WO2025221594A1 - Methods for generating dopaminergic neurons and organoids thereof - Google Patents

Abstract

Disclosed herein are methods for generating dopaminergic neurons and organoids comprising thereof. Particularly, the disclosure relates to methods for generating dopaminergic neurons by differentiating stem or precursor cells into Achaete-scute family bHLH transcription factor 1 (Ascl1)-driven mesenchymal progenitor cells and then terminally differentiating the Ascl1-driven mesenchymal progenitor cells into dopaminergic neurons alone or alongside astrocyte and hematopoietic precursor cells.

Description

METHODS FOR GENERATING DOPAMINERGIC NEURONS AND ORGANOIDS THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/634,506, filed April 16, 2024, the content of which is herein incorporated by reference in its entirety.

FIELD

[0002] The present disclosure relates to methods for generating dopaminergic neurons and organoids comprising thereof. Particularly, the disclosure relates to methods for generating dopaminergic neurons by differentiating stem or precursor cells into achaete-scute family bHLH transcription factor 1 (Ascll)-driven mesenchymal progenitor cells and then terminally differentiating the Ascii -driven mesenchymal progenitor cells into dopaminergic neurons alone or alongside astrocyte and hematopoietic precursor cells.

BACKGROUND

[0003] Human induced pluripotent cells have been successfully used to derive different cell types of the brain to mimic and study human neuronal network the complexity of which cannot be sufficiently replicated using animal models or animal-based cell culture systems. Neurons have been traditionally developed in vitro from human embryonic or induced pluripotent stem cells by providing patterning molecules in the culture medium that can signal the stem cells to express genes involved in neuronal differentiation. Moreover, exposing stem cells to specific ligands triggers precise developmental pathways involved in the generation of progenitor cells residing in specific brain regions, capable of differentiating into both neurons and glia.

Prolonged exposure to neurotrophic growth factors further enhances the differentiation and maturation of neurons in culture. The process can be time-consuming and can often result in a mixed population of neurons, progenitors, and glial cells.

[0004] An approach to rapidly produce large-scale high quantity neurons by directly overexpressing global neuronal transcription factor, Neurogenin 2 (NGN2) in human eSCs or iPSc, called induced neurons (or iNs) has vastly overcome the shortcomings of former method. This method has been further manipulated by overexpressing different transcription factors or combination of transcription factors to potentially develop specific neuronal types. Further, transcription factor overexpression techniques have been reported to vastly produce heterogenous neuronal population often representing the peripheral nervous system as a result of a lack of regional specification. Although the process is rapid, the percentage of dopaminergic neurons is still lower than those produced via traditionally patterned dopaminergic neurons.

SUMMARY

[0005] Disclosed herein are methods for generating dopaminergic neurons. In some embodiments, the methods comprise differentiating stem or precursor cells into mesenchymal progenitor cells; inducing expression of Achaete-scute homolog l(Ascll) gene in the mesenchymal progenitor cells to generate Ascii -driven mesenchymal progenitor cells; and differentiating the Ascii -driven mesenchymal progenitor cells to dopaminergic neurons. In some embodiments, the stem or precursor cells are induced pluripotent stem cells (iPSCs).

[0006] In some embodiments, differentiating the stem or precursor cells comprises: contacting the stem or precursor cells in cell culture with one or more patterning factors over a first period of time; and one or more midbrain-specific neurotrophic and pro-survival factors for a second period of time. In some embodiments, the second period of time follows the first period of time at least partially overlaps with the end of the first period of time. In some embodiments, the first period of time is about 10 to about 15 days. In some embodiments, the second period of time is about 1 to about 4 days.

[0007] In some embodiments, the one or more patterning factors are selected from Small Mothers Against Decapentaplegic (SMAD) inhibitors, sonic hedgehog (SHH) pathway activators, Wnt signaling activators, Fibroblast growth factor 8 (FGF8) signaling activators, and combinations thereof.

[0008] In some embodiments, the concentration of each of the one or more the patterning factors may increase, decrease, or remain constant over the first and/or second period of time. In some embodiments, the one or more Wnt signaling activators are increased after the start of the first period of time. In some embodiments, any or all of the SMAD inhibitors and SHH pathway activators are removed in a later portion of the first period of time. In some embodiments, the one or more FGF8 signaling activators are added in a later portion of the first period of time. [0009] In some embodiments, the neurotrophic and pro-survival factors comprise brain- derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, transforming growth factor beta-3 (TGF03), dibutyryl-cAMP, or any combination thereof. [0010] In some embodiments, the methods further comprise contacting the stem or precursor cells with a cell death inhibitor for any or all of the first and/or second period of time. [0011] In some embodiments, the methods further comprise transfecting the stem or precursor cells with a nucleic acid encoding the Ascii gene operably linked to an inducible promoter. In some embodiments, inducing the expression of the Ascii gene comprise contacting the mesenchymal progenitor cells with an inducing agent.

[0012] In some embodiments, differentiating the Ascii -driven mesenchymal progenitor cells to dopaminergic neurons comprises contacting the Ascii -driven mesenchymal progenitor cells for a third period of time with one or more of: neurotrophic and maturation factors and Notch pathway inhibitors. In some embodiments, the third period of time is one day to one month.

[0013] In some embodiments, the one or more neurotrophic and maturation factors comprise brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, transforming growth factor beta-3 (TGF|33), dibutyryl-cAMP, or any combination thereof.

[0014] In some embodiments, greater than 80% of cells generated by the method are terminally differentiated into dopaminergic neurons. In some embodiments, greater than 95% of cells generated by the method are terminally differentiated into dopaminergic neurons.

[0015] In some embodiments, the dopaminergic neurons, or any stem or precursor cell thereof, comprise a disease-associated genetic abnormality. In some embodiments, the disease is a neurological or neurodegenerative disease or disorder. In some embodiments, the dopaminergic neurons, or any stem or precursor cell thereof, comprise a Parkinson’s disease associated mutation.

[0016] Also disclosed herein are methods for generating three-dimensional midbrain assembled organoid comprising patterned Ascii -driven dopaminergic neurons. In some embodiments, the methods comprise contacting a first population of cells comprising Ascll- driven mesenchymal progenitor cells and iPSC-derived astrocyte precursor cells (APCs) with one or more neurotrophic factors to differentiate the Ascii -driven mesenchymal progenitor cells and the APCs and form aggregates; adding iPSC-derived hematopoietic precursor cells (HPCs) to the aggregates to generate a second population of cells; contacting the second population of cells with a microglial differentiation stimulating agent; and incubating the second population of cells to form assembled three-dimensional organoids comprising patterned Ascii -driven dopaminergic neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia. [0017] In some embodiments, the Ascii -driven mesenchymal progenitor cells are in excess to the APCs in the first population of cells. In some embodiments, the Ascii -driven mesenchymal progenitor cells and the APCs are at about a 10:1 ratio in the first population of cells. In some embodiments, the HPCs are added at an about equal ratio to the APCs in the first population of cells.

[0018] In some embodiments, one or more of the Ascii -driven mesenchymal progenitor cells, iPSC-derived APCs, and iPSC-derived HPCs comprise a disease-associated genetic abnormality. [0019] In some embodiments, the Ascii -driven mesenchymal progenitor cells are generated by: contacting induced pluripotent stem cells (iPSCs) with one or more patterning factors over a first period of time, and one or more midbrain-specific neurotrophic and pro-survival factors for a second period of time; and inducing expression of Achaete-scute homolog 1 (Ascii) gene.

[0020] In some embodiments, the second period of time follows the first period of time or partially overlaps with the end of the first period of time. In some embodiments, the first period of time is about 10 to about 15 days. In some embodiments, the second period of time is about 1 to about 4 days.

[0021] In some embodiments, the one or more patterning factors are selected from SMAD inhibitors, SHH pathway activators, Wnt signaling activators, FGF8 signaling activators, and combinations thereof. In some embodiments, the concentration of each of the one or more the patterning factors may increase, decrease, or remain constant over the first and/or second period of time. In some embodiments, one or more Wnt signaling activators are increased after the start of the first period of time. In some embodiments, any or all of the SMAD inhibitors and SHH pathway activators are removed in a later portion of the first period of time. In some embodiments, the one or more FGF8 signaling activators are added in a later portion of the first period of time.

[0022] In some embodiments, the methods further comprise contacting the stem or precursor cells with a cell death inhibitor for any or all of the first and/or second period of time.

[0023] In some embodiments, the microglial differentiation stimulating agent comprises one or more cytokines.

[0024] In some embodiments, the Ascii -driven dopaminergic neurons, midbrain iPSC- derived astrocytes, and iPSC-derived microglia are at about a 10: 1 : 1 ratio in the three- dimensional midbrain assembled organoid. [0025] Further disclosed herein are compositions of cells comprising a population of patterned Ascii -driven dopaminergic neurons. In some embodiments, the population of patterned Ascii -driven dopaminergic neurons is greater than 80% of total cells in the composition. In some embodiments, the population of patterned Ascii -driven dopaminergic neurons is greater than 95% of total cells in the composition.

[0026] In some embodiments, all cells in the composition are neurons.

[0027] In some embodiments, the composition is a three-dimensional midbrain assembled organoid.

[0028] Additionally disclosed are three-dimensional midbrain assembled organoid comprising patterned Ascii -driven dopaminergic neurons. In some embodiments, the organoids further comprise midbrain iPSC-derived astrocytes and iPSC-derived microglia. In some embodiments, the Ascii -driven dopaminergic neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia are at about a 10: 1 : 1 ratio in the organoid.

[0029] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIGS. 1 A-1E show the characterization of the differentiation of iPSCs into dopaminergic neurons. FIG. 1 A is a schematic depicting the timeline to differentiate iPSCs into floor plate mesenchymal progenitor cells in 11 days (in blue), followed by Ascii expression from Day 12 onwards (in light green) for terminal differentiation (in dark green) up to 14 days. FIG. IB is representative images of 7-day old dopaminergic neurons immunostained with substantia nigral A9 specific markers, ALDH1 Al (green; upper panel) and SOX6 (red; upper panel); and dopaminergic markers, Nurrl (Magenta; upper panel), TH (green; lower panel) and Alpha Synuclein (ASyn, Magenta; lower panel). FIG. 1C is a plot depicting -80% cell positive for TH and over 90% cells positive for Sox6 and Nurrl. FIG. ID is representative images of 14- day old dopaminergic neurons showing immunostaining against transporters of dopamine VMAT2 (red) and DAT (green), as well as Alpha Synuclein (ASyn, Magenta). FIG. IE is graphs of VMAT2 in Day 14 versus Day 21 dopaminergic neurons. Scale bar 10pm.

[0031] FIGS. 2A and 2B are images showing Ascii is sufficient to differentiate dopaminergic neurons from prepattemed iPSCs. FIG. 2 A is shows Day 9; FIG. 2B is Day 15 showing TH (green), MAP2 (red) with DAPI and Trans as labeled. Scale bar 10pm. [0032] FIG. 3A shows patterning followed by Ascii expression provides A9-midbrain specificity by expression of midbrain-specific transcription factors (Sox6, ALDH1A1, Enl, LMX1, FoxA2 and Nurrl, as labeled), and only low expression of AlO-marker CALB1. Scale bar 10pm. FIG. 3B is a plot depicting the high percentage of A9 midbrain markers (SOX6, ALDH1 Al, Enl, LMX1, Nurrl and FOXA2) and low percentage of A10 marker, CALB1.

[0033] FIGS. 4A and 4B show generation of high percentage (-80%) TH-positive dopaminergic neurons over Days 2, 7 and 14. Scale bar 10pm.

[0034] FIGS. 5 A and 5B are representative images of a 12 days old 3D assembled organoid containing TH-positive iPSC-derived A9-like patterned DA neurons (green), and GFAP-positive iAstrocytes (red) in ratio of 10:1 (FIG. 5A), or P2RY12-positive iMicroglia (red) and GFAP- positive iAstrocytes (Magenta) in the ratio of 10:1 :1 (FIG. 5B). Scale bar 50pm.

[0035] FIG. 6 shows representative images of high yield TH-positive neurons in 3D assembled organoids that mimic cell-autonomous and non-cell autonomous pathways in human SNc and show A9-midbrain specificity by expression of A9-midbrain-specific transcription factors (Sox6, Enl, LMX1, FoxA2 and Nurrl, as labeled), low levels of A10 marker (CALB1), and high levels of DA maturation markers (VMAT2, A-synuclein). Scale bar 50pm.

[0036] FIG. 7 A and 7B show depletion of TH-positive dopaminergic neurons (7 -day old) in

Parkinson’s Disease associated mutants: A-synuclein A53T (late onset Parkinson’s) and Synaptojanin-1 R219Q (juvenile and early onset Parkinson’s).

[0037] FIGS. 8A-8C show loss of TH-positive dopaminergic neurons in 12 day old mutant (as indicated) midbrain 3D organoids generated with iAstrocytes.

DETAILED DESCRIPTION

[0038] Disclosed herein are methods for rapidly generating dopaminergic neurons in vitro. Prior methods can suffer from being time-consuming (greater than 50 days for differentiated dopaminergic neurons) and labor-intensive due to slow maturation which results in only mid- to high-yield (50-80%) TH-positive neurons in a mixed population with astrocytes, radial glial cells, and progenitors. Rapid alternatives exist, but generate a low yield (20-50%) of TH-positive neurons with non-neuronal cell differentiation. The methods described herein generate a high percentage (>80%) of dopaminergic neurons in a fairly efficient time period.

[0039] The methods combine midbrain specification and achaete-scute family bHLH transcription factor 1 (Ascii) expression to rapidly generate high-quality and high-percentage mature A9 SNc dopaminergic neurons. The patterning process utilizes cell medium supplementation with midbrain-specific effectors and ligands modulating developmental pathways such as sonic hedgehog, Wnt, FGF8, for a short period of time to generate floor plate mesenchymal progenitor cells. The differentiation of A9 SNc dopaminergic neurons is then enhanced by inducing expression of Ascii.

[0040] Also shown herein are A9-specific 3D midbrain assembled organoids containing patterned Ascii -driven DA neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia and methods of generating thereof. The organoids described herein are generated having precise ratios of the neurons, astrocytes and microglia that reflect the ratios present in the human midbrain rather than a heterogeneous collection of cell types which do not often represent midbrain cell constitutions unlike published methods for organoid generation. The disclosed organoids show enhanced maturation of TH-positive DA neurons that express A9 markers such as SOX6 and ALDH1 Al, and distinct morphology of GF AP -positive astrocytes as well as P2RY 12-positive microglia. These defined midbrain-specific organoids facilitate investigation of the effects of neuroinflammation, oxidative and proteostatic stress on DA neuronal health and survival. For example, these organoids can be used to study neurodegenerative causing genetic abnormalities, such as those in Parkinson’s disease, that cause proteostatic failure in subtypes of human DA neurons.

[0041] These methods for generating DA neurons and related organoids provide a platform to perform large-scale screens (e.g., CRISPR screens) and drug discovery.

[0042] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

1. Definitions

[0043] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s) ,” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. [0044] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, e.g., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0045] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of’ or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (e.g., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of’ “only one of’ or “exactly one of.”

[0046] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0047] The term “stem cell” refers to a cell that retains the ability to renew itself through mitotic cell division and that can differentiate into a diverse range of specialized cell types. Mammalian stem cells can be divided into three broad categories: embryonic stem cells, which are derived ftom blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be derived, for example, from adult somatic cells such as peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes.

[0048] A “progenitor cell” is a cell that, like a stem cell, has the ability to differentiate into a specific type of cell, with limited options to differentiate, with usually only one target cell. A progenitor cell is usually a unipotent cell, it may also be a multipotent cell.

[0049] The term “differentiated cell” refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) in a cellular differentiation process.

[0050] As used herein, the term “somatic cell” refers to any cells forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as “gametes”) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body — apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells — is a somatic cell type: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a “non-embryonic somatic cell,” by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell” by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro. [0051] The term “organoid” refers to a three-dimensional organ-bud grown in vitro and in isolation from an intact organism.

[0052] As used herein, the term “contacting” is intended to include incubating agent to which the action refers and the cell together (e.g., adding to the cell culture medium or to cells in culture). The step of contacting can be conducted in any suitable manner. For example, the cells may be treated in adherent culture, or in suspension culture. It is understood that the cells contacted with a cell culture medium and/or agent can also be simultaneously or subsequently contacted with another agent, such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further.

[0053] The terms “cell culture medium,” “culture medium,” and “medium” refer to any media for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art.

[0054] The term “operably linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence.

[0055] A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA.

[0056] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, a patient may include either adults or juveniles (e.g., children).

Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs, and the like. Examples of nonmammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0057] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0058] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

2. Dopaminergic Neurons

[0059] Embodiments of the present disclosure include methods for generating dopaminergic neurons. The methods comprise differentiating stem or precursor cells into mesenchymal progenitor cells; inducing expression of Achaete-scute homolog l(Ascll) gene in the mesenchymal progenitor cells to generate Ascii -driven mesenchymal progenitor cells; and differentiating the Ascii -driven mesenchymal progenitor cells to dopaminergic neurons. [0060] Any stem or precursor cell that can be made to differentiate into neurons is suitable for the disclosed methods. In select embodiments, the stem or precursor cells are induced pluripotent stem cells (iPSCs). In some embodiments the iPSCs are derived from somatic cells obtained from a subject. Thus, the iPSCs may be subject-derived iPSCs. The somatic cells may include, without limitation, peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, and hepatocytes, and other cell types capable of generating patient-derived IPSCs that can be differentiated into mature neurons.

[0061] In some embodiments, the subject is healthy. In some embodiments, the subject has a diseases and disorder relating to the brain, such as central nervous system (CNS) disorders, neurological disorders, neurodegenerative diseases. In some embodiments, the subject has at least one genetic abnormality (e.g., mutation, deletion, substitution) associated with a neurological or neurodegenerative disease or disorder. In some embodiments, the subject has at least one associated genetic abnormality associated with dopaminergic neuron dysfunction.

[0062] Alternatively, disease-associated genetic abnormalities (e.g., mutations associated with a neurological or neurodegenerative disease or disorder) can be introduced into the iPSCs, or precursors thereof, through known genetic engineering techniques (e.g., techniques utilizing CRISPR/Cas systems, zinc finger nucleases (ZFN), and transcription activator-like effector nucleases (TALENs)).

[0063] Neurological disorders may be any disease affecting neuronal network connectivity, synaptic function and activity. A neurodegenerative disease refers to a central nervous system disease characterized by progressive, normally gradual, loss of functional neural tissue. Nonlimiting examples of neurodegenerative diseases include frontotemporal dementia (FTD), Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Multiple sclerosis, Niemann-Pick disease, Huntington's disease, transmissible spongiform encephalopathy, Charcot-Marie-Tooth disease, dementia with Lewy bodies (DLB), corticobasal degeneration, progressive supranuclear palsy, Bell’s palsy, neuronal ceroid lipofuscinoses, and hereditary spastic paraparesis.

[0064] In select embodiments, the iPSCs, or precursors thereof, comprise a genetic abnormality (e.g., mutation, deletion, substitution) associated with Parkinson’s Disease (PD). One or more genetic mutations associated with Parkinson’s disease include mutations in genes including, but not limited to, SNCA, PARK3, UCHL1, LRRK2, GIGYF2, HTRA2, EIF4G1, TMEM230, CHCHD2, RIC3, VPS35, PRKN, PINK1, PARK2, PARK7, PARK10, PARK12, PARK16, ATP13A2 (PARK9), PLA2G6, FBX07, DNAJC6, SYNJ1, and VPS13C.

[0065] The methods comprise differentiating stem or precursor cells into mesenchymal progenitor cells. In some embodiments, differentiation of the stem or precursor cells into mesenchymal progenitor cells comprises contacting the stem or precursor cells with one or more patterning factors over a first period of time; and one or more midbrain-specific neurotrophic and pro-survival factors for a second period of time. For example, the one or more patterning factors and/or the one or more midbrain-specific neurotrophic and pro-survival factors may be added to media in which the cells are being cultured. Alternatively, the media in which the cell are being cultured may be fully or partially exchanged with media comprising the one or more patterning factors and/or the one or more midbrain-specific neurotrophic and pro-survival factors. In either method, the cells are contacted and exposed to the one or more patterning factors and/or the one or more midbrain-specific neurotrophic and pro-survival factors.

[0066] The first period of time may be from about 10 to about 15 days. In some embodiments, the first period of time is at least about 11 days. For example, in some embodiments, the first period of time is about 10, about 11, about 12, about 13, about 14, or about 15 days. In some embodiments, following the first period of time the cells will form rosettes as an indication of patterning.

[0067] The second period of time may range from about 1 to about 4 days. In some embodiments, the second period of time is about 1, about 2, about3 or about 4 days. In exemplary embodiments, the second period of time is up to 2 days.

[0068] The first period of time and the second period of time may be consecutive or fully or partially overlapping. In some embodiments, the second period of time follows the first period of time. In some embodiments, the second period of time at least partially overlaps with the first period of time. In select embodiments, the second period of time at least partially overlaps with the end of the first period of time.

[0069] In some embodiments, the one or more patterning factors are selected from SMAD inhibitors, sonic hedgehog (SHH) pathway activators, Wnt signaling activators, Fibroblast growth factor 8 (FGF8) signaling activators, and combinations thereof. [0070] Exemplary SMAD inhibitors include, but are not limited to: A-77-01, A-83-01, BT173, Dorsomorphin, EW-7197, FK506-binding protein 12 (FKBP12), GW788388, Heparins, IN-1130, Isorhamnetin, K02288, LDN193189, LY2109761, LY2157299, LY3200882, LY364947, Momelotinib, Pirfenidone/fluorofenidone (PFD/FD), Progesterone, R-268712, RepSox, SB431542, SB-505124, SB-525334, SD-208, SIS3, Testosterone, transmembrane serine protease 6 (TMPRSS6), TP-0427736, and Valproic acid. In some embodiments, the SMAD inhibitors comprise SB431542 and LDN193189.

[0071] SHH pathway activators may be a recombinant SHH polypeptide, or active portion or variant thereof, and small molecule activators. Exemplary sonic hedgehog (SHH) pathway activators include, but are not limited to: SHH protein, purmorphamine, or an analog thereof (e.g., SAG, 3-chloro-N-[(lr,4r)-4 (methylamino)cyclohexyl]-N-[3-(pyridin-4- yl)benzyl]benzo[b]thiophene-2-carboxamide). In some embodiments, the SHH pathway activator comprises an SHH protein, or active portion or variant thereof and/or purmorphamine.

[0072] Wnt signaling activators include Wnt ligands, Wnt proteins, Wnt mimics, R-spondin (RSPO), Norrin, and inhibitors of known Wnt inhibitors. Exemplary Wnt signaling activators include, but are not limited to: TWS119, IQ-1, arylindolemaleimide (SB-216763), anilinomaleimide (SB-415286), CHIR99021, L807mts, Riluzole, BAY 36-7620, LY2090314, WAY-316606, ABC99, QS11, BIO(6-bromoindirubin-3'-oxime), and DCA.

[0073] Exemplary Fibroblast growth factor 8 (FGF8) signaling activators, includes recombinant Fibroblast Growth Factor 8b (FGF8b).

[0074] The concentration of each of the one or more patterning factors may be modulated over the first period of time. For example, for any given patterning factor, the concentration may increase or decrease over time, may start at an initial concentration and be increased or decreased to one or more subsequent concentrations at certain intervals over the first period of time, or may be removed for a portion of the first period of time. In certain embodiments, one or more Wnt signaling activators start at an initial concentration and are then increased to a second concentration for the remainder of the first period of time. For example, after 2-4 days the concentration of the Wnt signaling activators being contacted with the cells in increased 3 to 5 fold to a second concentration for the remainder of the first and/or second period of time. In certain embodiments, any or all of the SMAD inhibitors and SHH pathway activators are removed in a later portion of the first period of time. For example, after 6-9 days the SMAD inhibitors and SHH pathway activators are no longer contacted with the cells for the remainder of the first and/or second period of time. In certain embodiments, any or all of FGF8 signaling activators are added in a later portion of the first period of time, for example after removal of the SMAD inhibitors and SHH pathway activators.

[0075] The one or more neurotrophic and pro-survival factors may comprise brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, transforming growth factor beta-3 (TGF 3), dibutyryl-cAMP, or any combination thereof.

[0076] In some embodiments, differentiating stem or precursor cells into mesenchymal progenitor cells may further comprise contacting the stem or precursor cells with a cell death inhibitor for any or all of the first and/or second period of time. Cell death inhibitors can be used to confer a pro-survival effect after dissociation of the stem or precursor cells from their typical colony formation to single cells during culture and differentiation into the desired cell type. Cell death inhibitors can assist in biasing the differentiation potential in the absence of a physical microenvironment changes. In select embodiments, a cell death inhibitor may be added after the start of the first period of time and be included in the media for the later portion of the first period of time and all of the second period of time. In some embodiments, the cell death inhibitor is a Rho-associated protein kinase (ROCK) inhibitor.

[0077] Following generation of mesenchymal progenitor cells, the methods comprise inducing expression of the Achaete-scute homolog 1 (Ascii) gene in the mesenchymal progenitor cells to generate Ascii -driven mesenchymal progenitor cells.

[0078] In some embodiments, the stem or precursor cells include a nucleic acid encoding the Ascii gene operably linked to an inducible promoter. Thus, the expression of the Ascii gene can be induced by activating the inducible promoter. Promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter/regulatory sequence capable of driving expression of the Ascii gene operably linked thereto. In select embodiments, inducing the expression of the Ascii gene comprise contacting the mesenchymal progenitor cells with an inducing agent to activate the inducible promoter. The inducing agent may be added to and replenished in media in which the cells are being cultured. The inducing agent may be supplemented to the media for any length of time after the initial induction, e.g., to continue Ascii expression during differentiation to dopaminergic neurons during any or all of the third period of time.

[0079] The methods comprise differentiating the Ascii -driven mesenchymal progenitor cells to dopaminergic neurons. In some embodiments, the dopaminergic neurons are mature A9 SNc dopaminergic neurons. In some embodiments, differentiating the Ascii -driven mesenchymal progenitor cells to dopaminergic neurons comprises contacting the Ascii -driven mesenchymal progenitor cells for a third period of time with one or more of: neurotrophic and maturation factors and Notch pathway inhibitors. The third period of time may be from one day to one month.

[0080] The one or more neurotrophic and maturation factors may comprise brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, transforming growth factor beta-3 (TGFJ33), dibutyryl-cAMP, or any combination thereof.

[0081] Exemplary Notch pathway inhibitors include, but are not limited to: DAPT, LY-

411575, JI130, YO-01027, RO4929097, Limantrafin, Tangeretin, Carvacrol, RBPJ Inhibitor-1, Crenigacestat, IMR-1, Semagacestat, Psoralidin, Bruceine D, BMS -906024 (AL 101), FLI-06, Avagacestat, JI051, ZLDI-8, BMS-983970, LY900009, Z-Ile-Leu-aldehyde, BMS-986115, ASR-490, IMR-1 A, SAHM1, BT-GSI, MRK-560, andMRK-003. In select embodiments, the Notch pathway inhibitor comprises DAPT.

[0082] As described above for the one or more patterning factors, the concentration of each of the neurotrophic and maturation factors and Notch pathway inhibitors may be modulated over the third period of time as necessary to successfully produce a high-percentage of dopaminergic neurons. In some embodiments, a constant concentration of the neurotrophic and maturation factors and Notch pathway inhibitors is maintained in the culture by feeding or replenishing the cell culture media with the selected neurotrophic and maturation factors and Notch pathway inhibitors.

[0083] In some embodiments, a cell death inhibitor, as described elsewhere herein, is added with the initial contact of the neurotrophic and maturation factors and Notch pathway inhibitors but then subsequently removed for the remainder of the third period of time.

[0084] Progression to dopaminergic neurons can be monitored by expression of tyrosine hydroxylase (TH), an enzyme involved in dopamine production, substantia nigra specific genes such as SOX6 and Nurrl , dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2) and a-synuclein. Monitoring the progression can allow one to determine when during the third period of time the method has resulted in a high percentage of dopaminergic neurons. In some embodiments, the method results in 80% of cells generated being terminally differentiated into dopaminergic neurons. For example, after a period of 7 days following differentiating the Ascii -driven mesenchymal progenitor cells to dopaminergic neurons greater than approximately 80% of cells are terminally differentiated into dopaminergic neurons. In some embodiments, greater than 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the cells generated are differentiated into dopaminergic neurons after more than 7 days. In some embodiments, greater than 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the cells generated are differentiated into dopaminergic neurons after more than 14 days.

[0085] In some embodiments, the method further comprises introducing one or more genetic abnormalities into the differentiated dopaminergic neurons. In some embodiments, the genetic abnormality is a disease-associated genetic abnormality. In some embodiments, the disease- associated genetic abnormality is associated with a neurological or neurodegenerative disease or disorder, as described above. In some embodiments, the disease-associated genetic abnormality is associated with Parkinson’s Disease. In some embodiments, the disease-associated genetic abnormality is in a gene selected from the group including SNCA, PARK3, UCHL1, LRRK2, GIGYF2, HTRA2, EIF4G1, TMEM230, CHCHD2, RIC3, VPS35, PRKN, PINK1, PARK2, PARK7, PARK10, PARK12, PARK16, ATP13A2 (PARK9), PLA2G6, FBX07, DNAJC6, SYNJ1, and VPS13C.

[0086] Methods for preparing and culturing stem and precursor cells, specifically, and cells in general can be found in standard textbooks and reviews in cell biology, tissue culture, and embryology.

[0087] Culture vessels suitable for use for culturing the cell(s) include, but are not limited to: flask, flask for tissue culture, spinner flask, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK@ Chambers, culture bag, and roller bottle, as long as it is capable of culturing the cells therein. The cells may be cultured in any desired volume. The culture vessel surface can be prepared with cellular adhesive. The cellular adhesive culture vessel can be coated with any substrate for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells. The substrate used for cell adhesion can be any material intended to attach cells. Non-limiting substrates for cell adhesion include collagen, gelatin, poly-L-lysine, poly-D-lysine, poly-L-omithine, laminin, vitronectin, and fibronectin and mixtures thereof, for example, protein mixtures from Engelbreth-Holm-Swarm mouse sarcoma cells and lysed cell membrane preparations.

[0088] Also disclosed herein are compositions of cells comprising patterned Asci 1 -driven dopaminergic neurons. In some embodiments, the compositions are generated comprising the method disclosed above. In some embodiments, the compositions are organoids. In some embodiments, the organoids are generated by the methods disclosed elsewhere herein.

[0089] The composition may comprise one or more other cell types, for example, including but not limited to, microglia, oligodendrocytes, endothelial cells, cells of the immune system, stromal cells, differentiated human cell types such as cortical neurons, subcortical neurons, and sensory cells. In some embodiments, the composition comprises one or more of astrocytes, oligodendrocytes, ependymal cells, and microglia. In some embodiments, the composition comprises non-dopaminergic neurons.

3. Organoids

[0090] Embodiments of the present disclosure include three-dimensional midbrain assembled organoid comprising patterned Ascii -driven dopaminergic neurons and methods for generating three-dimensional midbrain assembled organoids comprising patterned Ascii -driven dopaminergic neurons.

[0091] The organoids comprising patterned Asci 1 -driven dopaminergic neurons may comprise one or more other cell types, for example, including but not limited to, microglia, oligodendrocytes, endothelial cells, cells of the immune system, stromal cells, differentiated human cell types such as cortical neurons, subcortical neurons, and sensory cells. In some embodiments, the composition comprises one or more of astrocytes and microglia.

[0092] Methods for generating three-dimensional midbrain assembled organoid comprising patterned Ascii -driven dopaminergic neurons comprise contacting a first population of cells comprising Ascii -driven mesenchymal progenitor cells and iPSC-derived astrocyte precursor cells (APCs) with one or more neurotrophic factors to differentiate the Ascii -driven mesenchymal progenitor cells and the APCs and form aggregates; adding iPSC-derived hematopoietic precursor cells (HPCs) to the aggregates to generate a second population of cells; contacting the second population of cells with a microglial differentiation stimulating agent; and incubating the second population of cells to form assembled three-dimensional organoids comprising patterned Ascii -driven dopaminergic neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia.

[0093] iPSC-derived astrocyte precursor cells (APCs) may be generated using methods known in the art. See for example, Krencik and Zhang, Nature protocols, 2011 and Calatayud, et al; protocols. io, 2022, incorporated herein by reference, and Example 2.

[0094] In some embodiments, Ascii -driven mesenchymal progenitor cells are generated by the methods described above. For example, the Ascii -driven mesenchymal progenitor cells may be generated by contacting induced pluripotent stem cells (iPSCs) with: one or more patterning factors over a first period of time, and one or more midbrain-specific neurotrophic and prosurvival factors for a second period of time; and inducing expression of Achaete-scute homolog 1 (Ascii) gene. Descriptions provided above for the iPSCs, patterning factors, midbrain-specific neurotrophic and pro-survival factors, first and second periods of time and induction of Ascii provided above are suitable and applicable to the methods generating the disclosed organoids. [0095] The one or more neurotrophic factors may comprise brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, transforming growth factor beta-3 (TGF03), dibutyryl-cAMP, or any combination thereof. The concentration of each of the one or more neurotrophic factors may be modulated over the course of time required to form aggregates. For example, initially the culture medium may contain BDNF, GDNF, Ascorbic acid, TGF03, and dibutyryl-cAMP, after a period of time, e.g., 24 hours, BDNF, GDNF, and ascorbic acid are added.

[0096] In some embodiments, a cell death inhibitor, as described above, may be included initially but then removed for aggregate formation to occur.

[0097] “Hematopoietic precursor cells (HPCs)” and “hematopoietic progenitors” refer to immature progenitor cells of the hematopoietic lineage. HPCs are characterized by surface expression of CD45 and, in some cases, CD34, and a capacity to differentiate into myeloid progenitors. HPCs are also known as hematovascular mesoderm progenitors. HPCs may be generated using methods known in the art. See for example, McQuade and Blurton- Jones, Methods in Molecular Biology, 2021 and Example 2. [0098] Microglial differentiation stimulating agents include any agent when which added to the medium aids in differentiation of the HPCs to microglia. In some embodiments, the microglial differentiation stimulation agent comprises one or more cytokines. In some embodiments, the one or more cytokines include any of IL-34, TGF01, M-CSF, CD200, and CX3CL1. In select embodiments, the microglial differentiation stimulation agent comprises IL- 34 and/or macrophage colony-stimulating factor (mCSF).

[0099] Incubating the second population of cells to form the assembled three-dimensional organoids can be monitored for phenotypic characteristics and for known markers indicative of Ascii -driven dopaminergic neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia, e.g., SOX6 and ALDH1A1; GFAP; and P2RY12, respectively. The time period of incubation may vary from about 7 to about 30 days, based on desired organoid cell distributions. [00100] The Ascii -driven mesenchymal progenitor cells are in excess to the APCs in the first population of cells to represent the human midbrain cell constitution. In some embodiments, the Ascii -driven mesenchymal progenitor cells and the APCs are at about a 10: 1 ratio in the first population of cells. In some embodiments, the HPCs are added at an about equal ratio to the APCs in the first population of cells.

[00101] The three-dimensional midbrain assembled organoid comprises cell populations similar to the human midbrain cell composition. In certain embodiments, the Ascii -driven dopaminergic neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia are at about a 10:1:1 ratio in the three-dimensional midbrain assembled organoid.

[00102] In some embodiments, one or more of the Ascii -driven mesenchymal progenitor cells, iPSC-derived APCs, and iPSC-derived HPCs comprise a disease-associated genetic abnormality. In some embodiments, the methods comprising introducing one or more genetic abnormalities in the Ascll-driven mesenchymal progenitor cells, iPSC-derived APCs, and iPSC-derived HPCs. Alternatively, one or more of the Ascll-driven dopaminergic neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia can be genetically engineered to introduce one or more genetic abnormalities.

[00103] The one or more genetic abnormalities may be associated with a neurological or neurodegenerative disease or disorder, as described above. In some embodiments, the one or more genetic abnormalities are associated with Parkinson’s Disease. In some embodiments, the one or more genetic abnormalities are in genes selected from the group including SNCA, PARK3, UCHL1, LRRK2, GIGYF2, HTRA2, EIF4G1, TMEM230, CHCHD2, RIC3, VPS35, PRKN, PINK1, PARK2, PARK7, PARK10, PARK12, PARK16, ATP13A2 (PARK9), PLA2G6, FBX07, DNAJC6, SYNJ1, and VPS13C.

4. Methods of Use

[00104] The disclosed dopaminergic neurons and compositions and organoids comprising three-dimensional midbrain assembled organoid comprising patterned Ascii -driven dopaminergic neurons may be suitable for use in methods for studying (e.g., modeling) diseases and screening for therapeutic and prophylactic interventions in such diseases. The generated neurons on organoids provide human-based disease models that accurately replicate molecular and cellular signatures of susceptible DA populations can be used to study underlying cell- autonomous and non-cell-autonomous cellular mechanisms of various neurological and neurodegenerative disorders, as outlined above.

[00105] As described above, the disclosed dopaminergic neurons, compositions, and organoids can comprise cells having a disease-associated genetic abnormality. Alternatively, disease or disease-like state may be induced through any method known in the art. For example, induction may be via a chemical or biological agent such as a virus, neurotoxin, bacteria, metal, small molecule, peptide, or polynucleotide.

[00106] The disclosed dopaminergic neurons and compositions and organoids can be subjected to a plurality of candidate agents or other therapeutic and prophylactic interventions. Candidate agents can include, but are not limited to, small molecules, genetic constructs that increase or decrease expression of an RNA of interest, CRISPR systems, optogenetic perturbation, electrical changes, and the like. Methods are also provided for determining the activity of a candidate agent on a disease-relevant cell, the method comprising contacting one or more cells of the organoid comprising at least one genetic abnormality associated with a neurological or neurodegenerative disease or disorder, with the candidate agent; and determining the effect of the agent on morphologic, genetic, and/or functional parameters.

5. Examples

Example 1

Generation of iPSC-derived patterned AsclI-driven dopaminergic neurons

[00107] Generation of floor plate mesenchymal progenitor cells [00108] The human KOL2.1 J iPSCs cultured in StemFlex medium (Stemcell technologies;

A3349401) are first stably transfected by a piggybac vector encoding the mouse Ascii gene (Ng, et al; Stem Cell Reports, 2021), which is highly conserved in humans, using the Lipofectamine Stem transfection reagent (Gibco; STEM00001) as per the manual’s instructions. The cells are maintained in StemFlex medium containing the selective antibiotic, G418, at the concentration of 50pg/ml. To pattern the iPSCs, on Day 0, the cells are dissociated using Accutase (Innovative cell technologies; AT104) for 10 minutes at 37 deg Celsius and plated at a density of 400k cells/sq. cm/well in a 6 well plate in 3ml of NB(N2B27) - Neurobasal-A medium (Gibco; 10- 888-022) containing 0.5x B27 supplement without Vitamin A (Gibco 12587010), lx N2 supplement (Gibco; 1702048), lx GlutaMAX (Gibco; 35050061), lx MEM Non-Essential Amino Acids (NEAA) (Gibco; 11140050). Patterning factors are added to this medium to modulate key midbrain-specific pathways: SMAD inhibitors [lOpM SB431542 (Tocris; 1614 - 50mg) and 500nM LDN193189 hydrochloride (SIGMA; SML0559-5MG)]; SHH pathway activators [200ng/ml Human SHH protein (Peprotech; 100-45), 0.7pM Purmorphamine (SIGMA; 540223-5MG)]; Wnt signaling activator [0.7pM CHIR99021 (StemCell Technologies; 72054)]; and cell death inhibitor [lOpM Y27632 dihydrochloride ROCK inhibitor (Tocris; 125410)]. Throughout the protocol, cells are rinsed with DPBS containing calcium and magnesium (Gibco; 14040141) before being fed with fresh medium. On Day 1, cells fed with the Day 0 medium without the ROCK inhibitor. On Day 3 and Day 5, cells are fed with the above described medium with the concentration of CHIR99021 increased to 3pM. On Day 7 and 8, SMAD inhibition and SHH activation are discontinued, the cells are fed with 4ml of NB(N2B27) with only 3pM CHIR99021 for continued Wnt activation. On Day 9, cells are fed with the above NB(N2B27) medium containing 3pM CHIR99021 and lOOng/ml Fibroblast Growth Factor 8b (FGF8b) for activate Wnt and FGF8 signaling, respectively. On Day 10, the cells are fed with NB(B27) - Neurobasal-A medium containing 0.5x B27 without vitamin A, lx GlutaMAX - with midbrain-specific neurotrophic and pro-survival factors [20ng/ml Brain-Derived Neurotrophic Factor (BDNF), 20ng/ml Glial cell line-Derived Neurotrophic Factor (GDNF), Ing/ml Transforming Growth Factor [33 (TGF03), 200pM dibutyryl-cAMP, 200pM Ascorbic acid, 3pM CHIR99021, lOOng/ml FGF8b] and cell death inhibitor [lOpM Y27632 dihydrochloride ROCK inhibitor]. Next day, the cells are dissociated with Accutase for 10 minutes at 37 deg Celsius to produce a single cell suspension. The cells are plated at a density of 800k cells/sq. cm/well in a 6 well plate in the same medium composition as Day 10, except the Wnt activator, CHIR99021. These 11 days old cells are specified for midbrain lineage.

[00109] Ascii expression and DA neuronal differentiation

[00110] On Day 12, the cells are rinsed and fed with Day 11 medium composition (without cell death inhibitor) containing 2pg/ml of doxycycline to induce the expression of Ascii. After 48 hours, the progenitor cells can be dissociated using accutase and frozen in NB(B27) + 10% DMSO in a cell freezing container in -80 deg freezer and then moved to liquid nitrogen for longterm storage. For differentiation, these 14 days old progenitors induced for Ascii expression are plated at a density of 100k cells/sq. cm in glass chamber slides coated with poly-L-omithine and human laminin in NB(B27) medium containing neurotrophic and maturation factors [20ng/ml BDNF, 20ng/ml GDNF, 200pM Ascorbic acid, lng/ml TGF 3, 200pM dibutyryl-cAMP]; Notch pathway inhibitor [lOpM DAPT], Ascii inducer [2pg/ml doxycycline]; and cell death inhibitor [lOpM Y27632 dihydrochloride ROCK inhibitor]. Next day, the cells are fed with the same medium as above without the cell death inhibitor. The developing neurons are fed every 3-4 days. Expression of tyrosine hydroxylase (TH), an enzyme involved in dopamine production, can be detected as early as Day 1 of differentiation using immunostaining. At Day 7, ~80% of the cells are TH-positive dopaminergic neurons, while above 90% cells express substantia nigra specific genes such as SOX6 andNurrl (Figure IB, C). As early as Day 14, these TH-positive cells also show expression of dopamine transporter (DAT), Vesicular monoamine transporter 2 (VMAT2) and a-synuclein, involved in dopamine release and recycling (Figure ID).

Example 2

Generation of mature 3D midbrain assembled organoids

[00111] Transcriptomic analysis of current models of human midbrain organoids shows a heterogenous collection of cell types including radial glial cells, progenitors, astrocytes, and oligodendrocytes apart from DA neurons. These often do not represent the human midbrain cell constitution. A method was developed to generate A9-specific 3D midbrain assembled organoids containing patterned Ascii -driven DA neurons, midbrain iPSC-derived astrocytes, and iPSC- derived microglia in precise numbers that reflect the ratios present in the human midbrain, unlike published methods. These 3D organoids show enhanced maturation of TH-positive DA neurons that express A9 markers such as SOX6 and ALDH1 Al, and distinct morphology of GFAP- positive astrocytes as well as P2RY 12 -positive microglia (FIGS. 2A-2B). Assembling mature 3D midbrain organoids containing A9-like DA neurons with glial cells serves as an excellent model to study intricate midbrain-specific cell-autonomous and non-cell-autonomous mechanisms in aging and disease progression.

[00112] Assembly of midbrain 3D organoids

[00113] A 96-well V-shaped bottom plates (Costar; 3894) are pre-treated with anti-adherent solution (Stem Cell Technologies; 07010) right before cell seeding. Day 14 patterned Ascll- driven progenitors and iPSC-derived astrocyte precursor cells (APCs) are seeded (10:1 iDA:iA ratio; 50,000 cells total per well) in lOOpl of NB(B27) medium containing neurotrophic factors [20ng/ml BDNF, 20ng/ml GDNF, 200pM Ascorbic acid, Ing/ml TGF03, 200 pM dibutyryl- cAMP]; Ascii inducer [2pg/ml doxycycline]; and cell death inhibitor [lOpM Y27632 dihydrochloride ROCK inhibitor]. After 24 hours, an additional 1 OOJJ.1 of NB(B27) medium containing only 20ng/ml BDNF, 20ng/ml GDNF, 200 M Ascorbic acid is added and the organoids are allowed to aggregate. To generate triple cell type 3D organoids, five days after aggregation, iPSC-derived hematopoietic precursor cells (HPCs) are added to integrate into the immature assembled organoids at a ratio of 10: 1 : 1 iDA:iA:iM. Two cytokines, IL-34 (lOOng/ml) and mCSF (25ng/ml) are added to the medium to aid microglial differentiation. A 50% media change is performed every 4-5 days for continued maturation of 3D assembled organoids. 12 day old mature 3D midbrain assembled organoids show over 80% TH-positive dopaminergic neurons, with successful integration of P2RY12-positive microglia and mature GFAP-positive astrocytes.

[00114] APCs

[00115] The APCs are generated based on the method described in Krencik and Zhang, Nature protocols, 2011. Briefly, iPSCs are patterned for 11 days to make floor plate mesenchymal cells, as described in Calatayud, et al; protocols.io, 2022. The generated rosettes are then selected by incubation in the STEMdiff Neural Rosette Selection reagent (STEMCELL technologies; 05832) for one hour. These rosettes are plated on Matrigel-coated plates for further maturation for 5-6 days, after which the rosette selection process is repeated. The rosettes are then matured in suspension in low-adherence flasks to form APCs in medium containing EGF (lOng/ml) and FGFb (lOng/ml) cytokines for 3-6 months based on the step-by-step protocol in the section ‘Astrosphere maintenance and maturation’ in Krencik and Zhang, Nature protocols, 2011. The 6- month-old APCs are directly used for 3D organoid formation and mature to astrocytes in the 3D environment.

[00116] HPCs

[00117] Hematopoietic precursor cells (HPCs) are generated from iPSCs using the STEMdiff Hematopoietic differentiation (STEMCELL technologies #05310) based on the step-by-step protocol described in Section 3.1 in McQuade and Blurton- Jones, Methods in Molecular Biology, 2021. Briefly, 20-40 iPSC clusters of approximately 100pm diameter are exposed to the STEMDiff Hematopoietic basal medium containing supplement ‘A’ for the first 4 days and supplement ‘B’ for the next 6-8 days. The floating HPCs are collected on day 10 and day 12 of the protocol and either frozen on Bambanker or used directly after collection.

[00118] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

[00119] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.

Claims

CLAIMS What is claimed is:

1. A method for generating dopaminergic neurons comprising: differentiating stem or precursor cells into mesenchymal progenitor cells; inducing expression of Achaete-scute homolog 1 (Ascii) gene in the mesenchymal progenitor cells to generate Ascii -driven mesenchymal progenitor cells; and differentiating the Ascii -driven mesenchymal progenitor cells to dopaminergic neurons.

2. The method of claim 1, wherein the stem or precursor cells are induced pluripotent stem cells (iPSCs).

3. The method of claim 1 or 2, wherein differentiating the stem or precursor cells comprises: contacting the stem or precursor cells in cell culture with: one or more patterning factors over a first period of time; and one or more midbrain-specific neurotrophic and pro-survival factors for a second period of time, wherein the second period of time follows the first period of time at least partially overlaps with the end of the first period of time.

4. The method of claim 3, wherein the first period of time is about 10 to about 15 days.

5. The method of claim 3 or 4, wherein the second period of time is about 1 to about 4 days.

6. The method of any of claims 3-5, wherein the one or more patterning factors are selected from Small Mothers Against Decapentaplegic (SMAD) inhibitors, sonic hedgehog (SHH) pathway activators, Wnt signaling activators, Fibroblast growth factor 8 (FGF8) signaling activators, and combinations thereof.

7. The method of any of claims 3-6, wherein the concentration of each of the one or more the patterning factors may increase, decrease, or remain constant over the first and/or second period of time.

8. The method of claim 7, wherein one or more Wnt signaling activators are increased after the start of the first period of time.

9. The method of claim 7 or 8, wherein any or all of the SMAD inhibitors and SHH pathway activators are removed in a later portion of the first period of time.

10. The method of any of claims 7-9, wherein the one or more FGF8 signaling activators are added in a later portion of the first period of time.

11. The method of any of claims 3-10, wherein the neurotrophic and pro-survival factors include brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, transforming growth factor beta-3 (TGFP3), dibutyryl-cAMP, or any combination thereof.

12. The method of any of claims 3-11, further comprising contacting the stem or precursor cells with a cell death inhibitor for any or all of the first and/or second period of time.

13. The method of any of claims 1-12, further comprising transfecting the stem or precursor cells with a nucleic acid encoding the Ascii gene operably linked to an inducible promoter.

14. The method of claim 13, wherein inducing the expression of the Ascii gene comprise contacting the mesenchymal progenitor cells with an inducing agent.

15. The method of any of claims 1-14, wherein differentiating the Ascii -driven mesenchymal progenitor cells to dopaminergic neurons comprises contacting the Ascii -driven mesenchymal progenitor cells for a third period of time with one or more of: neurotrophic and maturation factors and Notch pathway inhibitors.

16. The method of claim 15, wherein the third period of time is one day to one month.

17. The method of claim 15 or 16, wherein the one or more neurotrophic and maturation factors comprise brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ascorbic acid, transforming growth factor beta-3 (TGF03), dibutyryl-cAMP, or any combination thereof.

18. The method of any of claims 1-17, wherein greater than 80% of cells generated by the method are terminally differentiated into dopaminergic neurons.

19. The method of any of claims 1-18, wherein greater than 95% of cells generated by the method are terminally differentiated into dopaminergic neurons.

20. The method of any of claims 1-19, wherein the dopaminergic neurons, or any stem or precursor cell thereof, comprise a disease-associated genetic abnormality.

21. The method of any of claims 1-20, wherein the dopaminergic neurons, or any stem or precursor cell thereof, comprise a genetic abnormality associated with a neurological or neurodegenerative disease or disorder.

22. The method of any of claims 1-21, wherein the dopaminergic neurons, or any stem or precursor cell thereof, comprise a Parkinson’s disease associated genetic abnormality.

23. A method for generating three-dimensional midbrain assembled organoid comprising patterned Ascii -driven dopaminergic neurons comprising: contacting a first population of cells comprising Ascii -driven mesenchymal progenitor cells and iPSC-derived astrocyte precursor cells (APCs) with one or more neurotrophic factors to differentiate the Ascii -driven mesenchymal progenitor cells and the APCs and form aggregates; adding iPSC-derived hematopoietic precursor cells (HPCs) to the aggregates to generate a second population of cells; contacting the second population of cells with a microglial differentiation stimulating agent; and incubating the second population of cells to form assembled three-dimensional organoids comprising patterned Ascii -driven dopaminergic neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia.

24. The method of claim 23, wherein the Ascii -driven mesenchymal progenitor cells and the APCs are at about a 10: 1 ratio in the first population of cells.

25. The method of claim 23 or 24, wherein the HPCs are added at an about equal ratio to the APCs in the first population of cells.

26. The method of any of claims 23-25, wherein one or more of the Ascii-driven mesenchymal progenitor cells, iPSC-derived APCs, and iPSC-derived HPCs comprise a disease-associated genetic mutation.

27. The method of any of claims 23-26, wherein the Ascii -driven mesenchymal progenitor cells are generated by: contacting induced pluripotent stem cells (iPSCs) with: one or more patterning factors over a first period of time, and one or more midbrain-specific neurotrophic and pro-survival factors for a second period of time, wherein the second period of time follows the first period of time or partially overlaps with the end of the first period of time; and inducing expression of Achaete-scute homolog 1 (Ascii) gene.

28. The method of claim 27, wherein the first period of time is about 10 to about 15 days.

29. The method of claim 27 or 28, wherein the second period of time is about 1 to about 4 days.

30. The method of any of claims 27-29, wherein the one or more patterning factors are selected from SMAD inhibitors, SHH pathway activators, Wnt signaling activators, FGF8 signaling activators, and combinations thereof.

31. The method of any of claims 27-30, wherein the concentration of each of the one or more of the patterning factors may increase, decrease, or remain constant over the first and/or second period of time.

32. The method of claim 31, wherein one or more Wnt signaling activators are increased after the start of the first period of time.

33. The method of claim 31 or 32, wherein any or all of the SMAD inhibitors and SHH pathway activators are removed in a later portion of the first period of time.

34. The method of any of claims 31-33, wherein the one or more FGF8 signaling activators are added in a later portion of the first period of time.

35. The method of any of claims 31 -34, fiirther comprising contacting the stem or precursor cells with a cell death inhibitor for any or all of the first and/or second period of time.

36. The method of any of claims 23-35, wherein the microglial differentiation stimulating agent comprises one or more cytokines.

37. The method of any of claims 23-36, wherein the Ascii -driven dopaminergic neurons, midbrain iPSC-derived astrocytes, and iPSC-derived microglia are at about a 10: 1 : 1 ratio in the three-dimensional midbrain assembled organoid.

38. A composition of cells comprising a population of patterned Ascii -driven dopaminergic neurons.

39. The composition of claim 38, wherein the population of patterned Ascii -driven dopaminergic neurons is greater than 80% of total cells in the composition.

40. The composition of claim 38 or 39, wherein the population of patterned Ascll-driven dopaminergic neurons is greater than 95% of total cells in the composition.

41. The composition of any of claims 38-40, wherein all cells in the composition are neurons.

42. The composition of any of claims 38-41, wherein the composition is a three-dimensional midbrain assembled organoid.

43. A three-dimensional midbrain assembled organoid comprising patterned Ascii -driven dopaminergic neurons.

44. The organoid of claim 43, further comprising midbrain iPSC-derived astrocytes and iPSC- derived microglia.

45. The organoid of claim 44, wherein the Ascii -driven dopaminergic neurons, midbrain iPSC- derived astrocytes, and iPSC-derived microglia are at about a 10: 1 : 1 ratio in the organoid.