47.
  • the present disclosure provides an isolated SRE cell obtained by the methods of differentiation disclosed here (e.g., the immediately preceding aspect and embodiments).
  • the cell expresses transcription factors Pdxl and ISL1, and does not express the transcription factor Nkx6.1.
  • the present disclosure provides a population of the foregoing SRE cells.
  • the population may be substantially pure, meaning it includes at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% SRE cells.
  • the SRE cells may be formulated in with one or more therapeutically acceptable carriers.
  • FIG. 1 shows flow cytometry analysis of SRE clusters.
  • SRE clusters are comprised of cells that express predominantly Pdxl, ISL1, and insulin. A smaller proportion of cells express glucagon. In contrast Nkx6.1 expressing cells are almost undetectable.
  • FIG. 2 shows immunofluorescent images of SRE clusters. Fluorescent staining revealed that ISL1 is express in the nuclei of cells that express C-peptide (A). Nkx6.1 -expressing cells are nearly absent (B), but cells that do express Nkx6.1 do not co-express ISL-1 (C). ISL1 is expressed in the nuclei of cells that express the endocrine pancreas marker chromogranin A (D). C-peptide-expressing cells are more abundant that glucagon-expressing cells (E). C- peptide expressing cells do not express Nkx6.1 (F).
  • FIG. 3 shows that SRE clusters secrete C-peptide in response to glucose. Static incubation of 50 SRE clusters for 4X 20 min intervals in 2.2 mM glucose demonstrated a gradually decreasing C-peptide secretion. Exposure to 16.7 mM glucose caused increased C- peptide secretion by 40 min. Artificially stimulating C-peptide secretion in the presence of 30 mM KC1 shows a high reserve of C-peptide available for secretion.
  • FIG. 4 shows CIITA expression during P cell differentiation.
  • FIG. 4A shows a significant reduction in CIITA transcriptional activity across pancreatic progenitor (stage 3) to scP cell (stage 6) differentiation.
  • FIG. 4B shows analysis of scRNA-seq across differentiation of P cells from a different human ES cell line and demonstrates a similar significant reduction in CIITA activity during the differentiation of P cells (stage 7) from pancreatic progenitors (stage 4).
  • FIG. 5 shows protein expression of CIITA in SR1423 and SR1423-HI by using western blot as shown.
  • Cultures of SR1423 and SR1423-HI were exposed to TNF-a, a potent inducer of MHCII expression, for 3 days. Cells were collected, lysed and analyzed by western blot for the presence of MHCII.
  • FIG. 7 shows immunofluorescence analysis of expression of C-peptide, glucagon, SST (somatostatin), PPY (pancreatic polypeptide), and Ghrelin in differentiated cultures of SRI 423 and SR1423-HI at day 35, and native islets.
  • DAPI is used to stain the nucleus of the cells.
  • FIGS. 8A-8C show single cell sequencing of differentiated cultures of SR1423 and SR1423-HI to characterize the types of pancreatic cells generated from these stem cells.
  • FIG. 8A shows a map of the different types of pancreatic cells obtained from differentiated cultures of SR1423 and SR1423-HI stem cells.
  • FIG. 8B shows cell type prediction of cells obtained from SR1423 and SR1423-HI differentiation.
  • FIG. 8C shows average expression of markers of pancreatic cells in differentiated cultures of SR1423 and SR1423-HI.
  • the present disclosure was based on the surprising discovery that reducing or silencing expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) in stem cells improved the capacity of the stem cells to differentiate towards endocrine cells such as Synthetic Replacement Endocrine (SRE), and that had increased expression of insulin, and decreased expression of glucagon and somatostatin.
  • MHC major histocompatibility complex
  • SRE Synthetic Replacement Endocrine
  • the present disclosure provides Synthetic Replacement Endocrine (SRE) cells that can be used to treat diabetes, methods of generating such cells, populations of such cells, and methods of using the disclosed cells for treating diabetes. More specifically, the present disclosure provides differentiation and isolation of SRE cells that express transcription factors pancreatic duodenal homeobox-1 (Pdxl) and ISL LIN homeobox-1 (ISL1), but not homeobox protein NKX6.1 (Nkx6.1). Additionally, the disclosure also provides methods of producing cells with the foregoing expression profile.
  • SRE Synthetic Replacement Endocrine
  • Such protocols may include, among other steps, exposing stem cells (in particular, stem cells that exhibit a preference or predisposition toward an endoderm lineage) to retinoic acid, a hedgehog antagonist, and a bone morphogenetic protein (BMP) signaling inhibitor.
  • stem cells in particular, stem cells that exhibit a preference or predisposition toward an endoderm lineage
  • BMP bone morphogenetic protein
  • the cultures may be initiated in adhesion, allowing the cells to naturally and spontaneously form three-dimensional structures; and then transferring the three-dimensional structures to suspension culture.
  • the cells may not be exposed to Wnt3a during culture, either while grown in adhesion or in suspension.
  • the cells may not be exposed to tri-iodothyronine (T3) or analog thereof, a protein kinase C activator during culture, nicotinamide, transforming growth factor beta 1, or insulin-like growth factor- 1, either while grown in adhesion or in suspension.
  • T3 tri-iodothyronine
  • nicotinamide a protein kinase C activator during culture
  • nicotinamide transforming growth factor beta 1
  • insulin-like growth factor- 1 either while grown in adhesion or in suspension.
  • ISL1 transcription factor 1
  • Sharon 2019b Another marker of the pancreatic endocrine cell, in addition to the insulin-secreting beta cell and the glucagon-secreting alpha cell, is the transcription factor ISL1 (Sharon 2019b).
  • ISL1 -expressing cells that express insulin also express Nkx6.1.
  • the present disclosure describes a novel cell population derived from stem cells that express ISL1 and insulin, but not Nkx6.1. Other cells express ISL1 and glucagon.
  • a method is disclosed that generates a highly pure population of ISL1 -positive, Nkx6.1 -negative cells that express insulin or glucagon, and minimal to no non-pancreatic cells present.
  • This population is differentiated from stem cells by a differentiation protocol that employs a minimal amount of transcription factors, hormones, and chemical analogs. Eschewing the use of complex formulas of growth factors improves reproducibility and consistency.
  • This population is highly potent at creating stable, functional pancreatic grafts after implant to a suitable host. This cell population may activate expression of Nkx6.1 after implant to the host.
  • the term “substantially free of’ refers to the composition to which the agent has not been added, but it does not exclude that trace amounts of the agent exist.
  • the term “islet cell” refers to terminally differentiated pancreatic endocrine cells, and any precursor cell that is committed to form progeny normally classified as pancreatic endocrine.
  • the islet cell exhibits some of the morphological features and phenotypic markers (exemplified below) typical of an islet cell lineage. Mature alpha cells secrete glucagon; mature beta cells secrete insulin; mature delta cells secrete somatostatin; PP cells secrete pancreatic polypeptide.
  • “Synthetic Replacement Endocrine” or “SRE” cells are cells that are definitively identifiable as pancreatic endocrine cells based upon specific expression of the Pdxl, ISL1, and pancreatic hormones insulin or glucagon. These cells, upon implantation into a human subject, can mature or alter their gene expression profile to more closely resemble mature endocrine cells of the mammalian pancreas.
  • “SRE” cells that are differentiated according to the disclosed protocol express pancreatic transcription factors Pdxl and ISL1, but do not express the transcription factor Nkx6.1. After implantation into a suitable host, the SRE cell may ultimately express Nkx6.1.
  • pancreatic progenitors As used herein, “pancreatic progenitors,” “pancreatic precursors,” or “pancreatic stem cells” are pancreatic cells that may mature or change their pancreatic gene expression profile after implant to a suitable host. The matured cells may adopt a gene expression pattern that more closely resembles native pancreatic islets.
  • the term “stem cells” denotes undifferentiated cells that are able to differentiate into specialized cells (e.g., insulin-producing pancreatic cells).
  • the term “stem cell” can include pluripotent cells derived from pre- embryonic, embryonic, or fetal tissue after fertilization that are capable of producing progenitors of all of the three germinal layers (z.e., endoderm, mesoderm, and ectoderm); induced pluripotent cell (z.e., cells that have been transduced with reprogramming genes) that are capable of producing progenitors of all of the three germinal layers; and multipotent cells, such as reprogrammed cells (z.e., cells that have been transduced with reprogramming genes) that can differentiate into only one or two germ layers or that preferentially differentiate into a certain germ layer (e.g., reprogrammed cells that preferentially differentiate into ectoderm or end
  • PCT/US2013/047243 published as W02014/004341
  • PCT/US2019/017281 published as WO2019/157329.
  • the term includes both established lines of stem cells of various kinds (including cells obtained from primary tissue) that are pluripotent or multipotent in the manner described.
  • induced pluripotent cells or “induced pluripotent stem cells” (“iPS cells”) denote pluripotent cells derived by reprogramming of adult somatic cells, reproductive cells, multipotent cells, or other cell types, following standard art accepted methods (e.g., somatic-cell nuclear transfer, transduction with reprogramming genes, chemical inducement (see De Los Angeles et al., Cell Research, 23: 1337-1338 (2013); Federation et al., Trends in Cell Biology, 24: 179-187 (2013)), etc.).
  • the term includes both established induced pluripotent stem cells, and cells obtained from primary tissue that are pluripotent in the manner described.
  • non-pluripotent reprogrammed cells denote cells derived by reprogramming of adult somatic cells, reproductive cells, pluripotent cells, or other cell types, with known reprogramming methods, such as transduction/expression of reprogramming genes and other methods discussed above.
  • non-pluripotent reprogrammed cells or “multipotent reprogramed cells” may differentiate into only one or two germ layers or possess a preference to differentiate into a certain germ layer (e.g., reprogrammed cells that preferentially differentiate into ectoderm or endoderm cell types, but which cannot efficiently differentiate into mesoderm cell).
  • the term includes both established induced multipotent cells (e.g., SR1423), and cells obtained from primary tissue that are reprogrammed to be multipotent in the manner described.
  • suitable multipotent reprogramed cells are disclosed in PCT/US2013/047243 (published as W02014/004341) and PCT/US2019/017281 (published as WO2019/157329).
  • reprogramming genes denotes known genes and transcription factors that are commonly used in the art to induce pluripotency or multipotency in differentiated cells.
  • exemplary reprogramming genes include, but are not limited to, Oct4 (i.e., Oct-3/4 or Pou5fl); Sox family transcription factors such as Soxl, Sox2, Sox3, Soxl5, and Soxl8; Klf family transcription factors such as Klf4, Klfl, Klf2, and Klf5; Myc family transcription factors such as C-myc, N-myc, and L-myc; Nanog; LIN28; and Glisl.
  • Oct4 i.e., Oct-3/4 or Pou5fl
  • Sox family transcription factors such as Soxl, Sox2, Sox3, Soxl5, and Soxl8
  • Klf family transcription factors such as Klf4, Klfl, Klf2, and Klf5
  • Myc family transcription factors such as C-myc, N-myc, and L
  • the term “differentiate” or “differentiation” denotes a change in cell type from a less specific cell to a more specific cell. For example, any cell that has exited the pluripotent state and progressed along a developmental pathway toward a defined germ line has undergone differentiation.
  • the term “differentiated” is a relative term, so differentiating cells can be at different stages during their developmental path towards a mature functional cell type. A cell at a later stage of developmental progression can therefore be said to be more differentiated than a cell at an earlier stage.
  • differentiation inducing factors refers to one of a collection of compounds that are used in culture systems of this disclosure to induce differentiation of stem cells to differentiated cells of the islet lineage (including precursor cells, progenitor cells, cells with the ability to change their gene expression profile and terminally differentiated cells).
  • the agent may assist the differentiation process by inducing or assisting a change in phenotype, promoting growth of cells with a particular phenotype or retarding the growth of others. It may also act as an inhibitor to other factors that may be in the medium or synthesized by the cell population that would otherwise direct differentiation down the pathway to an unwanted cell type.
  • an “endoderm-inducing factor” can include, but is not limited to, Activin-A.
  • an “endocrine-inducing factor” can include, but is not limited to, retinoic acid and/or a hedgehog antagonist and/or a BMP inhibitor, either alone or in combination.
  • long-term when used in relation to the survival and functioning of foreign therapeutic cells used in a cell-based therapy/implant, means a period of at least six months or longer.
  • the phrases “therapeutically effective amount” means an amount of cells transplanted into a subject that provides the specific pharmacological effect for which the cells are transplanted, i.e. to produce insulin and lower blood glucose. It is emphasized that a therapeutically effective amount of cells will not always cause normal regulation of blood glucose in a given diabetic subject, even though such concentration is deemed to be a therapeutically effective amount by those of skill in the art. For convenience only, exemplary amounts are provided below. [0063] Those skilled in the art can adjust such amounts in accordance with standard practices as needed to treat a specific subject. The therapeutically effective amount may vary based on the site of implantation, the age and weight of the subject, and/or the subject’s condition, including the severity of the subject’s disease, the subject’s diet, and/or the subject’s overall health.
  • treatment refers to one or more of reducing, ameliorating or eliminating one or more symptoms or co-morbidities of diabetes, such as hyper- and hypo-glycemia, heart disease, renal disease, hepatic disease, retinopathy, neuropathy, non-healing ulcers, periodontal disease; reducing the subject’s reliance on exogenous insulin to regulate blood glucose, regulating the subject’s blood glucose without the use of exogenous insulin; reducing the subject’s percentage of glycosylated hemoglobin, or HbAlC levels; and/or reducing the subject’s reliance on other pharmaceutical interventions, such as insulin sensitizers, enhancers of glucose excretion, and other treatment modalities known in the art.
  • other pharmaceutical interventions such as insulin sensitizers, enhancers of glucose excretion, and other treatment modalities known in the art.
  • the terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to any individual mammalian subject, e.g., non-human primate, porcine, bovine, canine, feline, equine, or human.
  • the “individual,” “subject,” or “patient” is a human.
  • stem cell-based therapy One limitation of conventional stem cell-based therapy is that different stem cells possess different propensities to differentiate into mature cell types. For instance, it has been reported that epigenetic signatures of the starting cell population can persist in reprogrammed cells, a phenomenon called “epigenetic memory.” As a result, iPS cells and other reprogrammed cells may preferentially differentiate into cells that belong to the same germ layer from which they were derived. Accordingly, in some embodiments, the stem cells used in the disclosed methods for generating insulin-producing cells may be derived from mature endodermal cells that have been reprogrammed into pluripotent or multipotent stem cells. In some embodiments, the stem cells used in the disclosed methods may be derived from human pancreatic cells that have been reprogrammed.
  • Such donor pancreatic cells may come from the subject being treated for diabetes (ie., an autologous donor) or from a person that is not being treated for diabetes (z.e., an allogeneic donor).
  • the stem cells used in the disclosed methods may be reprogrammed primary cells from the islets of Langerhans of consented healthy adult donor pancreata.
  • Primary cells grown in cell culture can become homogenous and lose functional mature traits over time, possibly as a result of adaptation to artificial culture conditions or genetic drift. Accordingly, when primary cells are used as a starting cell population, it may be advantageous to reprogram the primary cells within, for example, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or within 1 day of cell harvest or isolation. For examples, isolated primary cells may be transduced with reprogramming genes within 5 days of cell harvest.
  • a primary cell can be reprogrammed via transduction with Oct4, Sox2, Klf4, and L-Myc.
  • a primary cell can be reprogrammed via transduction with Oct4, Sox2, Klf4, and C-Myc.
  • a primary cell can be reprogrammed via transduction with LIN28, Oct4, Sox2, and Nanog.
  • a primary cell can be reprogrammed via transduction with Glisl, Oct-3/4, Sox2, and Klf4.
  • the cells used in the disclosed differentiation and treatment methods may possess a preference for differentiating toward one germ line over another.
  • a primary cell or stem cell e.g., SR1423
  • SR1423 may efficiently differentiate to ectoderm or endoderm lineages, but be substantially unable to differentiate into a mesodermal lineage. This could be determined by, for example, employing differentiation protocols or kits to push a stem cell toward a specific germ line, yet failing to detect a germ line marker (e.g., OTX2 for ectoderm, Soxl7 for endoderm, or Brachyury for mesoderm).
  • OTX2 for ectoderm
  • Soxl7 for endoderm
  • Brachyury for mesoderm
  • a stem cell or primary cell that preferentially differentiates along an endodermal lineage can be identified by certain molecular markers.
  • a stem cell or primary cell that preferentially differentiates along an endodermal lineage may express markers typical of pluripotency and have a normal karyotype, yet even if markers typical of pluripotency are expressed, the stem cells may be only multipotent, and therefore not fit the accepted criteria for pluripotency.
  • a stem cell or primary cell that preferentially differentiates along an endodermal lineage may also possess a unique gene expression profile.
  • a stem cell or primary cell that preferentially differentiates along an endodermal lineage may down-regulate expression of BHMT2, Cox7Al, and HSPB2 relative to a control level or control cell.
  • a stem cell or primary cell that preferentially differentiates along an endodermal lineage may up-regulate expression of NAP IL 1 relative to a control level or control cell.
  • cells that preferentially differentiate along an endodermal lineage may up-regulate expression of GLIS2, CCDC58, and MTX3 and down-regulate expression of C7orf29 relative to a control cell.
  • Expression levels may be determined by any means known in the art, such as qRT-PCR or microarray analysis, and the control cells used as a standard of comparison may include pluripotent cells that do not exhibit preferential differentiation to the endodermal lineage or a substantial inability to differentiate to the mesodermal lineage, such as the standard embryonic stem cell lines found in the NIH registry. While not being bound by theory, it is believed that at least BHMT2 and NAP IL 1 play roles in DNA modification and may contribute to epigenetic memory.
  • the differential expression of BHMT2, Cox7Al, HSPB2, and/or NAP1L1 may be at least about 1 log, at least about 2 logs, or at least about 3 logs increased (for BHMT2, Cox7Al, and HSPB2) or decreased (forNAPILl) relative to pluripotent cell that does not display preferential differentiation to the endodermal lineage and is not substantially unable to differentiate to the mesodermal lineage, or a stem cell that meets the standard criteria for pluripotency.
  • Identifying a stem cell with the disclosed expression profile indicates a preference for differentiating into an endodermal lineage and thereafter an insulin-producing cell.
  • Direct testing of differentiation preference to specific germ layers increases efficiency of generating cell lines inclined to a particular fate and therefore are suitable for cell-based therapy.
  • Mature endocrine cells of native human islets express the transcription factors Pdxl, ISL1 and Nkx6.1. It is generally believed that Nkx6.1 expression is limited to the insulin- secreting beta cells. For example, in rodents each of these three transcription factors are essential for endocrine pancreatic development. Isolated mature islets containing Pdxl+/Nkx6.1+/Insulin+ beta cells are capable of regulating blood glucose after transplant into insulin-dependent diabetic subjects (Marfil-Garza et al., The Lancet Diabetes & Endocrinology, 10(7): 519-532 (2022)).
  • Pluripotent human stem cells are capable of maturing into pancreatic endocrine cells in the laboratory (Silva et al., Stem cell Research and Therapy, 13:308 (2022)). To verify their authenticity, the gene and protein expression of pluripotent stem cell-derived human pancreatic endocrine cells are benchmarked against mature and developing pancreatic cells of rodents and humans (Alvarez Dominguez et al., Cell, 185(2): 235-249 (2022); Silva et al., Stem cell Research and Therapy, 13:308 (2022); Yoshihara, Frontiers in Cell and Developmental Biology, 10 (2022)).
  • the state-of-the-art describes the main stem cell-derived, anti-diabetes cell type as the insulin-secreting beta cell that progresses along a maturation timeline in which pancreatic cells express Pdxl and Nkx6.1, followed by gain of expression of insulin and C- peptide (a by-product of insulin synthesis) (Pagliuca FW, et al. Cell, 154(2): 428-439 (2014); Velazco-Cruz et al., Stem Cell Reports, 12(2): 351-365 (2019); Hogrebe 2020, Nostro 2015).
  • This order of expression is the same as during native development of islets in rodents and humans, although the timeline of these events, the spatial organization of the developing cells and their cellular environments are vastly different to stem cell-derived islets grown in the laboratory.
  • pancreatic cells that can be implanted to treat diabetes must express Nkx6.1 and Pdxl, and this same literature is silent with respect to ISL1 expression in stem cell- derived islets or pancreatic precursors.
  • Pluripotent stem cell-derived islets that express Nkx6.1 and C-peptide have been found to make and secrete insulin, but at levels that are far lower than native mature pancreatic islets (Fantuzzi et al., Frontiers in Cell and Developmental Biology, 10 (2022); Veres el al., Nature 569: 368-373 (2019)).
  • pluripotent stem cell-derived islets lack potency compared to native islets.
  • cell sources may include, but are not limited to, human embryonic stem cells, induced pluripotent stem cells, non-pluripotent or multipotent reprogrammed cells (e.g., SR1423), and other conventional cell sources known in the art.
  • the disclosed methods of generating isolated insulin-producing SRE cells from stem cells comprise a multi-step process wherein endoderm differentiation is first initiated, followed by differentiation towards the endocrine cell lineage to generate hormone-secreting cells.
  • the hormone-secreting cells then undergo further maturation or adoption of functional endocrine cell characteristics post-transplantation.
  • the endoderm differentiation is typically initiated by contacting the stem cells with an endoderm-inducing agent, such as Activin-A.
  • an endocrine-inducing agent such as retinoic acid and/or a hedgehog antagonist and/or a BMP inhibitor, to further differentiate the cells into the SRE cells.
  • Hedgehog antagonists include SANT1 and cyclopamine, among others.
  • BMP signaling inhibitors include LDN193189, among others.
  • stem cells are cultured in a first medium comprising an endoderm-inducing agent.
  • the endoderm-inducing agent comprises at least Activin-A.
  • the endoderm-inducing agent comprises Activin-A and Wortmannin.
  • the disclosed methods do not employ or include use of an activator of Wnt signaling, such as CHIR-99021 (a small molecule activator of Wnt signaling) and/or the growth factor Wnt3A. Exposure of the stem cells to an endoderm-inducing agent results in differentiation of the cells into endoderm cells.
  • the disclosed differentiation method exposes the cells long-term to retinoic acid (RA).
  • the disclosed differentiation protocol exposes the cells long-term to a Hedgehog antagonist such as cyclopamine or SANT1.
  • the disclosed differentiation protocol exposes the cells long-term to a BMP inhibitor such as LDN193189.
  • the disclosed methods do not employ or include use of tri-iodothyronine (T3).
  • T3 tri-iodothyronine
  • the disclosed methods do not employ a protein kinase C activator or analog thereof.
  • the disclosed methods do not employ nicotinamide, a form of vitamin B3.
  • the disclosed methods do not employ transforming growth factor beta 1 (TGFbetal) protein.
  • the disclosed methods do not employ insulin-like growth factor 1 (IGF-1).
  • the disclosed differentiation protocol does not employ the creation of three-dimensional suspension cultures through dissociation of adhesion cultures and re-aggregation of cells in suspension.
  • the stem cells are differentiated into endoderm cells in the presence of Activin A at a concentration of about 1 to about 200 ng/mL, about 25 to about 175 ng/mL, about 50 to about 150 ng/mL, or about 75 to about 125 ng/mL.
  • the Activin A concentration may be about 1 ng/mL, about 10 ng/mL, about 20 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 160 ng/mL, about 170 ng/mL, about 180 ng/mL, about 190 ng/mL, or about 200 ng/mL.
  • the stem cells are differentiated into endoderm cells in the presence of Wortmannin at a concentration of about 0.1 to about 2.0 pM, about 0.25 to about 1.75 pM, about 0.5 to about 1.5 pM, or about 0.75 to about 1.25 pM.
  • the Wortmannin concentration may be about 0.1 pM, about 0.5 pM, about 1.0 pM, about 1.5 pM, or about 2.0 pM.
  • the medium used to differentiate endoderm cells to SRE cells can comprise KGF, but in some embodiment, the medium used to differentiate endoderm cells to SRE cells can comprise retinoic acid, a Hedgehog antagonist (e.g., cyclopamine and/or SANT1), and a BMP signaling inhibitor (e.g., LDN193189) and combinations thereof without KGF.
  • a Hedgehog antagonist e.g., cyclopamine and/or SANT1
  • BMP signaling inhibitor e.g., LDN193189
  • the step of differentiating endoderm cells to SRE cells may comprise culturing the cells for 1-5 days in a medium comprising retinoic acid, a Hedgehog antagonist (e.g., cyclopamine or SANT1), and a BMP signaling inhibitor (e.g., LDN193189), with or without KGF.
  • a Hedgehog antagonist e.g., cyclopamine or SANT1
  • a BMP signaling inhibitor e.g., LDN193189
  • the cells are differentiated in the presence of retinoic acid for at least twenty (20) days at a concentration of about 0.05 pM, about 0.1 pM, about 0.5 pM, about 1.0 pM, about 1.5 pM, or about 2.0 pM.
  • the cells are differentiated in the presence of Hedgehog antagonist cyclopamine for at least twenty (20) days at a concentration of about 0.05 pM, about 0.1 pM, about 0. pM, or about 0.5 pM.
  • the cells are differentiated in the presence of a chemical analog of cyclopamine SANT-1 ((4-Benzyl-piperazin-l-yl)-(3,5-dimethyl-l-phenyl-lH-pyrazol-4- ylmethylene)-amine) at a concentration of about 0.05 pM, about 0.1 pM, about 0.25 pM, or about 0.5 pM.
  • cyclopamine SANT-1 ((4-Benzyl-piperazin-l-yl)-(3,5-dimethyl-l-phenyl-lH-pyrazol-4- ylmethylene)-amine
  • the cells are differentiated in the presence of LDN193189 at a concentration of about 0.05 pM, about 0.1 pM, about 0. pM, or about 0.5 pM.
  • the endoderm cells are differentiated into SRE cells in the presence of retinoic acid at a concentration of about 1.0 to about 10.0 pM, about 2.0 to about 8.0 pM, or about 3.0 to about 5.0 pM.
  • the retinoic acid concentration may be about 1.0 pM, about 1.5 pM, about 2.0 pM, about 2.5 pM, about 3.0 pM, about 3.5 pM, about 4.0 pM, about 4.5 pM, about 5.0 pM, about 5.5 pM, about 6.0 pM, about 6.5 pM, about 7.0 pM, about 7.5 pM, about 8.0 pM, about 8.5 pM, about 9.0 pM, about 9.5 pM, or about 10.0 pM.
  • the endoderm cells are differentiated into SRE cells in the presence of cyclopamine at a concentration of about 0.1 to about 1.0 pM or about 0.25 to about 0.75 pM.
  • the cyclopamine concentration may be about 0.1 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.9 pM, or about 1.0 pM.
  • the endoderm cells are differentiated into SRE cells in the presence of LDN193189 at a concentration of about 0.1 to about 1.0 pM or about 0.25 to about 0.75 pM.
  • the cyclopamine concentration may be about 0.1 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.9 pM, or about 1.0 pM.
  • the endoderm cells are differentiated into SRE cells in the presence of KGF at a concentration of 1 to about 100 ng/mL, about 25 to about 75 ng/mL, or about 60 to about 70 ng/mL.
  • the KGF concentration may be about 1 ng/mL, about 10 ng/mL, about 20 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, or about 100 ng/mL.
  • the cells are not exposed to KGF until after the cells have been differentiated from endoderm cells to endocrine cells.
  • the SRE cells can be further cultured in the presence of additional growth factors and/or hormones.
  • the SRE cells may be cultured in a medium comprising Noggin, EGF, y-secretase inhibitor XXI, Alk5i II, and/or combinations thereof.
  • the SRE cells may be cultured in a medium comprising Noggin, EGF, y-secretase inhibitor XXI, Alk5i II, and combinations thereof and further comprising retinoic acid and/or cyclopamine (e.g., cyclopamine KAAD).
  • cyclopamine e.g., cyclopamine KAAD
  • the pancreatic progenitor cells may be cultured in the presence of these agents for about 1, about 2, about 3, about 4, or about 5 days, thereby differentiating the pancreatic progenitor cells into a pancreatic lineage.
  • the endoderm cells are differentiated into SRE cells in the presence of Noggin at a concentration of about 1 to about 100 ng/mL, about 25 to about 75 ng/mL, or about 60 to about 70 ng/mL.
  • the Noggin concentration may be about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, or about 100 ng/mL.
  • the SRE cells are further cultured in the presence of EGF at a concentration of about 1 to about 100 ng/mL, about 25 to about 75 ng/mL, or about 60 to about 70 ng/mL.
  • the EGF concentration may be about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, or about 100 ng/mL.
  • the SRE cells are further cultured in the presence of IGF-I at a concentration of about 1 to about 100 ng/mL, about 25 to about 75 ng/mL, or about 60 to about 70 ng/mL.
  • the IGF-I concentration may be about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, or about 100 ng/mL.
  • the stem cells or endoderm cells are differentiated on an adhesive substrate comprised of vitronectin and/or laminin and/or collagen.
  • the stem cells or endoderm cells can spontaneously and naturally form three- dimensional structures are collected and transferred to suspension culture.
  • the stem cells used in the disclosed differentiation method are derived from pancreatic primary tissue. In some embodiments, the stem cells used in the disclosed differentiation method are embryonic stem cells. In some embodiments, the stem cells used in the disclosed differentiation method are induced pluripotent stem cells. In some embodiments, the stem cells used in the disclosed differentiation method are non-pluripotent reprogrammed cells. In some embodiments, the stem cells are human stem cells.
  • reprogramming genes it may be desirable to reprogram cells by expressing reprogramming genes in the cell without incorporating the reprogramming genes into the genome of the cell.
  • transduced genes can be expressed in a cell without incorporating those genes into the genome using, for example, episomal expression plasmids.
  • the reprogramming genes may be expressed on at least 1, at least 2, at least 3, or at least 4 or more episomal expression plasmids.
  • multiple reprogramming genes are known in the art and may be used for the purposed of the disclosed methods, but in some embodiments, the reprogramming genes comprise Oct4, Sox2, Klf4, and L-Myc.
  • the total culturing time required for differentiating cells from a stem cell into an insulin-producing cell may be about 30 days or less.
  • the cells may be cultured for about 30 days, about 29 days, about 28 days, about 27 days, about 26 days, about 25 days, or less.
  • the present disclosure provides a method of producing insulin-secreting pancreatic cells, comprising (a) culturing human stem cells in a first medium comprising Activin-A and Wortmannin, and optionally are not exposed to keratinocyte growth factors (KGF) prior to differentiation into endoderm cells, thereby differentiating the human stem cells into endoderm cells; and (b) culturing the endoderm cells from (a) in a second medium comprising retinoic acid, a Hedgehog antagonist, and a BMP signaling inhibitor and optionally comprising KGF, thereby differentiating the endoderm cells into endocrine cells.
  • KGF keratinocyte growth factors
  • the present disclosure provides a method of producing insulinsecreting SRE cells, comprising (a) culturing human stem cells in a first medium comprising Activin-A and Wortmannin, and optionally are not exposed to keratinocyte growth factors (KGF) prior to differentiation into endoderm cells, thereby differentiating the human stem cells into endoderm cells; and (b) culturing the endoderm cells from (a) in a second medium comprising retinoic acid, a Hedgehog antagonist, and a BMP signaling inhibitor and optionally comprising KGF, thereby differentiating the endoderm cells into SRE cells.
  • KGF keratinocyte growth factors
  • the SRE cells are further matured in a medium wherein the human cells are not exposed to T3.
  • the SRE cells are further matured in a medium wherein the human cells are not exposed to a protein kinase C activator.
  • the SRE cells are further matured in a medium wherein the human cells are not exposed to nicotinamide, a form of vitamin B3.
  • the SRE cells are further matured in a medium wherein the human cells are not exposed to transforming growth factor beta 1 (TGFbetal) protein.
  • TGFbetal transforming growth factor beta 1
  • the SRE cells are further matured in a medium wherein the human cells are not exposed to insulin-like growth factor 1 (IGF-1).
  • IGF-1 insulin-like growth factor 1
  • the total culturing time for steps (a) and (b) may be 30 days or less.
  • the cells may be cultured for about 30 days, about 29 days, about 28 days, about 27 days, about 26 days, about 25 days, or less.
  • step (a) may comprise days 1-3 of culture and step (b) may comprise days 4-27 of culture.
  • the present disclosure provides a method of producing insulinsecreting SRE cells, comprising (a) culturing human stem cells in a first medium comprising Activin-A and Wortmannin, thereby differentiating the human stem cells into endoderm cells; (b) exposing the cells during subsequent culture steps to retinoic acid for at least twenty (20) days; (c) exposing the cells during subsequent culture steps to a Hedgehog antagonist such as cyclopamine or a chemical analog for at least twenty (20) days; (d) exposing the cells during subsequent culture steps to a BMP signaling inhibitor such as LDN193189 for no more than six (6) days; (e) not exposing the cells to Tri-iodothyronine (T3), (f) not exposing the cells to a protein kinase C activator, (g) not exposing the cells to IGF, (h) not exposing the cells to TGFP, (i) not exposing the cells to nicotinamide, (j
  • the present disclosure provides methods of producing mammalian insulin-secreting cells, comprising: culturing mammalian stem cells in adhesion, thereby allowing the mammalian stem cells to spontaneously form three-dimensional structures; and culturing of the three-dimensional structures in suspension; wherein the culturing steps comprise at least a 20-day exposure to retinoic acid, a Hedgehog antagonist, and do not comprise exposing the stem cells of three-dimensional structures to T3, an activator of protein kinase C, IGF, TGFP, or Nicotinamide.
  • the present disclosure provides methods of producing insulinsecreting cells, comprising: culturing mammalian stem cells on an adhesive substrate in a first medium comprising Activin-A and Wortmannin, further culturing the cells in at least one additional medium comprising retinoic acid, a Hedgehog antagonist, and a BMP signaling inhibitor; and transferring the cells to a suspension culture when the cells form three- dimensional cell structures; wherein the cells are exposed to retinoic acid, a Hedgehog antagonist, for at least 20 days.
  • a starting stem cell or non- pluripotent progenitor cell that preferentially differentiates or is predisposed to differentiation to the endoderm lineage. This may allow for simpler differentiation and may achieve a purer and more mature culture of insulin-secreting cells compared to traditional methods.
  • the starting stem cell population is grown in adhesion, while the later stages of culture take place in suspension.
  • the phrases “grown in adhesion” or “cultured in adhesion” refer to standard cell culture wherein cells adhere to the surface of the culture dish.
  • the culture dish may be coated with a substrate to promote adhesion, and in some cases the dish may be given a net positive charge to promote adhesion.
  • culturing of stem cells, such as iPS cells requires an adhesive substrate, and various adhesion-promoting substrates are known in the art.
  • vitronectin or Matrigel can be applied to a cell culture vessel to promote adhesion, but Matrigel is harvested from mouse sarcoma cells and is therefore not preferred for clinical use.
  • differentiation is commenced by culturing the starting stem cell population with an endoderm-inducing media, and grown in adhesion. Formation of 3D structures (e.g., aggregates of differentiated/differentiating cells) on the substrate/plate may occur gradually as differentiation progresses. By day about 15, the 3D structures begin to detach from the plate, and these 3D structures can be transferred into vessels that are not coated with an adhesive substrate (e.g., vitronectin), such that the 3D structures are cultured in a free- floating suspension. This transition, from culturing in adhesion to a suspension culture is novel and allows for a more natural differentiation into an insulin-secreting cell.
  • an adhesive substrate e.g., vitronectin
  • the cells are preferably exposed to retinoic acid, a Hedgehog antagonist, and a BMP signaling inhibitor for at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, or at least about 28 days.
  • this continued exposure to retinoic acid, a Hedgehog antagonist, and a BMP signaling inhibitor may commence after the starting stem cell population has been forced toward an endodermal lineage, for example, after the starting stem cell population has been cultured in the presence of Activin A and wortmannin for about 1, about 2, about 3, about 4, or about 5 days.
  • mammalian stem cells such as human and non-human primate stem cells.
  • additional mammalian cells such as pig, cow, horse, sheep, dog, or cat stem cells may also be differentiated according to the disclosed methods.
  • the disclosed methods can employ various forms of cells culture including, for example, adherent cultures and/or suspension cultures.
  • the disclosed protocols for generating insulin-producing cells enhanced the yield of insulin-producing beta cell progenitor cells from both human embryonic stem cells and reprogrammed pancreatic tissue.
  • the protocol disclosed herein yields near homogeneous populations of insulin-producing cells. Producing a homologous cell population is not only important for therapeutic efficacy, but also for safety, as residual stems cells or other proliferative cells can form teratomas when transplanted and have tumorigenic potential.
  • the high degree of differentiation and homogeneity provided by the disclosed differentiation methods yields fewer cells with tumorigenic potential, which is essential in the development of a useful cell therapy.
  • stem cells that preferentially differentiate into endoderm cell may be identified according to the methods disclosed in Section II of this application. This can increase the overall efficiency of the differentiation process as well as increase the yield of insulin-producing cells.
  • the stem cells may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 99% reduction, or at least 100% reduction of CIITA expression compared to parental stem cells.
  • the expression of CIITA may be silenced. Silenced expression means that no expression can be detected.
  • the reduced expression of CIITA is sufficient to prevent expression of MCH class II.
  • MHC class II trans- activator regulates MHCII transcription.
  • MHCII is a transmembrane heterodimer expressed on phagocytes, dendritic cells, B cells and other immune cells that present extracellular antigens to initiate immune responses. Due to its expression in lymphocytes and its role in immunity, mutations to CIITA are associated with lymphomas and malignancies that are caused by viral infection (Machado and Steimle, Int J Mol Sci. 2021 Jan 22;22(3): 1074).
  • a “hypoimmune” cell is defined herein as a cell that has reduced immunogenicity, and the use of hypoimmune cells thus mitigates the risk of graft rejection or graft-vs-host disease after administration of the cells to a subject in need thereof.
  • CIITA may influence multiple genes in ways that are not yet elucidated. It is only the present disclosure that shows that CIITA has a role in differentiation of pancreatic endocrine cells, and that reducing CIITA expression in stem cells improves their capacity to differentiate towards pancreatic endocrine cells.
  • the present disclosure relates to an isolated synthetic replacement endocrine (SRE) cell obtained by differentiation of the modified stem cell has reduced or abolished expression of major histocompatibility complex (MHC) class II transactivator (CIITA).
  • SRE synthetic replacement endocrine
  • the SRE may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 99% reduction, or at least 100% ,or about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, or about 70% to about 100% reduction of CIITA expression compared to those obtained from parental stem cells with native expression of CIITA.
  • At least about 50%, about 60%, about 80%, about 90%, about 95%, about 99%, about 100%, about 50% to about 100%, about 50% to about 75%, or about 75% to about 99% of the population of SRE cells have at least about 2 fold to 20 fold, about 2 fold to 10 fold, or about 2 fold to 5 fold reduction in expression of MHC class II compared to SRE cells generated from wild type stem cells. In some embodiments, 100% of cells of the population of the SRE cells generated from the modified stem cell that has reduced or abolished expression of CIITA do not have expression of MHC class II molecules.
  • the isolated SRE cell obtained by differentiation of the modified stem cell has reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) expresses somatostatin at lower levels than an SRE cells obtained from a stem cell that expresses CIITA sufficient to induce MHCII expression.
  • MHC major histocompatibility complex
  • CIITA major histocompatibility complex
  • the isolated SRE with reduced CIITA expression have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, or about 70% to about 100% reduction of somatostatin expression compared to a SRE obtained from parental stem cells with native expression of CIITA.
  • At least about 50%, about 60%, about 80%, about 90%, about 95%, about 99%, about 100%, about 50% to about 100%, about 50% to about 75%, or about 75% to about 99% of the population of SRE cells have at least about 1.5 fold to 10 fold, about 1.5 fold to 5 fold, or about 1.5 fold to 3 fold reduction in expression of somatostatin compared to SRE cells generated from wild type stem cells.
  • the isolated SRE cell obtained by differentiation of the modified stem cell has reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) secretes insulin at higher levels than an SRE cells obtained from a stem cell that expresses CIITA sufficient to induce MHCII expression.
  • MHC major histocompatibility complex
  • CIITA major histocompatibility complex
  • SRE cells differentiated from the modified line secreted more insulin into the media in response to glucose exposure than the parent line.
  • the isolated SRE with reduced CIITA expression have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100% increased expression of insulin compared to a SRE obtained from parental stem cells with native expression of CIITA.
  • At least about 50%, about 60%, about 80%, about 90%, about 95%, about 99%, about 100%, about 50% to about 100%, about 50% to about 75%, or about 75% to about 99% of the population of SRE cells have at least about 1.5 fold to 10 fold, about 1.5 fold to 5 fold, or about 1.5 fold to 3 fold increased secretion of insulin compared to SRE cells generated from wild type stem cells.
  • the isolated SRE cell obtained by differentiation of the modified stem cell has reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) expresses glucagon at lower levels than an SRE cells obtained from a stem cell that expresses CIITA sufficient to induce MHCII expression.
  • MHC major histocompatibility complex
  • CIITA major histocompatibility complex
  • the isolated SRE with reduced CIITA expression have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, or about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100% reduction of glucagon expression compared to a SRE obtained from parental stem cells with native expression of CIITA.
  • At least about 50%, about 60%, about 80%, about 90%, about 95%, about 99%, about 100%, about 50% to about 100%, about 50% to about 75%, or about 75% to about 99% of the population of SRE cells have at least about 1.5 fold to 10 fold, about 1.5 fold to 5 fold, or about 1.5 fold to 3 fold reduction in expression of glucagon compared to SRE cells generated from wild type stem cells.
  • the stem cells having reduced or abolished expression of major histocompatibility complex (MHC) class II trans-activator (CIITA) may be obtained by any method known in the art to be able to reduce gene expression or genetically silencing genes.
  • MHC major histocompatibility complex
  • CIITA gene expression can be altered using zinc-finger nucleases (ZFN), transcription activator-like effector nuclease (TALEN), a CRISPR/Cas system or traditional homologous recombination techniques.
  • the CIITA expression can be altered using a regulator such as a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a single-stranded RNA (ssRNA), a double-stranded RNA (dsRNA), an mRNA, a micro-RNA (miRNA), an miRNA mimic, an antisense RNA, a ribozyme, an antisense oligonucleotide, a bifunctional antisense oligonucleotide, a short hairpin RNA (shRNA), an antagomir, an aptamer, a small interfering RNA (siRNA), a dsDNA, a DNAzyme, a ssDNA, polypeptide or active fragment thereof, an antibody, an intrabody, a transbody, a protein, an enzyme, a peptidomimetic, a peptoid, a transcriptional factor, or a small organic molecule.
  • the regulator such as a
  • the SRE cells disclosed herein can be used to treat diabetes in a subject in need thereof.
  • the subject in need of treatment is a mammal, for example, a human subject with insulin-dependent diabetes.
  • the present disclosure provides methods for producing a population of substantially homologous SRE cells, which can be incorporated into a cell-based composition for treating diabetes. Accordingly, provided herein are cell-based compositions for treating diabetes, comprising a population of SRE cells and a suitable carrier for implantation into a human subject in need thereof, wherein at least 66% of the cells are insulin-producing beta pancreatic cells.
  • the cell-based composition may comprise at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% insulin-producing pancreatic cells that express the transcription factors Pdxl and ISL1, and do not express the transcription factor Nkx6.1.
  • Suitable carriers for implanting therapeutic cells are known in the art and may include but are not limited to hydrogels, natural and synthetic polymer scaffolds, extracellular matrix (which may comprise, e.g., collagen, laminin, fibronectin, etc.), hyaluronic acid, biomimetic scaffolds, polylactide (PLA) scaffolds, polyglycolide (PGA) scaffolds, PLA-PGA copolymer (PLGA) scaffolds, as well as hydroxyapatite scaffolds, and macro-porous cryogels.
  • the carrier suitable for transplantation may comprise encapsulating the SRE cells in macro-capsules, such as macro-capsules comprising alginate, cellulose sulfate, glucomannan, or a combination thereof.
  • At least 66% of the SRE cells express Pdxl. In some embodiments, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the SRE cells express Pdxl.
  • At least 66% of the SRE cells express ISLl. In some embodiments, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the SRE cells express ISLl.
  • At least about 68% of the SRE cells do not express Nkx6.1. In some embodiments, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the SRE cells do not express Nkx6.1.
  • At least 66% of the SRE cells express C-peptide. In some embodiments, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the SRE cells express C-peptide.
  • a population of the SRE cells disclosed herein may be incorporated into a cell-based composition as described above.
  • the population may be substantially pure, meaning it includes at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% SRE cells.
  • the SRE cells may be formulated in with one or more therapeutically acceptable carriers.
  • the cell-based compositions for treating diabetes may be prepared according to the methods disclosed herein.
  • the disclosed SRE cells of a cell-based composition may, for example, be derived according to a method comprising contacting a stem cell with a combination of growth factors to drive differentiation of the stem cell to an endocrine lineage, wherein the combination of growth factors comprises retinoic acid, a Hedgehog antagonist, and a bone morphogenetic protein (BMP) signaling inhibitor.
  • the Hedgehog antagonist is selected from SANT1 and cyclopamine.
  • the BMP signaling inhibitor is LDN193189.
  • the stem cell is not exposed to tri-iodothyronine (T3) or analog thereof during the differentiation process.
  • the stem cell is not exposed to a protein kinase C activator during the differentiation process.
  • endoderm-inducing factors are known in the art, including, but not limited to, Activin-A and Wortmannin.
  • various endocrine-inducing factor are known in the art, including, but not limited to, retinoic acid, Hedgehog antagonists such as cyclopamine and SANT1, and BMP signaling inhibitors such as LDN193189.
  • the source of the stem cells used for preparing the disclosed cell-based composition is not particularly limited; however, choosing a cell/cell line that preferentially differentiates into an endodermal lineage, as disclosed herein, may increase the yield of insulin-producing cells and increase differentiation efficiency.
  • the stem cells used in the disclosed differentiation methods for preparing a cell-based composition are derived from pancreatic primary tissue.
  • the stem cells used in the disclosed differentiation method are embryonic stem cells.
  • the stem cells used in the disclosed differentiation method are induced pluripotent stem cells.
  • the stem cells used in the disclosed differentiation method are non-pluripotent reprogrammed cells.
  • the stem cells are human stem cells.
  • the stem cells used in the disclosed differentiation methods are genetically modified to avoid activation of host lymphocytes. In some embodiments, the stem cells used in the disclosed differentiation methods are genetically modified to lack expression of MHC I and MHCII.
  • the stem cells used in the disclosed differentiation methods are genetically modified to avoid targeted destruction by natural killer cells. In some embodiments, the stem cells used in the disclosed differentiation methods are genetically modified to express CD47.
  • reprogramming genes when preparing insulin-producing cells for incorporation into a cell-based composition for treating diabetes, it may be desirable to reprogram cells by expressing reprogramming genes in the cell without incorporating the reprogramming genes into the genome of the cell.
  • transduced genes can be expressed in a cell without incorporating those genes into the genome using, for example, episomal expression plasmids.
  • the reprogramming genes may be expressed on at least 1, at least 2, at least 3, or at least 4 or more episomal expression plasmids.
  • multiple reprogramming genes are known in the art and may be used for the purposed of the disclosed methods, but in some embodiments, the reprogramming genes comprise Oct4, Sox2, Klf4, and L-Myc.
  • the cell-based composition is encapsulated in, for example, micro-capsules or macro-capsules.
  • the present disclosure also provides methods of treating diabetes using the disclosed cell-based compositions.
  • the methods of treating diabetes generally comprise implanting a therapeutically effective amount of the disclosed SRE cells into a subject in need thereof.
  • the therapeutically effective amount of SRE cells may be in the form of a cell-based composition, for instance, a population of SRE cells that are micro-encapsulated or macro-encapsulated.
  • the methods comprise implanting into an individual in need thereof a therapeutically effective amount of insulin-producing cells encapsulated in macro-capsules.
  • the composition of the macro-capsules is not particularly limited and those of skill in the art will understand that various materials can be used to encapsulate insulinproducing cells.
  • the capsules may comprise alginate, cellulose sulfate, glucomannan, or a combination thereof.
  • the macro-capsules may comprise at least one barrier in which the outer barrier is comprised of cellulose sulfate and glucomannan.
  • the macro-capsules may be formed in the shape of a cylindrical tube comprised of an inner capsule of alginate and an outer capsule of cellulose sulfate and glucomannan.
  • the methods comprise implanting into an individual in need thereof a therapeutically effective amount of insulin-producing cells encapsulated in the disclosed macro-capsules about once a year, once every two years, once every three years, once every four years, once every five years, or more.
  • the implanted cells will survive for at least six months after implantation.
  • the subject may require only one implant.
  • the cell-based composition may need to be replaced once every 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 or months, once every 1, 2, 3, 4, or 5 or more years or until the subject has recurring hyperglycemia, or a return to the diabetic state.
  • the cell-based composition can be implanted into the greater omentum of the subject.
  • the greater omentum also known as the great omentum, omentum majus, gastrocolic omentum, epiploon, or caul
  • the greater omentum is a large apron-like fold of visceral peritoneum that hangs down from the stomach and extends from the greater curvature of the stomach back to ascend to the transverse colon before reaching to the posterior abdominal wall.
  • the cell-based composition may be implanted into a pouch formed surgically from the omentum.
  • the cell-based composition is implanted into the peritoneal cavity. In some embodiments, cell-based composition is implanted into the peritoneal cavity and anchored to the omentum. In some embodiments, the cell-based composition is implanted into an omentum pouch.
  • Exemplary doses of insulin-producing cells can vary according to the size and health of the individual being treated. For example, in some embodiments, an exemplary implant of cells encapsulated in the disclosed cell-based composition may comprise 5 million cells to 30 million cells per Kg of body weight.
  • the disclosed methods of treatment can additionally comprise the administration of a second therapeutic in addition to the encapsulated therapeutic cells.
  • the additional therapeutic compound can include, but is not limited to, insulin injections, metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP- 4 inhibitors, GLP-1 receptor agonists, and SGLT2 inhibitors.
  • Particular treatment regimens comprising implanting the cell-based composition comprising insulin-producing cells may be evaluated according to whether they will improve a given patient’s outcome, meaning it will help stabilize or normalize the subject’s blood glucose levels or reduce the risk or occurrence of symptoms or co-morbidities associated with diabetes, including but not limited to, episodes of hypoglycemia, elevated levels of glycosylated hemoglobin (HbAlC levels), heart disease, retinopathy, neuropathy, renal disease, hepatic disease, periodontal disease, and non-healing ulcers.
  • the cell-based composition will be encapsulated, for example, in a capsule comprising alginate, cellulose sulfate, glucomannan, or a combination thereof.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from diabetes, increasing the quality of life of those suffering from diabetes, decreasing the dose of other medications required to treat diabetes, delaying or preventing complications associated with diabetes, and/or prolonging survival of individuals.
  • the subject of the methods is generally a subject with diabetes
  • the age of the patient is not limited.
  • the disclosed methods are useful for treating diabetes across all age groups and cohorts.
  • the subject may be a pediatric subject, while in other embodiments, the subject may be an adult subject.
  • Islet harvest Properly consented and anonymized whole human pancreata were obtained from registered organ donation.
  • the lobes were injected with collagenase P (Roche #1129 002 001) re-suspended to 1.4 mg/ml in islet isolation solution (Hanks Balanced Salt Solution (Invitrogen #14065-056) containing 0.35 g NaHCO3 per L and 1% Human Serum Albumin (Roche A9731)).
  • the inflated lobes were incubated at 37°C for 15-25 min with mild agitation.
  • the digest was diluted with cold islet isolation solution and centrifuged at 1500 RPM for 5 min.
  • the supernatant was discarded, and the pellet was washed in cold islet isolation solution with vigorous trituration.
  • the solution was filtered through a 420 pm sieve (Bellco Glass, Inc, Cat# 1985-00040) and centrifuged.
  • the pellet was re-suspended in 1.100 g/ml Histopaque (Sigma #10771, Sigma #11191) and centrifuged for 30 min at 1200 RPM.
  • the supernatant was collected, diluted 2X in islet isolation solution and centrifuged at 1500 RPM for 5 min.
  • the pellet was rinsed in islet isolation solution, centrifuged, and cultured in E8 medium (Gibco #A1517001) in a humidified incubator at 37°C and 5% CO2.
  • Reprogramming Cells were rinsed with PBS (Gibco #14190144) and incubated in TryplE select IX for 5 min at 37°C. Digestion was arrested with E8 medium and cells centrifuged at 1000 RPM for 5 min. Cells were re-suspended in BTX electroporation solution (VWR #89130-542) at 2xl0 6 cells per 200 pl and added to electroporation cuvette with 20 pg of reprogramming plasmids. Two reprogramming plasmids comprising EBNA episomal expression sequences, ampicillin resistance, and the reprogramming genes Oct4, Sox2, Klf4, and L-Myc under the control of the CMV promoter were constructed.
  • Electroporation cuvette was pulsed using a gene pulser XL (Bio-Rad).
  • Cells were transferred to vitronectin-coated dishes in E6 medium (Life Technologies # al516401) supplemented with 100 ng/ml bFGF (Life Technologies # PHG6015) and 1 pM hydrocortisone.
  • E6 medium Life Technologies # al516401
  • bFGF Life Technologies # PHG6015
  • hydrocortisone 1 pM hydrocortisone
  • Cells were cultured at 37°C in a humidified incubator with 5% CO2. After 24 h, media was changed with E6 supplemented with 100 ng/ml bFGF, and 1 pM hydrocortisone, and 100 pM sodium butyrate (Sigma # P1269) and changed every other day.
  • Stem cell colonies were manually detached and transferred to vitronectin-coated dishes in E8 medium.
  • 73 lines generated from the primary tissue of two donors were initially screened for the ability to express endodermal markers after 4 days exposure to endoderm-inducing agents Activin-A and Wortmannin. Cultures with the highest proportion of cells expressing endodermal markers were selected. Twenty-four cell lines having passed the first screen were subsequently screened for the ability to express pancreatic markers after exposure to a 12-day pancreatic differentiation protocol. The cell line that consistently generated the highest proportion of pancreatic cells was named SR1423 and was banked and used for all subsequent experiments.
  • SR1423 expressed markers typical of pluripotent cells and had a normal karyotype.
  • the DNA STR profile of SR1423 confirmed that it is a single cell line that matches the donor tissue. Additionally, SR1423 grows at a rate typical of pluripotent cell lines. It was observed that other induced pluripotent stem cell (hereinafter called “iPSC) lines from the same donor and reprogramming experiment demonstrated preferential differentiation as well.
  • the iPSC line “B” also differentiated well to endoderm while lines “C” and “D” showed no preference for differentiation to the endodermal lineage (data not shown).
  • Stem cell culture Undifferentiated iPS cells were maintained in 6-well tissue culture plates (Greiner Bio-One #657160) coated with vitronectin XF (Stem Cell Technologies #07180) or 17 pg/cm 2 Geltrex (Life Technologies #A1413301) following manufacturer’s instructions and fed daily with E8 medium. Cultures were passaged at 75-85% confluence every 3-5 days with 0.5 mMEDTA (Life Technologies #15575) and seeded at 7 x 10 3 cells/cm 2 .
  • Undifferentiated adherent cells were exposed to a multi-stage differentiation protocol as follows: Stage 1 (3 days) comprised of a 50:50 mixture of RPMTCMRL supplemented with B27, Activin A and Wortmannin; Stage 2 (2-8 days) comprised of low glucose DMEM supplemented with B27, SANT1, RA, LDN, and KGF; Stage 3 (4 days) comprised of low glucose DMEM supplemented with B27, SANT1, RA, KGF and EGF.
  • stage 3 the three-dimensional cell clusters are transferred to suspension culture;
  • Stage 4 (4 days) comprised of low glucose DMEM supplemented with B27, SANT1, RA, Gsxxi, Alk5i, zinc sulfate, and staurosporine;
  • Stage 5 (4-35 days) comprised of low glucose DMEM supplemented with zinc sulfate.
  • Cells were pelleted and re-suspended in BD Cytofix/Cytoperm and incubated at ambient temperature for 20-30 min. The cells were pelleted in a refrigerated centrifuge at 5°C for 2 min at 2000 RPM. The cell pellet was re-suspended in BD was buffer. The cells were pelleted again and re-suspended in PhosPerm buffer III for 20-30 min on ice. Cells were pelleted again and re-suspended in BD wash buffer. Cells were pelleted again and re-suspended in wash buffer supplemented with fluorophore conjugated primary antibodies or isotype controls.
  • FIG. 1 shows that SRE clusters are comprised of cells that express predominantly Pdxl, ISL1, and insulin. A smaller proportion of cells express glucagon. In contrast Nkx6.1 expressing cells are almost undetectable.
  • Sections on microscope slides were permeabilized in 0.5% Triton-X-100 in PBS for 20 min and rinsed in PBS. Sections were incubated in blocking buffer comprised of 10% horse serum in 0.1% TX-100 in PBS for 1 h. Sections were rinsed in PBS and incubated in primary antibodies diluted in 1% horse serum in 0.1% TX-100 in PBS. All primary antibodies were diluted 1 :00. Sections were incubated at 5°C overnight. Sections were rinsed in PBS and incubated for 2 h, at ambient temperature in secondary antibodies in 1% horse serum in 0.1% TX-100 in PBS.
  • FIG. 2 confirms that SRE clusters are comprised of cells that express predominantly Pdxl and ISL1, and that Nkx6.1 expressing cells are almost undetectable.
  • ISL1 is express in the nuclei of cells that express C- peptide and in the nuclei of cells that express the endocrine pancreas marker chromogranin A. Although Nkx6.1 -expressing cells are nearly absent, the few cells that do express Nkx6.1 do not co-express ISL-1.
  • Clusters were then re-suspended in 16.7 mM glucose for 20 min. This was repeated a second time. Clusters were then re-suspended in GSIS buffer supplemented with 16.7 mM glucose and 30 mM potassium chloride for 20 min. Supernatants were analyzed for C-peptide content using a Mercodia C-peptide ELISA kit and quantified using an absorbance plate reader.
  • FIG. 3 shows that incubation of 50 SRE clusters for 4, 20 min intervals in 2.2 mM glucose demonstrate a gradually decreasing C-peptide secretion. Exposure to 16.7 mM glucose causes increased C-peptide secretion by 40 minutes. Artificially stimulating C- peptide secretion in the presence of 30 mM KC1 shows a high reserve of C-peptide available for secretion.
  • MAD7 is a CRISPR-Cas endonuclease used for targeted deletions to the mammalian genome.
  • the sequence for MAD7 was downloaded from inscripta.com.
  • the MAD7 sequence was synthesized and cloned into backbone plasmid pcDNA3.1 downstream of a CMV promoter.
  • Guide RNAs were designed and synthesized that targeted mutation within the CIITa gene.
  • the MAD7-pCDNA3.1 expression plasmid and the guide RNAs were electroporated into undifferentiated SR1423 cultures. The electroporated cultures were plated onto vitronectin- coated dishes and cultured for 2 days in E8 culture media.
  • the guide RNAs were then introduced to the adherent cultures a second time using Lipofectamine. Cultures were maintained until surviving colonies grew large enough to passage, between five to ten days. During that time, colonies were selected and passaged to individual wells of a 6-well dish coated with vitronectin. Surviving, proliferative clones were cultured until adequate cell numbers were achieved for cry opreservation and PCR. DNA was isolated using ThermoFisher genomic DNA isolation kit. The CIITa gene sequence was amplified by PCR from genomic DNA using flanking primers. The PCR product was sequenced and analyzed for the presence of bi-allelic deletions that cause frameshift mutations.
  • Proteins in the polyacrylamide gel was transferred to a nitrocellulose membrane.
  • the nitrocellulose membrane was probed with an anti-MHCII antibody.
  • the presence of bound antibody was visualized using the ECL chemiluminescent secondary antibody system.
  • MHCII was detected in the parent line SR1423U, but not in clone EL3-14.
  • the EL3-14 clone was named SR1423- HI.
  • Stem cell lines SR1423 and SR1423-HI were differentiated to the pancreatic endocrine phenotype by exposure to a sequence of culture conditions comprising cocktails of growth factors, chemical agents, nutrients and buffers in cell culture media.
  • adherent, undifferentiated cultures were exposed for 3 days to DMEM low glucose/F-12 media supplemented with Activin A, Wortmannin and B27 nutrient mixture with daily media change.
  • Media was exchanged for DMEM low glucose supplemented with insulin-transferrin seleniumethanolamine (ITS-X), B27 nutrient mixture and KGF for two days with daily media change.
  • ITS-X insulin-transferrin seleniumethanolamine
  • the cultures lost adhesion to the culture dish and formed cell clusters.
  • Cell clusters were collected and transferred to suspension cultures.
  • pancreatic endocrine markers Clusters of cells were harvested on day 35, formalin-fixed and cryosectioned onto microscope slides for immunofluorescent analysis. Cluster sections were probed with antibodies that targeted the pancreatic hormones C-peptide (surrogate for insulin), glucagon, somatostatin, pancreatic polypeptide, and ghrelin. Primary antibodies were visualized by exposure to targeted secondary antibodies conjugated to fluorophores and visualized using a fluorescence microscope. Additional marker visualized were chromograninA and the transcription factor ISL1.
  • Detection of C-peptide secretion 50 clusters of differentiated SR1423 and SR1423HI were collected and cultured in bicarbonate buffer for one hour in a humidified tissue culture incubator at 37 degrees Celsius. Buffer was exchanged for 300uL bicarbonate buffer supplemented with 2mM glucose and culture for 20 minutes. Buffer was collected and exchanged for buffer supplemented with 16.7mM glucose for 20 minutes. Buffer was collected and exchanged for buffer supplemented with 2mM glucose for 20 minutes. This was repeated an additional 3 times. Buffer was collected and exchanged for buffer supplemented with 16.7mM glucose for 20 minutes. This was repeated one time. Buffer was exchanged for buffer supplemented with 16.7mM glucose and 30mM potassium chloride (KC1) for 20 minutes. Buffer was collected. All collected buffers were analyzed for C-peptide content in triplicate using an ELISA kit (Mercodia).
  • Raw expression matrices were imported into R and analyzed using Seurat workflows. Low quality cells were removed based on detection of fewer than 500 genes. Next, data were integrated and batch corrected using the reciprocal principal component analysis method in Seurat. Integrated data were visualized in low dimensional space via uniform manifold approximation projection (UMAP). Differential expression analysis between HI and parental cells was calculated via Wilcoxon rank sum tests with p-value adjustment for multiple comparisons. Unsupervised cell type prediction was performed using the Symphony package for R with pre-built adult human pancreas reference dataset provided by the package (Kang et al., Nat Comm, 2021).
  • RNA sequence of pluripotent stem cell-derived cultures showed that differentiation to the pancreatic endocrine phenotype is accompanied by a reduction in expression of the gene CIITa.
  • Previously published single cell RNA-sequencing (scRNA-seq) datasets covering pancreatic progenitor (Stage 3) to final differentiation of scP cells (Stage 6) was accessed via NCBI Gene Expression Omnibus (Accession GSE114412, Veres et al., Nature, 2019). Data were processed according to standard Seurat workflows including data integration and batch correction by reciprocal principal component analysis and dimensionality reduction by UMAP.
  • stage 4B showed a significant reduction in CIITA activity during the differentiation of P cells (stage 7) from pancreatic progenitors (stage 4) in vitro based on analysis of scRNA- seq data across differentiation of P cells from a different human ES cell line obtained from Weng et al., Nat Metabolism, 2020. These data showed that CIITA activity is downregulated during P cell differentiation.
  • pancreatic islets derived from SR1423-HI stem cells had increased expression of insulin, and decreased expression of glucagon and somatostatin compared to those obtained from SR1423 stem cells as shown in FIG. 7 and Table 1 below.
  • pancreatic islet hormone genes showed that delta and gamma assigned parental cells express insulin and somatostatin suggesting they are polyhormonal, a hallmark of immaturity. Moreover, parental line cells express higher levels and frequency of FEV, a marker of immature P cells. Interestingly, expression of glucagon, somatostatin and FEV was essentially absent in HI with silenced or reduced CIITA expression as shown in FIG. 8A, suggesting SR1423-HI cells with silenced or reduced CIITA expression generates a purer population of in vv/ra-derived P cells.
  • An adult human subject with insulin-dependent diabetes receives a transplant comprising a therapeutically effective amount of a composition comprising the disclosed SRE cells into the subject’s omentum pouch or peritoneal cavity.
  • the subject is evaluated for blood glucose levels.
  • the subject is monitored following the implant of a therapeutically effective number of SRE cells to ensure that the subject’s blood glucose levels have been stabilized.
  • the subject is further screened for glycosylated hemoglobin, and co-morbidities of diabetes over time.

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Abstract

La présente divulgation concerne une cellule souche modifiée utile pour générer des compositions à base de cellules afin de traiter le diabète, ainsi que des méthodes de préparation de telles cellules.
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WO2014004341A1 (fr) 2012-06-26 2014-01-03 Seraxis, Inc. Cellules souches et cellules pancréatiques utiles pour traiter le diabète sucré insulino-dépendant
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