Midbrain dopaminergic neuron compositions and methods of use

Abstract

The present disclosure, in certain embodiments, provides methods for generating midbrain dopamine neurons and precursors thereof, midbrain dopamine neurons and precursors thereof generated by such methods and compositions comprising such cells, and uses thereof for preventing, modeling, and / or treating a neurological disorder.

Description

[0001] Attorney Docket No. : 072734.1842

[0002] MIDBRAIN DOPAMINERGIC NEURON COMPOSITIONS AND METHODS OF

[0003] USE

[0004] 1. CROSS REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to United States Provisional Application No. 63 / 682,038, filed August 12, 2024, which is hereby incorporated by reference in its entirety, and to which priority is claimed.

[0006] 2. TECHNICAL FIELD

[0007] The present disclosure is directed to methods for generating midbrain dopamine (mDA) neurons and precursors thereof, mDA neurons and precursors thereof that are generated by such methods, and composition comprising such mDA neurons and precursors thereof. The present disclosure is also directed to uses of such mDA neurons, precursors thereof, as well as compositions comprising such mDA neurons and precursors thereof for the prevention, modeling, and / or treatment of neurological disorders.

[0008] 3. BACKGROUND

[0009] Parkinson’s disease (PD) is characterized by the loss of mDA neurons that lead to well- known motor symptoms such as tremor, rigor, and bradykinesia (Lees, et al. Lancet 373, 2055- 2066 (2009)). While other cell types such as enteric, olfactory, and cortical neurons are also affected (Del Tredici, et al. Neuropathol Appl Neurobiol 42, 33-50 (2016)), mDA neurons remain the key focus for developing novel cell-based treatments (Barker, et al. Nature reviews Neurology 11, 492-503 (2015); Tabar, et al. Nat Rev Genet 15, 82-92 (2014)) and for PD disease modeling (Sanchez-Danes, et al. EMBO Mol Med 4, 380-395 (2012); Miller, et al. Cell stem cell 13, 691-705 (2013); Chung, et al. Stem Cell Reports 7, 664-677 (2016); Reinhardt, et al. Cell stem cell 12, 354-367 (2013); Chung, et al. Science 342, 983-987 (2013); Cooper, et al. Sci Transl Med 4, 141ral90 (2012)). Human pluripotent stem cells (hPSCs), comprising both human ES and iPS cells, have become the cell type of choice for deriving mDA neurons in vitro (Alekseenko, et al. Nat Commun. 13:3046 (2022); Oosterveen, et al. Stem Cell Rep. 16:2718-2735 (2021); Kim, et al., Cell Stem Cell. 28:343-355 (2021)). Despite the current progress in human mDA derivation, there remains a need for novel protocols for the generation of mDA neurons and precursors thereof. The mDA neuron system is integral to movement control and behavior. It is comprised of diverse neuronal subtypes including A8, A9, and A10 mDA neurons. In PD, the A9 subtype is uniquely vulnerable to degeneration. Loss of A9 mDA Attorney Docket No. : 072734.1842 neurons and projections to the dorsolateral striatum result in the characteristic PD motor deficits. Despite rapid progress in generating clinically suitable mDA neurons from hPSCs, current attempts at cell replacement therapies employ heterogenous and insufficiently characterized populations of cells. Methods to selectively derive the A9 mDA subtype have remained elusive; however, they are critical for the development of next generation cell replacement therapies and faithful human PD disease models.

[0010] 4. SUMMARY OF THE INVENTION

[0011] The present disclosure is directed, in certain embodiments, to methods for generating mDA neurons and precursors thereof, mDA neurons and precursors thereof that are generated by such methods, compositions comprising such mDA neurons and precursors thereof, as well as uses of such mDA neurons and precursors thereof and compositions comprising such mDA neurons and precursors thereof for the prevention, modeling, and / or treatment of neurological disorders.

[0012] In certain embodiments, the present disclosure provides in vitro methods for inducing differentiation of stem cells to mDA neurons or precursors thereof. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling and at least one activator of Sonic hedgehog (SHH) signaling. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, and at least one activator of wingless (Wnt) signaling. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, at least one activator of Wnt signaling, and at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, at least one activator of Wnt signaling, at least one activator of (TGFP) / Activin-Nodal signaling, and at least one activator of bone morphogenetic protein (BMP) signaling. In certain embodiments, the in vitro methods for inducing differentiation comprises: contacting the stem cells with at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, and at least one activator of Wnt signaling; and contacting the cells with at least one activator of (TGFP) / Activin-Nodal Attorney Docket No. : 072734.1842 signaling and at least one activator of BMP signaling to obtain a population of differentiated cells expressing at least one marker indicating a mDA neuron or a precursor thereof.

[0013] In certain embodiments, the contact of the cells with the at least one activator of (TGFP) / Activin-Nodal signaling is initiated at least about 15 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the contact of the cells with the at least one activator of (TGFP) / Activin-Nodal signaling is initiated no later than about 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the contact of the cells with the at least one activator of (TGFP) / Activin-Nodal signaling is initiated about 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the contact of the cells with the at least one activator of (TGFP) / Activin-Nodal signaling is initiated 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the cells are contacted with the at least one activator of (TGFP) / Activin-Nodal signaling for at least about 1 day. In certain embodiments, the cells are contacted with the at least one activator of (TGFP) / Activin-Nodal signaling for up to about 9 days, or up to about 10 days, or up to about 25 days, or up to about 30 days. In certain embodiments, the cells are contacted with the at least one activator of (TGFP) / Activin-Nodal signaling for about 5 days, about 10 days, or about 20 days. In certain embodiments, the cells are contacted with the at least one activator of (TGFP) / Activin-Nodal signaling for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, or 20 days. In certain embodiments, the contact of the cells with the at least one activator of BMP signaling is initiated at least about 15 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the contact of the cells with the at least one activator of BMP signaling is initiated no later than about 16 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the contact of the cells with the at least one activator of BMP signaling is initiated about 16 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the contact of the cells with the at least one activator of BMP signaling is initiated 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the cells are contacted with the at least one activator of BMP signaling for at least about 1 day. In certain embodiments, the cells are contacted with the at least one activator of BMP signaling for up to about 9 days, or up to about 10 days, or up to about 25 days, or up to about 30 days. In certain embodiments, the cells are Attorney Docket No. : 072734.1842 contacted with the at least one activator of BMP signaling for about 5 days, about 10 days, or about 20 days. In certain embodiments, the cells are contacted with the at least one activator of BMP signaling for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, or 20 days.

[0014] In certain embodiments, the cells are contacted with the at least one inhibitor of SMAD signaling for about 5 days. In certain embodiments, the cells are contacted with the at least one inhibitor of SMAD signaling for 6 days or 7 days. In certain embodiments, the cells are contacted with the at least one activator of SHH signaling for about 5 days. In certain embodiments, the cells are contacted with the at least one activator of SHH signaling for 6 days or 7 days. In certain embodiments, the cells are contacted with the at least one activator of Wnt signaling for about 15 days. In certain embodiments, the cells are contacted with the at least one activator of Wnt signaling for 10 days or 11 days. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased about 4 days from its initial contact with the stem cells. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased by between about 200 % and about 1000 % from the initial concentration of the at least one activator of Wnt signaling. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased by about 500 % from the initial concentration of the at least one activator of Wnt signaling. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased to from about 1 pM to between about 5 pM and about 10 pM. In certain embodiments, the concentration of the at least one activator of Wnt signaling is increased to a concentration of about 6 pM.

[0015] In certain embodiments, the at least one activator of (TGFP) / Activin-Nodal signaling comprises Activin A. In certain embodiments, the at least one activator of BMP signaling comprises BMP7 or TCS21311.

[0016] In certain embodiments, the at least one inhibitor of SMAD signaling comprises an inhibitor of TGFp / Activin-Nodal signaling, an inhibitor of BMP signaling, or a combination thereof. In certain embodiments, the at least one inhibitor of TGFp / Activin-Nodal signaling comprises an inhibitor of ALK5. In certain embodiments, the at least one inhibitor of TGFp / Activin-Nodal signaling is selected from the group consisting of SB431542, derivatives of SB431542, and combinations thereof. In certain embodiments, the derivative of SB431542 comprises A83-01. In certain embodiments, the at least one inhibitor of TGFp / Activin-Nodal signaling comprises SB431542. In certain embodiments, the at least one inhibitor of BMP signaling is selected from the group consisting of LDN193189, Noggin, dorsomorphin, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphin, and Attorney Docket No. : 072734.1842 combinations thereof. In certain embodiments, the at least one inhibitor of BMP comprises LDN-193189. In certain embodiments, the at least one activator of Wnt signaling comprises an inhibitor of glycogen synthase kinase 3p (GSK3P) signaling. In certain embodiments, the at least one activator of Wnt signaling is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, deoxycholic acid, BIO, SB- 216763, Wnt3A, Wntl, Wnt5a, derivatives thereof, and combinations thereof. In certain embodiments, the at least one activator of Wnt signaling comprises CHIR99021. In certain embodiments, the at least one activator of SHH signaling is selected from the group consisting of SHH proteins, Smoothened agonists (SAG), and combinations thereof. In certain embodiments, the SHH protein is selected from the group consisting of recombinant SHHs, modified N-terminal SHHs, and combinations thereof. In certain embodiments, the modified N-terminal SHH comprises two Isoleucines at the N-terminus. In certain embodiments, the modified N-terminal SHH has at least about 90% sequence identity to an un-modified N- terminal SHH. In certain embodiments, the un-modified N-terminal SHH is an un-modified mouse N-terminal SHH or a un-modified human N-terminal SHH. In certain embodiments, modified N-terminal SHH comprises SHH C25II.

[0017] In certain embodiments, the SAG comprises purmorphamine. In certain embodiments, at least about 80% of the differentiated cells express FOXA2 and EN1 about 15 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, greater than about 80% or greater than about 90% of the differentiated cells express FOXA2 and EN1 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, the at least one marker indicating a mDA neuron or a precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LM03, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof. In certain embodiments, the differentiated cells do no express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0018] In certain embodiments, the method further comprises isolating cells that express at least one positive marker and do not express at least one negative marker. In certain embodiments, the at least one positive marker is selected from the group consisting of PITX3, ALDH1A1, TH, and combinations thereof. In certain embodiments, the at least one positive marker comprises ALDH1A1. In certain embodiments, the at least one negative marker is selected from CD49e, CD340, and combination thereof. Attorney Docket No. : 072734.1842

[0019] In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the stem cells are selected from the group consisting of nonembryonic stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof. In certain embodiments, the stem cells are human stem cells, non-human primate stem cells, or rodent stem cells. In certain embodiments, the stem cells are human stem cells.

[0020] In certain embodiments, the present disclosure provides an in vitro method for inducing differentiation of stem cells, comprising: (a) contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and (b) contacting the cells with at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling and at least one activator of bone morphogenetic protein (BMP) signaling for up to about 9 days; and (c) contacting the cells with at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling and at least one activator of bone morphogenetic protein (BMP) signaling for up to about 15 days to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof. In certain embodiments, the at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling of step (b) comprises Activin A. In certain embodiments, the at least one BMP of step (b) comprises BMP7, TCS21311, or a combination thereof. In certain embodiments, the at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling of step (c) comprises Activin A. In certain embodiments, the at least one BMP of step (c) comprises BMP4.

[0021] In certain embodiments, step (c) further comprises contacting the cells with at least one activator of estrogen-related receptor p (ESRRB). In certain embodiments, the at least one activator of ESRRB comprises DY-131.

[0022] In certain embodiments, the present disclosure, in certain embodiments, provides cell populations of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by a method as disclosed herein.

[0023] In certain embodiments, the present disclosure provides compositions comprising the cell population as disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0024] In certain embodiments, the present disclosure provides kits for inducing differentiation of stem cells to mDA neurons or precursors thereof, comprising: (a) at least one inhibitor of SMAD signaling; (b) at least one activator of SHH signaling; (c) at least one activator of Wnt signaling; (d) at least one activator of TGFp / Activin-Nodal signaling; and (e) at least one Attorney Docket No. : 072734.1842 activator of BMP signaling. In certain embodiments, the kit further comprises (f) instructions for inducing differentiation of the stem cells into a population of differentiated cells that express at least one marker indicating a mDA neuron or a precursor thereof.

[0025] In certain embodiments, the present disclosure provides methods of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder, comprising administering to the subject an effective amount of the cell population as disclosed herein. In certain embodiments, the methods of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder comprise administering to the subject an effective amount of the composition as disclosed herein. In certain embodiments, the present disclosure provides methods of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder, comprising administering to the subject an effective amount of one of the following: (a) the cell population as disclosed herein; or (b) the composition as disclosed herein. In certain embodiments, the neurological disorder is characterized by reduction of mDA neuron function. In certain embodiments, the reduction of mDA neuron function is age related. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and combinations thereof. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof. In certain embodiments, the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia.

[0026] In certain embodiments, the present disclosure provides cell populations or compositions as disclosed herein for use in preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder in a subject. In certain embodiments, the neurological disorder is characterized by reduction of mDA neuron function. In certain embodiments, the reduction of mDA neuron function is age related. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and combinations thereof. In certain embodiments, the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof. In certain embodiments, the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia. Attorney Docket No. : 072734.1842

[0027] 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows characterization of hPSC-mDA populations using scRNA-seq. Figure 1 A shows the schematic of the hPSC-mDA neuron differentiation protocol. Figure IB shows the UMAP plots of hPSC-derived mDA cells colored by the indicated stages (upper) and clusters (lower). Figure 1C shows the UMAP plots of joint expression of the indicated gene sets.

[0029] Figure 2 shows identification of pseudotime trajectories resembling A9 mDA and A10 mDA development. URD trajectories are colored by the indicated stages (left), CALB2 expression (middle) and LM03 expression (right).

[0030] Figures 3A-3B show identification of BMP and TGFP signaling networks in neuronal progenitors. Figures 3 A-3B show heatmaps showing BMP (Figure 3 A) and TGFP (Figure 3B) signaling networks in the indicated populations.

[0031] Figure 4 shows modulation of TGFP signaling pathways during mDA progenitor differentiation.

[0032] Figures 5A-5C show generation of A9-like mDA neurons from hPSCs by modulating Activin A and BMP7 signaling. Figure 5A shows UMAP plots showing the expression levels of CD99 and TH, grouped by the indicated stages. Figure 5B shows schematic of the PITX3::GFP ES line. Figure 5 C shows flow cytometry analyses of PITX3 and CD99 expression and ALDH activity of the day 25 populations.

[0033] Figures 6A-6D show characterization of A9-like mDA neurons. Figures 6A-6C show immunofluorescence analysis (Figures 6A and 6B) and quantification (Figure 6C) of the expression levels of PITX3, ALDH1A1, CALB1, and TH in the day 26 mDA populations. Unpaired t-test; n>3. Figure 6D shows RT-qPCR analysis of the expression levels of TH, NR4A2, CALB1, and ALDH1A1 in the day 25 populations. Unpaired t-test; n>3.

[0034] Figure 7 shows generation of A9-like mDA neurons from iPSCs. Flow cytometry analyses of CD99 and TH expression and ALDH activity of the day 25 populations generating using KOLF2.1 iPSCs.

[0035] Figures 8A-8G show characterization of Activin A and BMP7 induced mDA cells using scMultiomic-seq. Figure 8A shows UMAP plots of hPSC-derived mDA cells colored by the indicated treatment (left) and clusters (right). Figures 8B-8D show expression of mDA markers (Figure 8B), A9 markers (Figure 8C), and A10 markers (Figure 8D). Figure 8E shows differentially accessible peaks in the indicated clusters. Figures 8F-8G show differentially accessible peaks in cluster 6 (ALDH1A1 / A9; Figure 8F) and transcription factor motifs enriched in the cluster (Figure 8G) compared to cluster 5. Attorney Docket No. : 072734.1842

[0036] Figures 9A-9B show isolation of pure A9-like mDA neurons and neuroblasts. Figure 9A shows FACS sorting strategy to purify A9-like mDA neurons and neuroblasts based on CD99 expression and ALDH activity detected using the AldeRed™ ALDH Detection kit (Millipore Sigma; SCR150). Figure 9B shows immunofluorescence analysis of the expression levels of PITX3, ALDH1A1, and CALB1 in the day 26 mDA populations.

[0037] Figures 10A-10B show generation of mDA spheroids from the sorted day 25 CD99+ALDH+ and CD99-ALDH- populations. Figures 10A-10B show immunofluorescence analysis of the expression levels of ALDH1A1, TH, and PITX3 in the day 40 mDA spheroids generated from day 25 sorted CD99-ALDH- (Figure 10A) and day 25 CD99+ALDH+ populations (Figure 10B).

[0038] Figures 11A-11B show generation of mature A9-like mDA neurons from hPSCs by modulating Activin A and BMP7 signaling. Figures 11A-11B show immunofluorescence analysis (Figure 11 A) and quantification (Figure IB) of the expression levels of ALDH1A1, TH, CALB1, and FOXA2 in the day 40 mature mDA populations.

[0039] Figures 12A-12C show maintenance of ALDH1A1 expression in late-stage AO- enriched culture by activating BMP4 and Activin A signaling. Figure 12A shows schematic of the hPSC-mDA neuron differentiation protocol. Figure 12B shows flow cytometry analysis of CD99, CD56, and ALDH expression in the day 36 populations generated under the indicated conditions. Figure 12C shows immunofluorescence analysis of CALB1, ALDH1A1, and TH expression levels in the day 45 populations.

[0040] Figures 13A-13C show identification of positive regulations of A9 development. Figures 13A-13C depict heatmaps showing the expression of genes (Figure 13A), ATAC-seq peaks (Figure 13B), and enrichment of transcription factor motifs (Figure 13C) during A9 neuron development. Figure 13D shows plot highlighting candidate positive regulators of A9 development, including ESRRB.

[0041] Figures 14A-14D show that activation of ESRRB enhances ALDH activity at late stages of A9 development. Figure 14A shows schematic of the sorting strategy and downstream analyses of ALDH' and ALDH+populations isolated from day 25 A9-enriched cultures. Figure 14B shows flow cytometry analysis of CD99, CD56, ALDH1 Al, and TH expression in day 35 AlO-enriched (LDN-treated) cultures and in ALDH' and ALDH+populations from A9- enriched cultures treated with the indicated conditions. Figure 14C shows immunofluorescence analysis of DDC and ALDH1A1 expression in day 35 ALDH' and ALDH+populations following treatment with Activin A, BMP4, and the ESRRB agonist DY-131 from day 26 to day 35. Figure 14D shows immunofluorescence staining for PITX3, DDC, CALB1, and Attorney Docket No. : 072734.1842

[0042] ALDH1A1 in day 44 AlO-enriched (LDN-treated) cultures and purified A9 populations subjected to the indicated treatments.

[0043] Figures 15A-15F show single-cell sequencing profiling of day 36 A9 and A10 midbrain dopaminergic neurons. Figure 15A shows UMAP plots displaying cell identities (right) and corresponding differentiation conditions (left). Figure 15B shows UMAP feature plots showing expression of general mDA markers (LMX1A, NR4A2, FOXA2, EN1, TH), A9-specific markers (ALDH1A1, LM03), and A10 marker CALB1. Figure 15C shows UMAP highlighting bulk A9 and A10 mDA populations derived from hPSCs. Figure 15D shows heatmap of differentially expressed genes between A9 and A10 populations. Figure 15E shows dot plot illustrating enrichment of biological pathways and processes in A9 versus A10 neurons. Figure 15F shows violin plots showing transcriptional activity scores of selected transcription factors in A9 and A10 populations.

[0044] Figures 16A-16C show integration analysis revealing transcriptomic similarities between mDA subtypes generated in vitro and those identified in human fetal midbrain. Figure 16A shows UMAP plots displaying the data source (top) and identified clusters (bottom) following integration of hPSC-derived and fetal datasets. Figure 16B shows bar plots showing the proportional contributions of in vitro and fetal cell types to each cluster. Figure 16C shows UMAP plots illustrating overlapping mDA subtypes derived from hPSCs (top) and human fetal tissue (bottom). Fetal data: La Manno, 2016, Cell; DOI: 10.1016 / j.cell.2016.09.027.

[0045] Figures 17A-17D show that hPSC-derived A9 and A10 mDA neurons exhibit distinct functional properties. Figures 17A-17B show selective pigmentation observed in day 45 A9 mDA neurons following treatment with Levodopa (L-DOPA); pigmentation is observed through brightfield microscopy (Figure 17A) or images of cells in media (Figure 17B). Figures 17C-17D show HPLC quantification of intracellular dopamine (DA), 3,4- dihydroxyphenylacetic acid (DOPAC), and released DA in naive (Figure 17C) and L-DOPA- treated (Figure 17D) A9 and A10 neurons at days 55-60. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), p < ),0001 (****) by Mann-Whitney test.

[0046] 6. DETAILED DESCRIPTION

[0047] In certain embodiments, the present disclosure provides methods for generating mDA neurons and precursors thereof, mDA neurons and precursors thereof generated by such methods, compositions comprising such cells, and uses thereof for preventing and / or treating neurological disorders. Attorney Docket No. : 072734.1842

[0048] Non-limiting embodiments of the presently disclosed subject matter are described by the present specification and Examples.

[0049] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0050] 5.1. Definitions;

[0051] 5.2. Methods of Differentiating Stem Cells;

[0052] 5.3. Cell Populations and Compositions;

[0053] 5.4. Methods of Preventing, Modeling, and / or Treating Neurological Disorders; and

[0054] 5.5. Kits.

[0055] 5.1. Definitions

[0056] The terms used in this specification generally have their ordinary meanings in the art, within the context of the present disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0057] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold, or within 2-fold, of a value.

[0058] As used herein, the term “signaling” in reference to a “signal transduction protein” refers to a protein that is activated or otherwise affected by ligand binding to a membrane receptor protein or some other stimulus. Examples of signal transduction protein include, but are not limited to, a SMAD, a Wingless (Wnt) complex protein, including beta-catenin, NOTCH, transforming growth factor beta (TGFP), Activin, Nodal, glycogen synthase kinase 3P (GSK3P) proteins, bone morphogenetic proteins (BMP) and fibroblast growth factors (FGF). For many cell surface receptors or internal receptor proteins, ligand-receptor interactions are not directly linked to the cell’ s response. The ligand activated receptor can first interact with other proteins inside the cell before the ultimate physiological effect of the ligand on the cell’s behavior is produced. Often, the behavior of a chain of several interacting cell proteins is Attorney Docket No. : 072734.1842 altered following receptor activation or inhibition. The entire set of cell changes induced by receptor activation is called a signal transduction mechanism or signaling pathway.

[0059] As used herein, the term “signals” refer to internal and external factors that control changes in cell structure and function. They can be chemical or physical in nature.

[0060] As used herein, the term “ligands” refers to molecules and proteins that bind to receptors, e.g., transforming growth factor-beta (TFGP), Activin, Nodal, bone morphogenic proteins (BMPs), etc.

[0061] “Inhibitor” as used herein, refers to a compound or molecule (e.g., small molecule, peptide, peptidomimetic, natural compound, siRNA, anti-sense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, decreases, suppresses, eliminates, or blocks) the signaling function of the molecule or pathway (e.g., Wnt signaling pathway, and SMAD signaling). An inhibitor can be any compound or molecule that changes any activity of a named protein (signaling molecule, any molecule involved with the named signaling molecule, a named associated molecule, such as a GSK3P). (e.g., including, but not limited to, the signaling molecules described herein). For example, an inhibitor of SMAD signaling can function, for example, via directly contacting SMAD, contacting SMAD mRNA, causing conformational changes of SMAD, decreasing SMAD protein levels, or interfering with SMAD interactions with signaling partners, and affecting the expression of SMAD target genes.

[0062] Inhibitors also include molecules that indirectly regulate biological activity, for example, SMAD biological activity, by intercepting upstream signaling molecules (e.g., within the extracellular domain, examples of a signaling molecule and an effect include: Noggin which sequesters bone morphogenic proteins, inhibiting activation of ALK receptors 1,2,3, and 6, thus preventing downstream SMAD activation. Likewise, Chordin, Cerberus, Follistatin, similarly sequester extracellular activators of SMAD signaling. Bambi, a transmembrane protein, also acts as a pseudo-receptor to sequester extracellular TGFp signaling molecules). Antibodies that block upstream or downstream proteins are contemplated for use to neutralize extracellular activators of protein signaling, and the like. Although the foregoing example relates to SMAD signaling inhibition, similar or analogous mechanisms can be used to inhibit other signaling molecules. Examples of inhibitors include, but are not limited to: LDN193189 (LDN) and SB431542 (SB) (LSB) for SMAD signaling inhibition, and IWP2 for Wnt inhibition. Inhibitors are described in terms of competitive inhibition (binds to the active site in a manner as to exclude or reduce the binding of another known binding compound) and allosteric inhibition (binds to a protein in a manner to change the protein conformation in a manner which interferes with binding of a compound to that protein’ s active site) in addition Attorney Docket No. : 072734.1842 to inhibition induced by binding to and affecting a molecule upstream from the named signaling molecule that in turn causes inhibition of the named molecule. An inhibitor can be a “direct inhibitor” that inhibits a signaling target or a signaling target pathway by actually contacting the signaling target.

[0063] “Activators,” as used herein, refer to compounds that increase, induce, stimulate, activate, facilitate, or enhance activation the signaling function of the molecule or pathway, e.g., Wnt signaling, SHH signaling, etc.

[0064] As used herein, the term “Wnt” or “wingless” in reference to a ligand refers to a group of secreted proteins (e.g., integration 1 in humans) that are capable of interacting with a Wnt receptor, such as a receptor in the Frizzled and LRPDerailed / RYK receptor family. As used herein, the term “a Wnt or wingless signaling pathway refers to a signaling pathway composed of Wnt family ligands and Wnt family receptors, such as Frizzled and LRPDerailed / RYK receptors, mediated with or without P-catenin. The Wnt signaling pathway include canonical Wnt signaling (e.g., mediation by P-catenin) and non-canonical Wnt signaling (mediation without P-catenin).

[0065] As used herein, the term “derivative” refers to a chemical compound with a similar core structure.

[0066] As used herein, the term “a population of cells” or “a cell population” refers to a group of at least two cells. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells. The population may be a pure population comprising one cell type, such as a population of midbrain DA precursors, or a population of undifferentiated stem cells, e.g., a population of A9 subtype midbrain dopamine neurons. Alternatively, the population may comprise more than one cell type, for example a mixed cell population, e.g., a cell population mixed of A9 subtype midbrain dopamine neurons and A10 subtype midbrain dopamine neurons.

[0067] As used herein, the term “stem cell” refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

[0068] As used herein, the term “embryonic stem cell” and “ESC” refer to a primitive (undifferentiated) cell that is derived from preimplantation-stage embryo, capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers. A human embryonic stem cell refers to an embryonic stem cell that is from a human embryo. As used herein, the term “human embryonic stem cell” or “hESC” refers to a type of pluripotent stem cells derived from early stage human Attorney Docket No. : 072734.1842 embryos, up to and including the blastocyst stage, that is capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers.

[0069] As used herein, the term “embryonic stem cell line” refers to a population of embryonic stem cells which have been cultured under in vitro conditions that allow proliferation without differentiation for up to days, months to years.

[0070] As used herein, the term “pluripotent” refers to an ability to develop into the three developmental germ layers of the organism including endoderm, mesoderm, and ectoderm.

[0071] As used herein, the term “totipotent” refers to an ability to give rise to all the cell types of the body plus all of the cell types that make up the extraembryonic tissues such as the placenta.

[0072] As used herein, the term “multipotent” refers to an ability to develop into more than one cell type of the body.

[0073] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a type of pluripotent stem cell formed by the introduction of certain embryonic genes (such as but not limited to OCT4, SOX2, and KLF4 transgenes) (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), herein incorporated by reference) into a somatic cell.

[0074] As used herein, the term “somatic cell” refers to any cell in the body other than gametes (egg or sperm); sometimes referred to as “adult” cells.

[0075] As used herein, the term “somatic (adult) stem cell” refers to a relatively rare undifferentiated cell found in many organs and differentiated tissues with a limited capacity for both self-renewal (in the laboratory) and differentiation.

[0076] As used herein, the term “neuron” refers to a nerve cell, the principal functional units of the nervous system. A neuron consists of a cell body and its processes - an axon and at least one dendrite. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0077] As used herein, the term “proliferation” refers to an increase in cell number.

[0078] As used herein, the term “undifferentiated” refers to a cell that has not yet developed into a specialized cell type.

[0079] As used herein, the term “differentiation” refers to a process whereby an unspecialized embryonic cell acquires the features of a specialized cell such as a neuron, heart, liver, or muscle cell. Differentiation is controlled by the interaction of a cell’s genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface. Attorney Docket No. : 072734.1842

[0080] As used herein, the term “directed differentiation” refers to a manipulation of stem cell culture conditions to induce differentiation into a particular (for example, desired) cell type, such as midbrain dopamine neurons or precursors thereof. In references to a stem cell, “directed differentiation” refers to the use of small molecules, growth factor proteins, and other growth conditions to promote the transition of a stem cell from the pluripotent state into a more mature or specialized cell fate.

[0081] As used herein, the term “inducing differentiation” in reference to a cell refers to changing the default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, “inducing differentiation in a stem cell” refers to inducing the stem cell (e.g., human stem cell) to divide into progeny cells with characteristics that are different from the stem cell, such as genotype (e.g., change in gene expression as determined by genetic analysis such as a microarray) and / or phenotype (e.g., change in expression of a protein marker of mDA neurons or precursors thereof, such as EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LM03, SNCA, ADCAP1, CHRNA4, ALDH1A1, SOX6, WNT1, DAT, VMAT2, and GIRK2).

[0082] As used herein, the term “cell culture” refers to a growth of cells in vitro in an artificial medium for research or medical treatment.

[0083] As used herein, the term “culture medium” refers to a liquid that covers cells in a culture vessel, such as a Petri plate, a multi-well plate, and the like, and contains nutrients to nourish and support the cells. Culture medium may also include growth factors added to produce desired changes in the cells.

[0084] As used herein, the term “contacting” a cell or cells with a compound (e.g., at least one inhibitor, activator, and / or inducer) refers to providing the compound in a location that permits the cell or cells access to the compound. The contacting may be accomplished using any suitable method. For example, contacting can be accomplished by adding the compound, in concentrated form, to a cell or population of cells, for example in the context of a cell culture, to achieve the desired concentration. Contacting may also be accomplished by including the compound as a component of a formulated culture medium.

[0085] As used herein, the term “in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures.

[0086] As used herein, the term “in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neural tube formation, etc. Attorney Docket No. : 072734.1842

[0087] As used herein, the term “expressing” in relation to a gene or protein refers to making an mRNA or protein which can be observed using assays such as microarray assays, antibody staining assays, and the like.

[0088] As used herein, the term “marker” or “cell marker” refers to gene or protein that identifies a particular cell or cell type. A marker for a cell may not be limited to one marker, markers may refer to a “pattern” of markers such that a designated group of markers may identity a cell or cell type from another cell or cell type.

[0089] As used herein, the term “derived from” or “established from” or “differentiated from” when made in reference to any cell disclosed herein refers to a cell that was obtained from (e.g., isolated, purified, etc.) an ultimate parent cell in a cell line, tissue (such as a dissociated embryo, or fluids using any manipulation, such as, without limitation, single cell isolation, culture in vitro, treatment and / or mutagenesis using for example proteins, chemicals, radiation, infection with virus, transfection with DNA sequences, such as with a morphogen, etc., selection (such as by serial culture) of any cell that is contained in cultured parent cells. A derived cell can be selected from a mixed population by virtue of response to a growth factor, cytokine, selected progression of cytokine treatments, adhesiveness, lack of adhesiveness, sorting procedure, and the like.

[0090] An “individual” or “subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0091] As used herein, the term “disease” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0092] As used herein, the term “treating” or “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may Attorney Docket No. : 072734.1842 prevent the onset of the disorder or a symptom of the disorder in a subj ect at risk for the disorder or suspected of having the disorder.

[0093] 5.2. Method of Differentiating Stem Cells

[0094] The present disclosure provides, in certain embodiments, methods for inducing differentiation of stem cells, comprising contacting stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling (referred to as “SMAD inhibitor”), at least one activator of Sonic hedgehog (SHH) signaling (referred to as “SHH activator”), and at least one activator of wingless (Wnt) signaling (referred to as “Wnt activator”); and further contacting the cells with at least one activator of (TGFP) / Activin-Nodal signaling (referred to as “TGFp / Activin-Nodal activator”) and at least one activator of BMP signaling, to obtain a cell population comprising differentiated cells expressing at least one marker indicating a mDA neuron or a precursor thereof. In certain embodiments, the present disclosure provides in vitro methods for inducing differentiation of stem cells to mDA neurons or precursors thereof. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling and at least one activator of SHH signaling. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, and at least one activator of Wnt signaling. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, at least one activator of Wnt signaling, and at least one activator of (TGFP) / Activin-Nodal signaling. In certain embodiments, the in vitro methods for inducing differentiation comprise: contacting the stem cells with at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, at least one activator of Wnt signaling, at least one activator of (TGFP) / Activin-Nodal signaling, and at least one activator of BMP signaling In certain embodiments, the in vitro methods for inducing differentiation comprises: contacting the stem cells with at least one inhibitor of SMAD signaling, at least one activator of SHH signaling, and at least one activator of Wnt signaling; and contacting the cells with at least one activator of (TGFP) / Activin-Nodal signaling and at least one activator of BMP signaling to obtain a population of differentiated cells expressing at least one marker indicating a mDA neuron or a precursor thereof. Attorney Docket No. : 072734.1842

[0095] 5.2.1. Stem Cells

[0096] The presently disclosed subject matter, in certain embodiments, provides in vitro methods for inducing differentiation of stem cells to produce mDA neurons and precursors thereof. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and combinations thereof. In certain embodiments, the stem cells are multipotent stem cells. Non-limiting examples of stem cells that can be used with the presently disclosed methods include nonembryonic stem cells, embryonic stem cells, induced nonembryonic pluripotent cells, and engineered pluripotent cells. In certain embodiments, the stem cells are human stem cells. Non-limiting examples of human stem cells include human embryonic stem cells (hESC), human pluripotent stem cell (hPSC), human induced pluripotent stem cells (hiPSC), human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, F-class pluripotent stem cells, somatic stem cells, cancer stem cells, or any other cell capable of lineage specific differentiation. In certain embodiments, the stem cell is a human embryonic stem cell (hESC). In certain embodiments, the stem cell is a human induced pluripotent stem cell (hiPSC). In certain embodiments, the stem cells are non-human stem cells. In certain embodiments, the stem cell is a nonhuman primate stem cell. In certain embodiments, the stem cell is a rodent stem cell.

[0097] In certain embodiments, the stem cell or a progeny cell thereof contains an introduced heterologous nucleic acid, where said nucleic acid may encode a desired nucleic acid or protein product or have informational value (see, for example, U.S. Patent No. 6,312,911, which is incorporated by reference in its entirety). Non-limiting examples of protein products include markers detectable via in vivo imaging studies, for example receptors or other cell membrane proteins. Non-limiting examples of markers include fluorescent proteins (such as green fluorescent protein (GFP), blue fluorescent protein (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP)), P-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), enzymes (such as oxidases and peroxidases), and antigenic molecules. As used herein, the terms “reporter gene” or “reporter construct” refer to genetic constructs comprising a nucleic acid encoding a protein that is easily detectable or easily assayable, such as a colored protein, fluorescent protein such as GFP or an enzyme such as beta-galactosidase (lacZ gene). In certain embodiments, the reporter can be driven by a recombinant promoter of a premature post-mitotic mDA neuron marker gene, for example, NURR1. Attorney Docket No. : 072734.1842

[0098] 5.2.2. SMAD Inhibitors

[0099] In certain embodiments, the present disclosed subject matter provides compositions comprising at least one SMAD inhibitor, and methods of use. In certain embodiments, the methods comprise contacting cells with at least one SMAD inhibitor. In certain embodiments, the methods comprise contacting cells with a composition comprising at least one SMAD inhibitor. Non-limiting examples of SMAD inhibitors include inhibitors of transforming growth factor beta (TGFP) / Activin-Nodal signaling (referred to as “TGFp / Activin-Nodal inhibitor”), and inhibitors of bone morphogenetic proteins (BMP) signaling. In certain embodiments, the TGFp / Activin-Nodal inhibitor can neutralize the ligands including TGFPs, BMPs, Nodal, and activins, and / or block their signal pathways through blocking the receptors and downstream effectors. Non-limiting examples of TGFp / Activin-Nodal inhibitors include those disclosed in WO / 2010 / 096496, WO / 2011 / 149762, WO / 2013 / 067362, WO / 2014 / 176606, WO / 2015 / 077648, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1 ;30(7):715-20 (2012), all of which are incorporated by reference in their entireties herein for all purposes. In certain embodiments, the at least one TGFp / Activin-Nodal inhibitor is selected from inhibitors of ALK5, inhibitors of ALK4, inhibitors of ALK7, and combinations thereof). In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises an inhibitor of ALK5. In certain embodiments, the TGFp / Activin-Nodal inhibitor is a small molecule selected from SB431542, derivatives thereof, and mixtures thereof. “SB431542” refers to a molecule with a number CAS 301836-41-9, a molecular formula of C22H18N4O3, and a name of 4-[4-(l,3- benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]-benzamide, for example, see structure below:

[0100] In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542. In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises a derivative of SB431542. In certain embodiments, the derivative of SB431542 is A83-01.

[0101] In certain embodiments, the at least one SMAD inhibitor comprises an inhibitor of BMP signaling (referred to as “BMP inhibitor”). Non-limiting examples of BMP inhibitors include Attorney Docket No. : 072734.1842 those disclosed in WO2011 / 149762, Chambers et al., Nat BiotechnoL 2009 Mar;27(3):275-80, Kriks etal., Nature. 2011 Nov 6;480(7378):547-51, and Chambers et al., Nat Biotechno 2012 Jul l;30(7):715-20, all of which are incorporated by reference in their entireties. In certain embodiments, the BMP inhibitor is a small molecule selected from LDN193189, Noggin, dorsomorphin, derivatives thereof, and mixtures thereof. “LDN193189” refers to a small molecule DM-3189, IUPAC name 4-(6-(4-(piperazin-l-yl)phenyl)pyrazolo[l,5-a]pyrimidin- 3-yl)quinoline, with a chemical formula of C25H22N6 with the following formula.

[0102] LDN193189 is capable of functioning as a SMAD signaling inhibitor. LDN193189 is also highly potent small-molecule inhibitor of ALK2, ALK3, and ALK6, protein tyrosine kinases (PTK), inhibiting signaling of members of the ALK1 and ALK3 families of type I TGFP receptors, resulting in the inhibition of the transmission of multiple biological signals, including the bone morphogenetic proteins (BMP) BMP2, BMP4, BMP6, BMP7, and Activin cytokine signals and subsequently SMAD phosphorylation of Smadl, Smad5, and Smad8 (Yu et al. (2008) Nat Med 14: 1363-1369; Cuny et al. (2008) Bioorg. Med. Chem. Lett. 18:4388- 4392, herein incorporated by reference).

[0103] In certain embodiments, the BMP inhibitor comprises LDN193189. In certain embodiments, the BMP inhibitor comprises Noggin.

[0104] In certain embodiments, the stem cells are exposed to one SMAD inhibitor, e.g., one TGFp / Activin-Nodal inhibitor. In certain embodiments, the TGFp / Activin-Nodal inhibitor is SB431542. In certain embodiments, the TGFp / Activin-Nodal inhibitor is a derivative of SB431542. In certain embodiments, the TGFp / Activin-Nodal inhibitor is A83-01.

[0105] In certain embodiments, the stem cells are exposed to two SMAD inhibitors. In certain embodiments, the two SMAD inhibitors are a TGFp / Activin-Nodal inhibitor and a BMP inhibitor. In certain embodiments, the stem cells are exposed to SB431542 or A83-01, and LDN193189 or Noggin. In certain embodiments, the stem cells are exposed to SB431542 and LDN193189. In certain embodiments, the stem cells are exposed to A83-01 and LDN193189. Attorney Docket No. : 072734.1842

[0106] In certain embodiments, the stem cells are exposed to SB431542 and Noggin. In certain embodiments, the stem cells are exposed to A83-01 and Noggin.

[0107] In certain embodiments, the stem cells are exposed to or contacted with at least one SMAD inhibitor for at least about 5 days, or at least about 10 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for up to about 5 days, or up to about 10 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for between about 5 days and about 10 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for about 5 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for 6 days. In certain embodiments, the stem cells are contacted with or exposed to the at least one SMAD inhibitor for 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one SMAD inhibitor from day 0 through day 6. In certain embodiments, the at least one SMAD inhibitor is added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 6. In certain embodiments, the at least one SMAD inhibitor is added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day 6.

[0108] In certain embodiments, the cells are contacted with or exposed to a TGFp / Activin- Nodal inhibitor. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is between about 1 pM and about 20 pM, between about 1 pM and about 10 pM, between about 1 pM and about 15 pM, between about 10 pM and about 15 pM, between about 5 pM and about 10 pM, between about 5 pM and about 15 pM, between about 5 pM and about 20 pM, or between about 15 pM and about 20 pM. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is between about 1 pM and about 10 pM. In certain embodiments, the concentration of the TGFp / Activin-Nodal inhibitor contacted with or exposed to the cells is about 5 pM. about 10 pM. In certain embodiments, the concentration of the TGFp / Activin- Nodal inhibitor contacted with or exposed to the cells is about 10 pM. In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542 or a derivative thereof (e.g., A83-01). In certain embodiments, the TGFp / Activin-Nodal inhibitor comprises SB431542.

[0109] In certain embodiments, the cells are contacted with or exposed to a BMP inhibitor. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 50 nM and about 500 nM, or between about 100 nM and about 500 nM, or between about 200 nM and about 500 nM, or between about 200 and about 300 nM, or between about 200 nM and about 400 nM, or between about 100 nM and about 250 nM, or Attorney Docket No. : 072734.1842 between about 100 nM and about 250 nM, or between about 200 nM and about 250 nM, or between about 250 nM and about 300 nM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is between about 200 nM and about 300 mM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 150 nM, about 200 nM, about 250 nM, about 300 nM, or about 350 nM. In certain embodiments, the concentration of the BMP inhibitor contacted with or exposed to the cells is about 250 nM. In certain embodiments, the BMP inhibitor comprises LDN193189 or a derivative thereof. In certain embodiments, the BMP inhibitor comprises LDN193189.

[0110] In certain embodiments, the cells are contacted with or exposed to the TGFp / Activin- Nodal inhibitor and the BMP inhibitor simultaneously. In certain embodiments, the stem cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor for about 5 days. In certain embodiments, the stem cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor for 6 days. In certain embodiments, the stem cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor for 7 days. In certain embodiments, the cells are contacted with or exposed to the TGFp / Activin-Nodal inhibitor and the BMP inhibitor from day 0 through day 6. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day or every other day to a cell culture medium comprising the stem cells from day 0 through day 6. In certain embodiments, the TGFp / Activin-Nodal inhibitor and the BMP inhibitor are added every day (daily) to a cell culture medium comprising the stem cells from day 0 to day 6.

[0111] 5.2.3. Wnt Activators

[0112] In certain embodiments, the present disclosed subject matter provides compositions comprising at least one Wnt activator, and methods of use. In certain embodiments, the methods comprise contacting cells with at least one Wnt activator. In certain embodiments, the methods comprise contacting cells with a composition comprising at least one Wnt activator. In certain embodiments, the at least one Wnt activator lowers GSK3P activity resulting in activation of Wnt signaling. Thus, in certain embodiments, the Wnt activator is a GSK3P inhibitor. A GSK3P inhibitor is capable of activating a WNT signaling pathway, see e.g., Cadigan, etal., J Cell Sci. 2006;119:395-402; Kikuchi, et al, Cell Signaling. 2007; 19:659-671, which are incorporated by reference herein in their entireties. As used herein, the term “glycogen synthase kinase 3p inhibitor” or “GSK3P inhibitor” refers to a compound that inhibits a glycogen synthase kinase 3p enzyme, for example, see Doble, et al., J Cell Sci. 2003;116: 1175-1186, which is incorporated by reference herein in its entirety. Non-limiting Attorney Docket No. : 072734.1842 examples of GSK3P inhibitors include CHIR99021, BIO ((3E)-6-bromo-3-[3- (hydroxyamino)indol-2-ylidene]-lH-indol-2-one), AMBMP hydrochloride, LP 922056, SB- 216763, CHIR98014, Lithium, 3F8, deoxycholic acid, and those disclosed in WO2011 / 149762, WO13 / 067362, Chambers et al., Nat Biotechnol. 2012 Jul l;30(7):715-20, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Calder et al., J Neurosci. 2015 Aug 19;35(33): 11462-81, all of which are incorporated by reference in their entireties.

[0113] Non-limiting examples of Wnt activators include CHIR99021, Wnt3A, Wntl, Wnt5a, BIO ((3E)-6-bromo-3-[3-(hydroxyamino)indol-2-ylidene]-lH-indol -2-one), AMBMP hydrochloride, LP 922056, SB-216763, CHIR98014, Lithium, 3F8, deoxycholic acid, and those disclosed in WO2011 / 149762, WO13 / 067362, Chambers et al, Nat Biotechnol. 2012 Jul l;30(7):715-20, Kriks et al., Nature. 2011 Nov 6;480(7378):547-51, and Calder et al, J Neurosci. 2015 Aug 19;35(33): 11462-81, all of which are incorporated by reference in their entireties. In certain embodiments, the at least one Wnt activator is a small molecule selected from CHIR99021, Wnt3A, Wntl, Wnt5a, BIO, CHIR98014, Lithium, 3F8, deoxycholic acid, derivatives thereof, and mixtures thereof. In certain embodiments, the at least one Wnt activator comprises CHIR99021 or a derivative thereof. In certain embodiments, the at least one Wnt activator comprises CHIR99021. “CHIR99021” (also known as “aminopyrimidine” or “3-[3-(2-Carboxyethyl)-4-methylpyrrol-2-methylidenyl]-2-indolinone”) refers to IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl)pyrimidin-2-ylamino) ethylamino)nicotinonitrile with the following formula.

[0114] CHIR99021 is highly selective, showing nearly thousand-fold selectivity against a panel of related and unrelated kinases, with an IC50=6.7 nM against human GSK3P and nanomolar IC50 values against rodent GSK3P homologs.

[0115] In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator for at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 15 days, or at least about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt Attorney Docket No. : 072734.1842 activator for up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 15 days, or up to about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt activator for between about 5 days and about 20 days, between about 5 days and about 15 days, between about 10 days and about 20 days, between about 5 days and about 15 days, or between about 10 days and about 15 days. In certain embodiments, the cells are contacted with the at least one Wnt activator for between about 10 days and about 20 days. In certain embodiments, the cells are contacted with the at least one Wnt activator for about 10 days. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for 11 days. In certain embodiments, the cells are contacted with the at least one Wnt activator for about 12 days. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for 13 days. In certain embodiments, the cells are contacted with the at least one Wnt activator for about 14 days. In certain embodiments, the cells are contacted with the at least one Wnt activator for about 15 days. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for 16 days. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for 17 days. In certain embodiments, the cells are contacted with the at least one Wnt activator from day 0 through day 10. In certain embodiments, the at least one Wnt activator is added every day or every other day to a cell culture medium comprising the cells from day 0 through day 10. In certain embodiments, the at least one Wnt activator is added every day (daily) to a cell culture medium comprising the cells from day 0 through day 10.

[0116] In certain embodiments, the concentration of the at least Wnt activator is increased during its exposure to the cells (also referred to as “Wnt Boost”). In certain embodiments, the increase or Wnt Boost is initiated at least about 2 days, at least about 4 days, or at least about 5 days from the initial exposure of the cells to the at least one Wnt activator. In certain embodiments, the increase or Wnt Boost is initiated about 4 days from the initial exposure of the cells to the at least one Wnt activator.

[0117] In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for at least about 5 days, or at least about 10 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for at least about 5 days. In certain embodiments, the cells are contacted with the increased concentration of the at least one Wnt activator for up to about 5 days, up to about 10 days, or up to about 15 days. In certain embodiments, the cells Attorney Docket No. : 072734.1842 are contacted with the increased concentration of the at least one Wnt activator for up to about 10 days.

[0118] In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for between about 5 days and about 15 days, or between about 5 days and about 10 days, or between about 10 days and about 15 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for between about 5 days and about 10 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for about 5 days, about 10 days, or about 15 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for about 5 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for 5 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for 6 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator from day 4 through day 9. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for about 10 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for 12 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator for 13 days. In certain embodiments, the cells are contacted with or exposed to the increased concentration of the at least one Wnt activator from day 4 through day 10.

[0119] In certain embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt Boost is less than about 5 pM, less than about 3 pM, or less than about 1.5 pM, or less than about 1.5 pM, including, but not limited to, between about 0.01 pM and about 5 pM, between about 0.01 pM and about 3 pM, between about 0.05 pM and about 3 pM, between about 0.1 pM and about 3 pM, between about 0.5 pM and about 3 pM, between about 0.5 pM and about 2 pM, between about 0.5 pM and about 1 pM, or between about 0.5 pM and about 1.5 pM. In certain embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt Boost is about 1 pM. In certain embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt Boost is less than about 1.5 pM, e.g., about 1 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, or about 0.9 pM. In certain Attorney Docket No. : 072734.1842 embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt boost is about 1 pM. In certain embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt boost is about 0.5 pM. In certain embodiments, the initial concentration of the at least one Wnt activator contacted with or exposed to the cells prior to the Wnt boost is about 0.7 pM.

[0120] In certain embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is about 3 pM or greater, about 5 pM or greater, about 10 pM or greater, about 15 pM or greater, or about 20 pM or greater. In certain embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is between about 3 pM and about 15 pM, between about 3 pM and about 10 pM, or between about 5 pM and about 10 pM. In certain embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is between about 5 pM and about 10 pM. In certain embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, or aboutlO pM. In certain embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is about 3 pM. In certain embodiments, the increased concentration of the at least one Wnt activator post the Wnt boost is about 6 pM. In certain embodiments, the increased concentration of the at least one Wnt activator post the Wnt boost is about 7 pM. In certain embodiments, the increased concentration of the at least one Wnt activator post the Wnt Boost is about 7.5 pM.

[0121] In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by between about 50% and about 2000%, or between about 100% and about 1500%, or between about 150% and about 1500%, or between about 200% and about 1500%, or between about 250% and about 1500%, or between about 300% and about 1500%, or between about 300% and about 1000%, or between about 300% and about 400%, or between about 500% and about 1000%, or between about 800% and about 1000%, or between about 900% and about 1000%, or between about 950% and about 1000%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by between about 300% and about 1000%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by between about 300% and about 500%. In certain embodiments, the concentration Attorney Docket No. : 072734.1842 of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by between about 900% and about 1000%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 550%, about 600%, about 650%, about 700%, about 750%, about 800%, about 850%, about 900%, about 950%, about 1000%, about 1050%, or about 1100%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 200%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 300%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 350%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 500%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 950%. In certain embodiments, the concentration of the at least one Wnt activator is increased from the initial concentration contacted with or exposed to the cells by about 1000%.

[0122] In certain embodiments, the concentration of the at least one Wnt activator is increased from about 1 pM to between about 5 pM and about 10 pM. In certain embodiments, the concentration of the at least one Wnt activator is increased from about 1 pM to about 6 pM. In certain embodiments, the concentration of the at least one Wnt activator is increased from about 1 pM to between about 3 pM and about 5 pM. In certain embodiments, the concentration of the at least one Wnt activator is increased from about 1 pM to about 3 pM.

[0123] In certain embodiments, the at least one Wnt activator comprises a GSK3P inhibitor. In certain embodiments, the at least one Wnt activator comprises CHIR99021 or a derivative thereof. In certain embodiments, the at least one Wnt activator comprises CHIR99021.

[0124] 5.2.4. SHH Activators

[0125] As used herein, the term “Sonic hedgehog,” “SHH,” or “Shh” refers to a protein that is one of at least three proteins in the mammalian signaling pathway family called hedgehog, another is desert hedgehog (DHH) wile a third is Indian hedgehog (IHH). SHH interacts with at least two transmembrane proteins by interacting with transmembrane molecules Patched (PTC) and Smoothened (SMO). SHH typically binds to PTC, which then allows the activation of SMO as a signal transducer. In the absence of SHH, PTC typically inhibits SMO, which in Attorney Docket No. : 072734.1842 turn activates a transcriptional repressor so transcription of certain genes does not occur. When SHH is present and binds to PTC, PTC cannot interfere with the functioning of SMO. With SMO uninhibited, certain proteins are able to enter the nucleus and act as transcription factors allowing certain genes to be activated (see Gilbert, 2000 Developmental Biology (Sunderland, Mass., Sinauer Associates, Inc., Publishers). In certain embodiments, an SHH activator refers to any molecule or compound that is capable of activating a SHH signaling pathway, including a molecule or compound that is capable of binding to PTC or a SMO.

[0126] In certain embodiments, the present disclosed subject matter provides compositions comprising at least one SHH activator, and methods of use. In certain embodiments, the methods comprise contacting cells with at least one SHH activator. In certain embodiments, the methods comprise contacting cells with a composition comprising at least one SHH activator. In certain embodiments, the at least one SHH activator is selected from the group consisting of molecules that bind to PCT, molecules that bind to SMO, and combinations thereof. Non-limiting examples of SHH activators include those disclosed in WO 10 / 096496, WO13 / 067362, Chambers et al., Nat Biotechnol. 2009 Mar;27(3):275-80, and Kriks et al., Nature. 2011 Nov 6;480(7378):547-51. In certain embodiments, the at least one SHH activator is selected from the group consisting of a SHH protein, a SMO agonist, or a combination thereof. In certain embodiments, the SHH protein is selected from the group consisting of a recombinant SHH, a modified N-terminal SHH, or a combination thereof. In certain embodiments, the recombinant SHH comprises a N-terminal fragment and a C-terminal fragment. In certain embodiments, the modified N-terminal SHH comprises two Isoleucines at the N-terminus. In certain embodiments, the modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to an un-modified N-terminal SHH. In certain embodiments, the modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to an un-modified human N-terminal SHH. In certain embodiments, the modified N-terminal SHH has at least about 80%, about 85%, about 90%, about 95%, or about 99% sequence identity to an unmodified mouse N-terminal SHH. In certain embodiments, the modified N-terminal SHH comprises SHH C25II. In certain embodiments, the modified N-terminal SHH comprises SHH C24II.

[0127] Non-limiting examples of SMO agonists (SAGs) include purmorphamine, GSA10, and 20(S)- hydroxy Cholesterol. In certain embodiments, the SAG comprises purmorphamine.

[0128] In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for at least about 5 days, or at least about 10 days. In certain embodiments, the cells Attorney Docket No. : 072734.1842 are contacted with or exposed to the at least one SHH activator for up to about 5 days, or up to about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for between about 5 days and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for 6 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator for 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one SHH activator from day 0 through day 6. In certain embodiments, the at least one SHH activator is added every day or every other day to a cell culture medium comprising the cells from day 0 through day 6. In certain embodiments, the at least one SHH activator is added every day (daily) to a cell culture medium comprising the cells from day 0 through day 6.

[0129] In certain embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is between about 50 ng / mL and about 1000 ng / mL, between about 100 ng / mL and about 1000 ng / mL, between about 20 ng / mL and about 1000 ng / mL, between about 300 ng / mL and about 1000 ng / mL, between about 400 ng / mL and about 1000 ng / mL, between about 500 ng / mL and about 1000 ng / mL, between about 400 ng / mL and about 800 ng / mL, between about 400 ng / mL and about 700 ng / mL, between about 400 ng / mL and about 600 ng / mL, or between about 500 ng / mL and about 600 ng / mL. In certain embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is between about 400 ng / mL and about 600 ng / mL. In certain embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 550 ng / mL, or about 600 ng / mL. In certain embodiments, the concentration of the at least one SHH activator contacted with or exposed to the cells is about 500 ng / mL.

[0130] In certain embodiments, the at least one activator of SHH signaling comprises SHH C25II.

[0131] 5.2.5. FGF Activators

[0132] FGF family includes secreted signaling proteins (secreted FGFs) that signal to receptor tyrosine kinases. Phylogenetic analysis suggests that 22 Fgf genes can be arranged into seven subfamilies containing two to four members each. Branch lengths are proportional to the evolutionary distance between each gene.

[0133] In certain embodiments, the present disclosed subject matter provides compositions comprising a FGF activator, and methods of use. In certain embodiments, the methods comprise contacting cells with at least one FGF activator. In certain embodiments, the Attorney Docket No. : 072734.1842 methods comprise contacting cells with a composition comprising at least one FGF activator. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF8b, FGF2, FGF4, and derivatives thereof. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF17, FGF18, FGF2, FGF4, and derivatives thereof. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF17, and FGF18.

[0134] The FGF8 subfamily is comprised of FGF8a, FGF8b, FGF17, and FGF18. Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid / hindbrain organizer that produces FGF8a, FGF8b, FGF17 and FGF18. It has been shown that FGF8b functions differently from FGF8a, FGF 17, and FGF 18 (Liu et al., Development. 2003 Dec;130(25):6175- 85). FGF8b is the only protein that can induce the rl gene Gbx2 and strongly activate the pathway inhibitors Spry 1 / 2, as well as repress the midbrain gene Otx2 (Liu 2003). Moreover, FGF8b extends the organizer along the junction between the induced Gbx2 domain and the remaining Otx2 region in the midbrain, correlating with cerebellum development (Liu 2003). By contrast, FGF8a, FGF17, and FGF18 cause expansion of the midbrain and upregulating midbrain gene expression (Liu 2003).

[0135] In certain embodiments, the at least one FGF activator is capable of causing expansion of the midbrain and upregulating midbrain gene expression. In certain embodiments, the at least one FGF activator is capable of promoting midbrain development. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF 17, FGF 18, FGF2, FGF4, derivatives thereof, and combinations thereof. In certain embodiments, the at least one FGF activator is selected from the group consisting of FGF8a, FGF 17, FGF 18, and combinations thereof. In certain embodiments, the at least one FGF activator comprises or is FGF 18.

[0136] In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least about 1 day, at least about 3 days, at least about 5 days, at least about 8 days, or at least about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for up to about 5 days (e.g., up to 5 days, up to 6 days, or up to 7 days), or up to about 10 days (e.g., up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days), or up to about 15 days, or up to about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for at least 4 days and / or for up to 7 days. In certain embodiments, Attorney Docket No. : 072734.1842 the cells are contacted with or exposed to the at least one FGF activator for between about 1 days and about 20 days, between about 1 day and about 15 days, between about 1 day and about 5 days, between about 5 days and about 20 days, between about 5 days and about 15 days, or between about 5 days and about 10 days, between about 10 days and about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for between about 1 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 3 days, about 5 days, or about 8 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for between about 1 days and about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for about 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator for 5 days.

[0137] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated at least about 5 days, or at least about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated no later than about 15 days or no later than about 20 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated no later than 18 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated between about 5 days and about 20 days, between about 5 days and about 20 days, between about 10 days and about 15 days, between about 10 days and 18 days, between about 5 days and about 15 days, or between about 10 days and about 20 days, from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated between about 5 days and about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is Attorney Docket No. : 072734.1842 initiated 12 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated 13 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0138] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least FGF activator for about 5 days. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one FGF activator is initiated 12 days or 13 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least one FGF activator for 4 days or 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one FGF activator from day 12 through day 16. In certain embodiments, the at least one FGF activator is added every day or every other day to a cell culture medium comprising the cells from day 12 through day 16. In certain embodiments, the at least one FGF activator is added every day (daily) to a cell culture medium comprising the cells from day 12 through day 16.

[0139] In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is between about 10 ng / mL and about 500 ng / mL, between about 50 ng / mL and about 500 ng / mL, between about 100 ng / mL and about 500 ng / mL, between about 100 ng / mL and about 400 ng / mL, between about 100 ng / mL and about 300 ng / mL, between about 100 ng / mL and about 200 ng / mL, or between about 100 ng / mL and about 250 ng / mL. In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is between about 100 ng / mL and about 200 ng / mL. In certain embodiments, the concentration of the at least one FGF activator contacted with or exposed to the cells is about 100 ng / mL. In certain embodiments, concentration of the at least one FGF activator contacted with or exposed to the cells is about 200 ng / mL.

[0140] In certain embodiments, the at least one FGF activator comprises FGF18.

[0141] 5.2.6. Wnt Inhibitors

[0142] In certain embodiments, the present disclosed subject matter provides compositions comprising a Wnt inhibitor, and methods of use. In certain embodiments, the methods comprise contacting cells with at least one Wnt inhibitor. In certain embodiments, the methods comprise contacting cells with a composition comprising at least one Wnt inhibitor. Wnt signaling includes canonical Wnt signaling and non-canonical Wnt signaling. In certain embodiments, the at least one Wnt inhibitor is capable of inhibiting canonical Wnt signaling. Attorney Docket No. : 072734.1842

[0143] In certain embodiments, the at least one Wnt inhibitor is capable of inhibiting both canonical Wnt signaling and non-canonical Wnt signaling. Non-limiting examples of Wnt inhibitors that are capable of inhibiting both canonical Wnt signaling and non-canonical Wnt signaling include IWP2, IWRl-endo, IWP-01, Wnt-C59, IWP-L6, IWP12, LGK-974, IWR-1, ETC-159, iCRT3, IWP-4, Salinomycin, Pyrvinium Pamoate, iCRT14, FH535, CCT251545, Wogonin, NCB-0846, Hexachrorophene, KY02111, SO3031 (KY01-I), SO2031 (KY02-I), BC2059, PKF 115-584, Quercetin, NSC668036, G007-LK, and derivatives thereof. In certain embodiments, the at least one Wnt inhibitor is selected from the group consisting of IWP2, IWRl-endo, XAV939, IWP-01, Wnt-C59, IWP-L6, LGK-974, IWR-1, Wnt-C59, ETC-159, iCRT3, IWP-4, ICG-001, Salinomycin, Pyrvinium Pamoate, iCRT14, FH535, CCT251545, KYA1797K, Wogonin, NCB-0846, Hexachrorophene, PNU-74654, KY02111, SO3031 (KY01-I), SO2031 (KY02-I), Triptonide, IWP12, BC2059, PKF115-584, Quercetin, NSC668036, G007-LK, MSAB, LF3, JW55, Isoquercitrin, WIKI4 (Wnt Inhibitor Kinase Inhibitor 4), derivatives thereof, and combinations thereof. In certain embodiments, the at least one inhibitor of Wnt signaling is selected from the group consisting of IWP2, IWRl-endo, IWP-01, IWP12, Wnt-C59, IWP-L6, LGK-974, IWR-1, ETC-159, iCRT3, IWP-4, Salinomycin, Pyrvinium Pamoate, iCRT14, FH535, CCT251545, Wogonin, NCB-0846, Hexachrorophene, KY02111, SO3031 (KY01-I), SO2031 (KY02-I), BC2059, PKF115-584, Quercetin, NSC668036, G007-LK, derivatives thereof, and combinations thereof. In certain embodiments, the at least one inhibitor of Wnt signaling is selected from the group consisting of XAV939, ICG-001, PNU-74654, Triptonide, KYA1797K, MSAB, LF3, JW55, Isoquercitrin, WIKI4, derivatives thereof, and combinations thereof. In certain embodiments, the at least one Wnt inhibitor comprises IWP2 or a derivative thereof.

[0144] In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for at least about 1 day, at least about 3 days, at least about 5 days, at least about 8 days, at least about 10 days, at least about 15 days, or at least about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for up to about 5 days, or up to about 10 days, or up to about 15 days, up to about 20 days, up to about 25 days, or up to about 30 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for between about 1 days and about 20 days, between about 1 day and about 15 days, between about 1 day and about 5 days, between about 5 days and about 20 days, between about 5 days and about 15 days, or between about 5 days and about 10 days, between about 10 days and about 20 days, between about 10 days and about 15 days, or between about 15 days and about 20 days, between about 10 days and about 30 days, between Attorney Docket No. : 072734.1842 about 10 days and about 25 days, between about 15 days and about 30 days, between about 15 days and about 25 days, between about 20 days and about 30 days, between about 20 days and about 25 days, or between about 25 days and about 30 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for between about 1 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for between about 10 day and about 15 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for between about 15 day and about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for about 15 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 6 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 14 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 15 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 19 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor for 16 days, 17 days, 18 days, 21 days, 22 days, or 23 days.

[0145] In certain embodiments, the cells that are contacted with the at least one Wnt inhibitor comprise mDA neuron precursors and mDA neurons.

[0146] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated at least about 5 days, or at least about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated no later than about 15 days or no later than about 20 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated between about 5 days and about 20 days, between about 5 days and about 20 days, between about 10 days and about 15 days, between about 5 days and about 15 days, or between about 10 days and about 20 days, from the initial Attorney Docket No. : 072734.1842 contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated between about 5 days and about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated 11 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated 12 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated 13 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0147] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated about 10 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least Wnt inhibitor for about 5 days. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one Wnt inhibitor is initiated 12 days or 13 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least one Wnt inhibitor for 4 days or 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor from day 12 through day 16. In certain embodiments, the at least one Wnt inhibitor is added every day or every other day to a cell culture medium comprising the cells from day 12 through day 16. In certain embodiments, the at least one Wnt inhibitor is added every day (daily) to a cell culture medium comprising the cells from day 12 through day 16. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor from day 12 through day 25. In certain embodiments, the at least one Wnt inhibitor is added every day or every other day to a cell culture medium comprising the cells from day 12 through day 25. In certain embodiments, the at least one Wnt inhibitor is added every day (daily) to a Attorney Docket No. : 072734.1842 cell culture medium comprising the cells from day 12 through day 25. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor from day 12 through day 30. In certain embodiments, the at least one Wnt inhibitor is added every day or every other day to a cell culture medium comprising the cells from day 12 through day 30. In certain embodiments, the at least one Wnt inhibitor is added every day (daily) to a cell culture medium comprising the cells from day 12 through day 30. In certain embodiments, the cells are contacted with or exposed to the at least one Wnt inhibitor and the at least one FGF activator simultaneously. In certain embodiments, the at least one Wnt inhibitor and the at least one FGF activator together are added to a cell culture medium comprising the cells.

[0148] In certain embodiments, the concentration of the at least one Wnt inhibitor contacted with or exposed to the cells is between about 0.5 pM and about 20 pM, between about 0.5 pM and about 10 pM, between about 0.5 pM and about 5 pM, between about 0.5 pM and about 1 pM, between about 0.5 pM and about 2 pM, between about 5 pM and about 10 pM, between about 10 pM and about 20 pM, between about 1 pM and about 2 pM, or between about 1 pM and about 5 pM. In certain embodiments, the concentration of the at least one Wnt inhibitor contacted with or exposed to the cells is between about 0.5 pM and about 2 pM. In certain embodiments, the concentration of the at least one Wnt inhibitor contacted with or exposed to the cells is about 1 pM.

[0149] In certain embodiments, the at least one Wnt inhibitor comprises IWP2.

[0150] 5.2.7. TGFp / Activin-Nodal Activators

[0151] In certain embodiments, the present disclosed subject matter provides compositions comprising a TGFp / Activin-Nodal activator, and methods of use. In certain embodiments, the methods comprise contacting cells with at least one TGFp / Activin-Nodal activator. In certain embodiments, the methods comprise contacting cells with a composition comprising at least one TGFp / Activin-Nodal activator.

[0152] In certain embodiments, the method comprises inducing activation of TGFp / Activin- Nodal signaling in the cells. In certain embodiments, the stem cells are further contacted with at least an activator of TGFp / Activin-Nodal signaling.

[0153] In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least Attorney Docket No. : 072734.1842 about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, or at least about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for up to about 1 days, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, or up to about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for between about 1 days and about 10 days, between about 1 day and about 5 days, between about 5 days and about 10 days, between about 1 days and about 24 days, between about 1 days and about 25 days, between about 5 days and about 24 days, between about 5 days and about 25 days, between about 10 days and about 24 days, between about 10 days and about 25 days, between about 15 days and about 24 days, between about 15 days and about 25 days, between about 20 days and about 24 days, or between about 20 days and about 25 days.

[0154] In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for between about 1 day and about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for between about 1 day and about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for between about 5 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for between about 1 day and about 24 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin- Nodal activator for between about 1 day and about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 1 day. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 2 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 3 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 6 days. In certain embodiments, the cells are contacted with or exposed to Attorney Docket No. : 072734.1842 the at least one TGFp / Activin-Nodal activator for about 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 8 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 9 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 11 day. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 12 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 13 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 14 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 15 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 16 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 17 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 18 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 19 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 21 day. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 22 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 23 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 24 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator for about 25 days.

[0155] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one TGFp / Activin-Nodal activator is initiated at least about 15 days, at least about 16 days, or at least about 17 days, or at least about 18 days, or at least about 19 days, or at least about 20 days, or at least about 21 days, or at least about 22 days, or at least about 23 days, or at least about 24 days, or at least about 25 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one TGFp / Activin-Nodal Attorney Docket No. : 072734.1842 activator is initiated no later than about 15 days or no later than about 20 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one TGFp / Activin-Nodal activator is initiated between about 15 days and about 20 days, or between about 2 days and about 25 days, from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one TGFp / Activin-Nodal activator is initiated about 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one TGFp / Activin-Nodal activator is initiated 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0156] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one TGFp / Activin-Nodal activator is initiated about 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least one TGFp / Activin-Nodal activator for about 9 days. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one TGFp / Activin-Nodal activator is initiated 15 days or 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least one TGFp / Activin-Nodal activator for 9 days or 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin- Nodal activator from day 16 through day 25. In certain embodiments, the at least one TGFp / Activin-Nodal activator is added every day or every other day to a cell culture medium comprising the cells from day 16 through day 25. In certain embodiments, the at least one TGFp / Activin-Nodal activator is added every day (daily) to a cell culture medium comprising the cells from day 16 through day 25. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator from day 16 through day 40. In certain embodiments, the at least one TGFp / Activin-Nodal activator is added every day or every other day to a cell culture medium comprising the cells from day 16 through day 40. In certain embodiments, the at least one TGFp / Activin-Nodal activator is added every day (daily) to a cell culture medium comprising the cells from day 16 through day 40.

[0157] In certain embodiments, the concentration of the at least one TGFp / Activin-Nodal activator contacted with or exposed to the cells is between about 1 ng / ml and about 50 ng / ml, between about 1 ng / ml and about 40 ng / ml, between about 1 ng / ml and about 30 ng / ml, between Attorney Docket No. : 072734.1842 about 1 ng / ml and about 20 ng / ml, between about 1 ng / ml and about 10 ng / ml, between about 10 ng / ml and about 50 ng / ml, between about 10 ng / ml and about 40 ng / ml, between about 10 ng / ml and about 30 ng / ml, between about 10 ng / ml and about 20 ng / ml, between about 20 ng / ml and about 50 ng / ml, between about 20 ng / ml and about 40 ng / ml, between about 20 ng / ml and about 30 ng / ml, between about 30 ng / ml and about 50 ng / ml, between about 30 ng / ml and about 40 ng / ml, or between about 40 ng / ml and about 50 ng / ml. In certain embodiments, the concentration of the at least one TGFp / Activin-Nodal activator contacted with or exposed to the cells is 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, or about 50 ng / ml.

[0158] In certain embodiments, the at least one TGFp / Activin-Nodal activator comprises Activin A.

[0159] 5.2.8. BMP Activators

[0160] In certain embodiments, the present disclosed subject matter provides compositions comprising at least one BMP activator, and methods of use. In certain embodiments, the methods comprise contacting cells with at least one BMP activator. In certain embodiments, the methods comprise contacting cells with a composition comprising at least one BMP activator. Non-limiting examples of BMP include BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMPIO, BMP11, BMP15, TCS21311 and combinations thereof.

[0161] In certain embodiments, the method comprises inducing activation of BMP signaling in the cells. In certain embodiments, the stem cells are further contacted with at least a BMP.

[0162] In certain embodiments, the cells are contacted with or exposed to the at least one BMP for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, 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, or at least about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for up to about 1 days, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to Attorney Docket No. : 072734.1842 about 19 days, up to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, or up to about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for between about 1 days and about 10 days, between about 1 day and about 5 days, between about 5 days and about 10 days, between about 1 days and about 24 days, between about 1 days and about 25 days, between about 5 days and about 24 days, between about 5 days and about 25 days, between about 10 days and about 24 days, between about 10 days and about 25 days, between about 15 days and about 24 days, between about 15 days and about 25 days, between about 20 days and about 24 days, or between about 20 days and about 25 days.

[0163] In certain embodiments, the cells are contacted with or exposed to the at least one BMP for between about 1 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for between about 1 day and about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for between about 5 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for between about 1 day and about 24 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for between about 1 day and about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 1 day. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 2 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 3 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 6 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 8 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 9 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 11 day. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 12 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 13 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 14 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 15 days. In certain embodiments, the cells are contacted with or exposed Attorney Docket No. : 072734.1842 to the at least one BMP for about 16 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 17 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 18 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 19 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 21 day. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 22 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 23 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 24 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP for about 25 days.

[0164] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one BMP is initiated at least about 15 days, at least about 16 days, or at least about 17 days, or at least about 18 days, or at least about 19 days, or at least about 20 days, or at least about 21 days, or at least about 22 days, or at least about 23 days, or at least about 24 days, or at least about 25 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one BMP is initiated no later than about 15 days or no later than about 20 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one BMP is initiated between about 15 days and about 20 days, or between about 2 days and about 25 days, from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one BMP is initiated about 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one BMP is initiated 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0165] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one BMP is initiated about 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least one BMP for about 9 days. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one BMP is initiated 15 days or 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, Attorney Docket No. : 072734.1842 and the cells are contacted with the at least one BMP for 9 days or 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one BMP from day 16 through day 25. In certain embodiments, the at least one BMP is added every day or every other day to a cell culture medium comprising the cells from day 16 through day 25. In certain embodiments, the at least one BMP is added every day (daily) to a cell culture medium comprising the cells from day 16 through day 25. In certain embodiments, the cells are contacted with or exposed to the at least one BMP from day 16 through day 40. In certain embodiments, the at least one BMP is added every day or every other day to a cell culture medium comprising the cells from day 16 through day 40. In certain embodiments, the at least one BMP is added every day (daily) to a cell culture medium comprising the cells from day 16 through day 40. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP simultaneously.

[0166] In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP simultaneously for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP simultaneously for up to about 1 days, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin- Nodal activator and the at least one BMP simultaneously for between about 1 days and about 10 days, between about 1 day and about 5 days, between about 5 days and about 10 days. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator and the at least one BMP is initiated about 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator and the at least one BMP is initiated about 15 days or 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP simultaneously for about 9 days or 10 days. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator and the at least one BMP from day 16 through day 25. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one Attorney Docket No. : 072734.1842

[0167] TGFp / Activin-Nodal activator and the at least one BMP is initiated about 24 days or about 25 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP simultaneously for about 15 days or about 16 days. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator and the at least one BMP from day 25 through day 40. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator and the at least one BMP is initiated about 15 days or about 16 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP simultaneously for about 24 days or about 25 days. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator and the at least one BMP from day 16 through day 40.

[0168] In certain embodiments, the at least one TGFp / Activin-Nodal activator and the at least one BMP together are added to a cell culture medium comprising the cells.

[0169] In certain embodiments, the concentration of the at least one BMP contacted with or exposed to the cells is between about 1 ng / ml and about 50 ng / ml, between about 1 ng / ml and about 40 ng / ml, between about 1 ng / ml and about 30 ng / ml, between about 1 ng / ml and about 20 ng / ml, between about 1 ng / ml and about 10 ng / ml, between about 10 ng / ml and about 50 ng / ml, between about 10 ng / ml and about 40 ng / ml, between about 10 ng / ml and about 30 ng / ml, between about 10 ng / ml and about 20 ng / ml, between about 20 ng / ml and about 50 ng / ml, between about 20 ng / ml and about 40 ng / ml, between about 20 ng / ml and about 30 ng / ml, between about 30 ng / ml and about 50 ng / ml, between about 30 ng / ml and about 40 ng / ml, or between about 40 ng / ml and about 50 ng / ml. In certain embodiments, the concentration of the at least one BMP contacted with or exposed to the cells is 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, or about 50 ng / ml.

[0170] In certain embodiments, the at least one BMP comprises BMP4, BMP7, or TCS21311.

[0171] 5.2.8. ESRRB Activators

[0172] In certain embodiments, the present disclosed subject matter provides compositions comprising at least one estrogen-related receptor p (ESRRB) activator, and methods of use. In certain embodiments, the methods comprise contacting cells with at least one ESRRB activator. Attorney Docket No. : 072734.1842

[0173] In certain embodiments, the methods comprise contacting cells with a composition comprising at least one ESRRB activator. Non-limiting examples of ESRRB activators include DY-131.

[0174] In certain embodiments, the method comprises inducing activation of ESRRB signaling in the cells. In certain embodiments, the stem cells are further contacted with at least one ESRRB activator.

[0175] In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, 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, or at least about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for up to about 1 days, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, or up to about 25 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for between about 1 days and about 10 days, between about 1 day and about 5 days, between about 5 days and about 10 days, between about 1 days and about 24 days, between about 1 days and about 25 days, between about 5 days and about 24 days, between about 5 days and about 25 days, between about 10 days and about 24 days, between about 10 days and about 25 days, between about 15 days and about 24 days, between about 15 days and about 25 days, between about 20 days and about 24 days, or between about 20 days and about 25 days.

[0176] In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for between about 1 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for between about 1 day and about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for between about 5 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for between about 1 day and about 24 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for between about 1 day and about 25 days. In certain Attorney Docket No. : 072734.1842 embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 1 day. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 2 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 3 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 6 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 8 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 9 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 11 day. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 12 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 13 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 14 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 15 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 16 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 17 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 18 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 19 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 21 day. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 22 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 23 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 24 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator for about 25 days.

[0177] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one ESRRB activator is initiated at least about 15 days, at least about 16 days, or at Attorney Docket No. : 072734.1842 least about 17 days, or at least about 18 days, or at least about 19 days, or at least about 20 days, or at least about 21 days, or at least about 22 days, or at least about 23 days, or at least about 24 days, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor.

[0178] In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one ESRRB activator is initiated about 25 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least one ESRRB activator for about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator from day 25 through day 35. In certain embodiments, the at least one ESRRB activator is added every day or every other day to a cell culture medium comprising the cells from day 25 through day 35. In certain embodiments, the at least one ESRRB activator is added every day (daily) to a cell culture medium comprising the cells from day 25 through day 35. In certain embodiments, the contact of the cells with or the exposure of the cells to the at least one ESRRB activator is initiated about 25 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with the at least one ESRRB activator for about 19 days or about 20 days. In certain embodiments, the cells are contacted with or exposed to the at least one ESRRB activator from day 25 through day 44. In certain embodiments, the at least one ESRRB activator is added every day or every other day to a cell culture medium comprising the cells from day 25 through day 44. In certain embodiments, the at least one ESRRB activator is added every day (daily) to a cell culture medium comprising the cells from day 25 through day 44.

[0179] In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator simultaneously. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator simultaneously for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator simultaneously for up to about 1 days, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days. In certain embodiments, the cells Attorney Docket No. : 072734.1842 are contacted with or exposed to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator simultaneously for between about 1 days and about 10 days, between about 1 day and about 5 days, between about 5 days and about 10 days. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator is initiated about 25 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator is initiated about 25 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator simultaneously for about 10 days. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator is from day 25 through day 35.

[0180] In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator from day 25 through day 40. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator is initiated about 25 days from the initial contact of the cells with or the initial exposure of the cells to the at least one SMAD inhibitor, and the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator simultaneously for about 19 or about 20 days. In certain embodiments, the simultaneous contact or exposure of the cells to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator from day 25 through day 44.

[0181] In certain embodiments, the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator together are added to a cell culture medium comprising the cells.

[0182] In certain embodiments, the concentration of the at least one ESRRB activator contacted with or exposed to the cells is between about 1 ng / ml and about 50 ng / ml, between about 1 ng / ml and about 40 ng / ml, between about 1 ng / ml and about 30 ng / ml, between about 1 ng / ml and about 20 ng / ml, between about 1 ng / ml and about 10 ng / ml, between about 10 ng / ml and about 50 ng / ml, between about 10 ng / ml and about 40 ng / ml, between about 10 Attorney Docket No. : 072734.1842 ng / ml and about 30 ng / ml, between about 10 ng / ml and about 20 ng / ml, between about 20 ng / ml and about 50 ng / ml, between about 20 ng / ml and about 40 ng / ml, between about 20 ng / ml and about 30 ng / ml, between about 30 ng / ml and about 50 ng / ml, between about 30 ng / ml and about 40 ng / ml, or between about 40 ng / ml and about 50 ng / ml. In certain embodiments, the concentration of the at least one ESRRB activator contacted with or exposed to the cells is 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, or about 50 ng / ml.

[0183] In certain embodiments, the at least one ESRRB activator comprises DY-131.

[0184] 5.2.9. Exemplary Methods

[0185] In certain embodiments, the stem cells are contacted with or exposed to at least one TGFp / Activin-Nodal inhibitor (e.g., SB431542, e.g., at a concentration of about 10 pM), at least one BMP inhibitor (e.g., LDN193189, e.g., at a concentration of about 250 nM), and at least one SHH activator (e.g., SHH C25II, e.g., a concentration of about 500 ng / mL) for about

[0186] 5 days (e.g., 7 days, e.g., from day 0 to day 6), and the cells are contacted with the at least one Wnt activator (e.g., CHIR99021, e.g., at a concentration of about 1 pM for about 5 days (e.g., 4 days, e.g., from day 0 to day 3), and at a concentration of about 6 pM for about 5 days (e.g.,

[0187] 6 days, e.g., from day 4 to day 9), and at a concentration of about 3 pM for about 5 days (e.g.,

[0188] 7 days, e.g., from day 10 to day 16). The cells are contacted with or exposed to the at least one FGF activator (e.g., FGF18, e.g., at a concentration of about 100 ng / ml), wherein the contact of the cells with the at least one FGF activator is initiated about 10 days (e.g., 10 days or 12 days) from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one FGF activator for about 5 days (e.g., 5 days (from day 12 to day 16) or 7 days (e.g., from day 10 to day 16). The cells are contacted with or exposed to the at least one Wnt inhibitor (e.g., IWP2, e.g., at a concentration of about 1 pM), wherein the contact of the cells with the at least one Wnt inhibitor is initiated about 10 days (e.g., 10 days or 12 days) from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one Wnt inhibitor for about 5 days (e.g., 5 days (from day 12 to day 16), 7 days (e.g., from day 10 to day 16), about 15 days (e.g., 14 days (from day 12 to day 25), or about 20 days (e.g., 19 days (from day 12 to day 30). The cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator (e.g., Activin A, e.g., at a concentration of about 10 ng / ml), wherein the contact of the cells with the at least one TGFp / Activin-Nodal activator is initiated about 15 days (e.g., 15 days or 16 days) from the Attorney Docket No. : 072734.1842 initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one TGFp / Activin-Nodal activator for about 9 days (e.g., from day 16 to day 25).

[0189] The cells are contacted with or exposed to the at least one BMP (e.g., BMP7, e.g., at a concentration of about 20 ng / ml), wherein the contact of the cells with the at least one BMP is initiated about 15 days (e.g., 15 days or 16 days) from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one BMP for about 9 days (e.g., from day 16 to day 25).

[0190] In certain embodiments, the cells are contacted with or exposed to at least one TGFp / Activin-Nodal activator (e.g., Activin A) and at least one BMP (e.g., BMP7) concurrently for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP concurrently for up to about 1 days, or up to about 2 days, or up to about 3 days, or up to about

[0191] 4 days, or up to about 5 days, or up to about 6 days, or up to about 7 days, up to about 8 days, up to about 9 days, or up to about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for between about 1 days and about 10 days, between about 1 day and about 5 days, between about 5 days and about 10 days.

[0192] In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for between about 1 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for between about 1 day and about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for between about

[0193] 5 day and about 10 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for about 1 day. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for about 2 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for about 3 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for about 4 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently Attorney Docket No. : 072734.1842 for about 5 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for about 6 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin- Nodal activator the at least one BMP concurrently for about 7 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for about 8 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for about 9 days. In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator the at least one BMP concurrently for about 10 days.

[0194] In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP simultaneously, wherein the contact of the cells with the at least one TGFp / Activin-Nodal activator and the at least one BMP is initiated about 16 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one TGFp / Activin-Nodal activator and the at least one BMP for about 9 days. In certain embodiments, the at least one TGFp / Activin-Nodal activator comprises Activin A. In certain embodiments, the at least one BMP comprises BMP7, TCS21311, or combination thereof.

[0195] In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator and the at least one BMP simultaneously, wherein the contact of the cells with the at least one TGFp / Activin-Nodal activator and the at least one BMP is initiated about 25 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one TGFp / Activin-Nodal activator and the at least one BMP for about 15 days. In certain embodiments, the at least one TGFp / Activin-Nodal activator comprises Activin A. In certain embodiments, the at least one BMP comprises BMP4.

[0196] In certain embodiments, the cells are contacted with or exposed to Activin A and BMP7 simultaneously, wherein the contact of the cells with Activin A and BMP7 is initiated about 16 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with Activin A and BMP7 for about 9 days. In certain embodiments, the cells are contacted with or exposed to Activin A and TCS21311 simultaneously, wherein the contact of the cells with Activin A and TCS21311 is initiated about 16 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with Activin A and TCS21311 for about 9 days. Attorney Docket No. : 072734.1842

[0197] In certain embodiments, the cells are contacted with or exposed to Activin A and BMP4 simultaneously, wherein the contact of the cells with Activin A and BMP4 is initiated about 25 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with Activin A and BMP4 for about 15 days.

[0198] In certain embodiments, the cells are contacted with or exposed to the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the ESRRB activator simultaneously, wherein the contact of the cells with the at least one TGFp / Activin-Nodal activator, the at least one BMP, and the at least one ESRRB activator is initiated about 25 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with the at least one TGFp / Activin-Nodal activator and the at least one BMP for about 10 days. In certain embodiments, the at least one TGFp / Activin-Nodal activator comprises Activin A. In certain embodiments, the at least one BMP comprises BMP4. In certain embodiments, the at least one ESRRB activator comprises DY-131.

[0199] In certain embodiments, the cells are contacted with or exposed to Activin A, BMP4, and DY-131 simultaneously, wherein the contact of the cells with Activin A, BMP4, and DY- 131 is initiated about 25 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with Activin A and BMP4 for about 10 days.

[0200] The present disclosure provides an in vitro method for inducing differentiation of stem cells, comprising: (a) contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and (b) contacting the cells with at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling and at least one activator of bone morphogenetic protein (BMP) signaling for up to about 9 days; and (c) contacting the cells with at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling and at least one activator of bone morphogenetic protein (BMP) signaling for up to about 15 days to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof. In certain embodiments, the at least one activator of transforming growth factor beta (TGFP) / Activin- Nodal signaling of step (b) comprises Activin A. In certain embodiments, the at least one BMP of step (b) comprises BMP7, TCS21311, or a combination thereof. In certain embodiments, the at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling of step (c) comprises Activin A. In certain embodiments, the at least one BMP of step (c) comprises BMP4. In certain embodiments, step (c) further comprises contacting the cells with Attorney Docket No. : 072734.1842 at least one activator of estrogen-related receptor p (ESRRB). In certain embodiments, the at least one activator of ESRRB at step (c) comprises DY-131.

[0201] 5.2.10. Cell Culture Media

[0202] In certain embodiments, the above-described inhibitors and activators are added to a cell culture medium comprising the cells. Suitable cell culture media include, but are not limited to, Knockout® Serum Replacement (“KSR”) medium, Neurobasal® medium (NB), N2 medium, B-27 medium, and Essential 8® / Essential 6® (“E8 / E6”) medium, and combinations thereof. KSR medium, NB medium, N2 medium, B-27 medium, and E8ZE6 medium are commercially available. KSR medium is a defined, serum-free formulation optimized to grow and maintain undifferentiated hESCs in culture.

[0203] In certain embodiments, the cell culture medium is a KSR medium. The components of a KSR medium are disclosed in WO2011 / 149762. In certain embodiments, a KSR medium comprises Knockout DMEM, Knockout Serum Replacement, L-Glutamine, Pen / Strep, MEM, and 13 -mercaptoethanol. In certain embodiments, 1 liter of KSR medium comprises 820 mL of Knockout DMEM, 150 mL of Knockout Serum Replacement, 10 mL of 200 mM L- Glutamine, 10 mL of Pen / Strep, 10 mL of 10 mM MEM, and 55 pM of 13 -mercaptoethanol.

[0204] In certain embodiments, the cell culture medium is an E8ZE6 medium. E8ZE6 medium is a feeder-free and xeno-free medium that supports the growth and expansion of human pluripotent stem cells. E8ZE6 medium has been proven to support somatic cell reprogramming. In addition, E8ZE6 medium can be used as a base for the formulation of custom media for the culture of PSCs. One example E8ZE6 medium is described in Chen et al., Nat Methods 2011 May;8(5):424-9, which is incorporated by reference in its entirety. One example E8ZE6 medium is disclosed in WO 15 / 077648, which is incorporated by reference in its entirety. In certain embodiments, an E8ZE6 cell culture medium comprises DMEM / F12, ascorbic acid, selenium, insulin, NaHCOs, transferrin, FGF2 and TGFp. The E8 / E6 medium differs from a KSR medium in that E8ZE6 medium does not include an active BMP ingredient. Thus, in certain embodiments, when an E8ZE6 medium is used to culture the presently disclosed stem cells to differentiate into mDA neurons or precursors thereof, at least one BMP inhibitor is not required to be added to the E8ZE6 medium. In certain embodiments, the when an E8ZE6 medium is used to culture the presently disclosed stem cells to differentiate into mDA neurons or precursors thereof, at least one BMP inhibitor is added to the E8ZE6 medium.

[0205] 5.2.11. Differentiated Cells

[0206] In certain embodiments, the method comprises obtaining a cell population of the differenced cells, wherein at least about 10%, at least about 20%, at least about 30%, at least Attorney Docket No. : 072734.1842 about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the differentiated cells express at least one marker indicating a mDA neuron or a precursor thereof. Non-limiting examples of markers indicating a mDA neuron or a precursor thereof include engrailed- 1 (EN1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor related- 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1 A), PITX3, LM03, SNCA, ADCAP1, CHRNA4, ALDH1A1, DAT, VMAT1, SOX6, WNT1, and GIRK2.

[0207] In certain embodiments, the differentiated cells express the at least one marker indicating a mDA neuron or a precursor thereof at least about 10 days (e.g., about 15 days, about 20 days, about 30 days, about 40 days, or about 50 days) from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the differentiated cells express the at least one marker indicating a mDA neuron or a precursor thereof about 15 days (e.g., 15 days, 16 days, or 17 days) from the initial contact of the cells with the at least one SMAD inhibitor.

[0208] The treatment of the cells with at least Wnt inhibitor can improve mDA neuron derivation. In certain embodiments, the treatment of the cells with at least Wnt inhibitor increases expression of at least one of A9 subtype mDA neuron markers, A10 subtype mDA neuron markers, and mDA neuron maturity markers. In certain embodiments, the treatment of the cells with at least Wnt inhibitor increases expression of DAT. In certain embodiments, the treatment of the cells with at least Wnt inhibitor increases expression of VMAT2. In certain embodiments, the treatment of the cells with at least Wnt inhibitor increases expressions of DAT and VMAT2.

[0209] In certain embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%) of the differentiated cells express ALDH1 Al about 15 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%) of the differentiated cells express ALDH1 Al 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%) of the differentiated cells express ALDH1 Al about 25 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least Attorney Docket No. : 072734.1842 about 90%) of the differentiated cells express ALDH1 Al about 30 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.

[0210] Furthermore, the mDA neurons or precursors thereof generated by the methods disclosed herein have improved fiber outgrowth, reduced remaining Ki67+proliferating cells, and improved in vivo survival, which make these cells more suitable for therapeutic uses. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker at least about 2 weeks post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein can have a detectable expression level of at least one mDA neuron marker up to about 1 month, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 1 year, up to about 2 years, up to about 3 years, up to about 4 years, or up to about 5 years post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker about 1 month post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one mDA neuron marker about 2 months post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1A1 at least about 1 month post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1A1 about 2 months post in vivo transplantation. In certain embodiments, the mDA neurons or precursors thereof generated by the methods disclosed herein have a detectable expression level of at least one marker selected from the group consisting of TH, EN1, NURR1, and ALDH1A1 at least about 2 months post in vivo transplantation. Attorney Docket No. : 072734.1842

[0211] In certain embodiments, the differentiated cells derived from the presently disclosed methods do not express or have a low expression of at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0212] In certain embodiments, the cells are contacted with the activators and inhibitors described herein at a concentration and time effective to decrease expression of SMA, SIX1, PITX2, SIM1, POU4F1, and / or PHOX2A. In certain embodiments, the cells are contacted with the activators and inhibitors described herein at a concentration and time effective to decrease expression of PAX6, BARHL1, and / or BARHL2.

[0213] In certain embodiments, at least about 80% of the differentiated cells express FOXA2 and EN1 about 15 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling. In certain embodiments, greater than about 80% (e.g., greater than about 85% or greater than about 90%) of the differentiated cells express FOXA2 and EN1 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.

[0214] 5.2.12. Sorting Methods

[0215] In certain embodiments, the differentiation methods disclosed herein further comprise isolating mDA neurons and precursors thereof based on at least one or at least two markers. In certain embodiments, the marker is a negative marker, wherein the cells do not express a detectable level of the negative marker. In certain embodiments, the marker is a positive marker, wherein the cells express a detectable level of the positive marker.

[0216] In certain embodiments, the differentiation methods disclosed herein further comprise isolating cells that do not express a detectable level of at least one negative marker. In certain embodiments, the differentiation methods disclosed herein further comprise isolating cells that express a detectable level of at least one positive marker. In certain embodiments, the differentiation methods disclosed herein further comprise isolating cells that do not express a detectable level of at least one negative marker and express a detectable level of at least one positive marker.

[0217] In certain embodiments, the at least one negative marker is selected from the group consisting of CD49e, CD99, CD340, and combinations thereof. In certain embodiments, the at least one negative marker comprises CD49e. In certain embodiments, the differentiation methods disclosed herein further comprise isolating mDA neurons that do not express a detectable level of CD49e, CD340, and combination thereof. In certain embodiments, the differentiation methods disclosed herein further comprise isolating mDA neurons that do not express a detectable level of CD49e, CD99, CD340, and combinations thereof. In certain Attorney Docket No. : 072734.1842 embodiments, the at least one positive marker is selected from the group consisting of PITX3, ALDH1A1, TH, and combinations thereof. In certain embodiments, the at least one positive marker comprises ALDH1A1. In certain embodiments, the differentiation methods disclosed herein further comprise isolating mDA neuron precursors (e.g., neuroblasts) that express a detectable level of CD99.

[0218] Any surface-marker based cell isolation technology known in the art can be used in the presently disclosed methods. In certain embodiments, flow cytometry is used to the presently disclosed isolation methods.

[0219] 5.2.13. Differentiation of mDA Precursors to mDA neurons

[0220] In certain embodiments, the cells (e.g., mDA precursors) are further contacted with DA neuron lineage specific activators and inhibitors, for example, L-glutamine, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), Cyclic adenosine monophosphate (cAMP), Transforming growth factor beta (TGFP, for example, TGFP3), ascorbic acid (AA), and DAPT (which is also known as, N-[(3,5-Difluorophenyl)acetyl]-L- alanyl-2-phenyl]glycine-l,l-dimethylethyl ester; LY-374973, N-[N-(3,5-Difluorophenacetyl)- L-alanyl]-S-phenylglycine t-butyl ester; or N-[N- (3,5-difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester). In certain embodiments, the cells are contacted with the foregoing DA neuron lineage specific activators and inhibitors for at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 or more days, for example, between about 2 days and about 20 days, between about 3 days and about 19 days, between about 4 days and about 18 days, between about 5 days and about 17 days, between about 6 days and about 16 days, between about 7 days and about 15 days, between about 8 days and about 15 days, between about 9 days and about 14 days, or between about 10 days and about 13 days. In certain embodiments, the cells are contacted with the foregoing DA neuron lineage specific activators and inhibitors for up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, or up to about 10 days or more days. In certain embodiments, the cells are contacted with the foregoing DA neuron lineage specific activators and inhibitors for about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0221] In certain embodiments, the cells are contacted with L-glutamine at a concentration of between about 0.5 mM and about 5 mM, or between about 1 mM and about 5 mM, or between about 1.5 mM and about 2.5 mM, or between about 1 mM and about 2 mM. In certain embodiments, the cells are contacted with L-glutamine at a concentration of about 2 mM. Attorney Docket No. : 072734.1842

[0222] In certain embodiments, the cells are contacted with BDNF at a concentration of between about 5 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 40 ng / mL, or between about 20 ng / ml and about 50 ng / mL, or between about 20 ng / ml and about 40 ng / mL, or between about 10 ng / ml and about 30 ng / mL, or between about 10 ng / ml and about 20 ng / mL, or between about 20 ng / ml and about 30 ng / mL. In certain embodiments, the cells are contacted with BDNF at a concentration of about 20 ng / mL.

[0223] In certain embodiments, the cells are contacted with ascorbic acid (AA) at a concentration of between about 50 nM and about 500 nM, or between about 100 nM and about 500 nM, or between about 100 nM and about 400 nM, or between about 200 nM and about 400 nM, or between about 200 nM and about 300 nM, or between about 100 nM and about 300 nM. In certain embodiments, the cells are contacted with AA at a concentration of about 200 nM.

[0224] In certain embodiments, the cells are contacted with GDNF at a concentration of between about 5 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 50 ng / mL, or between about 10 ng / ml and about 40 ng / mL, or between about 20 ng / ml and about 50 ng / mL, or between about 20 ng / ml and about 40 ng / mL, or between about 10 ng / ml and about 30 ng / mL, or between about 10 ng / ml and about 20 ng / mL, or between about 20 ng / ml and about 30 ng / mL. In certain embodiments, the cells are contacted with GDNF at a concentration of about 20 ng / mL.

[0225] In certain embodiments, the cells are contacted with cAMP at a concentration of between about 200 nM and about 800 nM, or between about 200 nM and about 700 nM, or between about 300 nM and about 700 nM, or between about 300 nM and about 600 nM, or between about 400 nM and about 600 nM, or between about 450 nM and about 550 nM. In certain embodiments, the cells are contacted with cAMP at a concentration of about 500 nM.

[0226] In certain embodiments, the cells are contacted with TGFP3 at a concentration of between about 0.01 ng / ml and about 5 ng / mL, or between about 0.1 ng / ml and about 4 ng / mL, or between about 0.5 ng / ml and about 5 ng / mL, or between about 1 ng / ml and about 3 ng / mL, or between about 1 ng / ml and about 2 ng / mL. In certain embodiments, the cells are contacted with TGFP3 at a concentration of about 1 ng / mL.

[0227] In certain embodiments, the cells are contacted with DAPT at a concentration of between about 1 nM and about 50 nM, or between about 5 nM and about 50 nM, or between about 1 nM and about 20 nM, or between about 5 nM and about 20 nM, or between about 1 nM and about 10 nM, or between about 5 nM and about 10 nM, or between about 5 nM and about 15 nM, or between about 10 nM and about 20 nM, or between about 10 nM and about Attorney Docket No. : 072734.1842

[0228] 30 nM, or between about 30 nM and about 50 nM,. In certain embodiments, the cells are contacted with DAPT at a concentration of about 10 nM.

[0229] In certain embodiments, the differentiated midbrain DA precursors are further cultured as described by U.S. Publication No. 2015 / 0010514, which is incorporated by reference in its entirety.

[0230] 5.3. Cell Populations and Compositions

[0231] The presently disclosure provides a cell population of in vitro differentiated cells obtained by the methods disclosed herein, for example, in Section 5.2.

[0232] The presently disclosure provides a cell population of in vitro differentiated cells, wherein at least about 50% (e.g., at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of the cells express at least one marker indicating a mDA neuron or a precursor thereof. Non-limiting examples of markers indicating a mDA neuron or a precursor thereof include EN1, OTX2, TH, NURR1, FOXA2, LMX1A, PITX3, LM03, SNCA, ADCAP1, CHRNA4, SOX6, ALDH1A1, WNT1, DAT, VMAT1, and GIRK2. The presently disclosure also provides compositions comprising such cell populations. In certain embodiments, the in vitro differentiated cells are obtained by the differentiation methods described herewith, for example, in Section 5.2.

[0233] In certain embodiments, less than about 50% (e.g., less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1%) of the differentiated cells express at least one marker selected from PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, H0XA1, H0XA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.

[0234] In certain embodiments, the present disclosure, provides compositions comprising any of the cell populations disclosed herein.

[0235] In certain embodiments, the cells are comprised in a composition that further comprises a biocompatible scaffold or matrix, for example, a biocompatible three-dimensional scaffold that facilitates tissue regeneration when the cells are implanted or grafted to a subject. In certain embodiments, the biocompatible scaffold comprises extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogel. (See, e.g., U.S. Publication Nos. 2015 / 0159135, Attorney Docket No. : 072734.1842

[0236] 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entireties). In certain embodiments, the composition further comprises growth factors for promoting maturation of the implanted / grafted cells into midbrain DA cells.

[0237] In certain embodiments, the composition comprises a cell population of from about 1 x

[0238] 104to about 1 x 1010, from about 1 x 104to about 1 x 105, from about 1 x 105to about 1 x 109, from about 1 x 105to about 1 x 106, from about 1 x 105to about 1 x 107, from about 1 x 106to about 1 x 107, from about 1 x 106to about 1 x 108, from about 1 x 107to about 1 x 108, from about 1 x 108to about 1 x 109, from about 1 x 108to about 1 x 1010, or from about 1 x 109to about 1 x 1010the cells are administered to a subject. In certain embodiments, from about 1 x

[0239] 105to about 1 x 107the cells thereof are administered to a subject.

[0240] In certain embodiments, said composition is frozen. In certain embodiments, said composition further comprises at least one cryoprotectant, for example, but not limited to, dimethylsulfoxide (DMSO), glycerol, polyethylene glycol, sucrose, trehalose, dextrose, or a combination thereof.

[0241] In certain embodiments, the composition further comprises a biocompatible scaffold or matrix, for example, a biocompatible three-dimensional scaffold that facilitates tissue regeneration when the cells are implanted or grafted to a subject. In certain embodiments, the biocompatible scaffold comprises extracellular matrix material, synthetic polymers, cytokines, collagen, polypeptides or proteins, polysaccharides including fibronectin, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin, and / or hydrogel. (See, e.g., U.S. Publication Nos. 2015 / 0159135, 2011 / 0296542, 2009 / 0123433, and 2008 / 0268019, the contents of each of which are incorporated by reference in their entireties).

[0242] In certain embodiments, the composition is a pharmaceutical composition that comprises a pharmaceutically acceptable carrier. The compositions can be used for preventing and / or treating a neurodegenerative disorder include Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and multiple sclerosis.

[0243] In certain embodiments, the presently disclosed subject matter also provides a device comprising the differentiated cells or the composition comprising thereof, as disclosed herein. Non-limiting examples of devices include syringes, fine glass tubes, stereotactic needles and cannulas. Attorney Docket No. : 072734.1842

[0244] 5.4. Method of Preventing, Modeling, and / or Treating Neurological Disorders

[0245] The cell populations and compositions disclosed herein (e.g., those disclosed in Section 5.3) can be used for preventing, modeling, and / or treating at least a symptom in a subject having a neurological disorder. In certain embodiments, the presently disclosed subject matter provides for methods of preventing, modeling, and / or treating at least a symptom in a subject having a neurological disorder. In certain embodiments, the present disclosure provides methods of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder, comprising administering to the subject an effective amount of the cell population as disclosed herein. In certain embodiments, the methods of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder comprise administering to the subject an effective amount of the composition as disclosed herein. In certain embodiments, the present disclosure provides methods of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder, comprising administering to the subject an effective amount of one of the following: (a) the cell population as disclosed herein; or (b) the composition as disclosed herein. In certain embodiments, the method comprises administering an effective amount of the presently disclosed stem-cell- derived mDA neurons or a composition comprising thereof into a subject suffering from a neurological disorder. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0246] In certain embodiments, the neurological disorder is characterized by reduction of mDA neuron function. The reduction of mDA neuron function can be age related.

[0247] In certain embodiments, the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia.

[0248] Non-limiting examples of neurological disorders include Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and multiple sclerosis. In certain embodiments, the neurological disorder is Parkinsonism or Parkinson’s disease.

[0249] In certain embodiments, the neurological disorder is Parkinson’s disease. Primary motor signs of Parkinson’s disease include, for example, but not limited to, tremor of the hands, arms, legs, jaw and face, bradykinesia or slowness of movement, rigidity or stiffness of the limbs and trunk and postural instability or impaired balance and coordination.

[0250] In certain embodiments, the neurological disorder is a parkinsonism disease, which refers to diseases that are linked to an insufficiency of dopamine in the basal ganglia, which is a part of the brain that controls movement. Symptoms include tremor, bradykinesia (extreme Attorney Docket No. : 072734.1842 slowness of movement), flexed posture, postural instability, and rigidity. Non-limiting examples of parkinsonism diseases include corticobasal degeneration, Lewy body dementia, multiple systematrophy, and progressive supranuclear palsy.

[0251] The cells or compositions can be administered or provided systemically or directly to a subject for preventing, modeling, and / or treating a neurological disorder. In certain embodiments, the cells or compositions are directly injected into an organ of interest (e.g., the central nervous system (CNS)). In certain embodiments, the cells or compositions are directly injected into the striatum.

[0252] The cells or compositions can be administered in any physiologically acceptable vehicle. The cells or compositions can be administered via localized injection, orthotopic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In certain embodiments, the cells or compositions are administered to a subject suffering from a neurodegenerative disorder via orthotopic (OT) injection.

[0253] The cells or compositions can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g., a composition comprising the presently disclosed stem-cell-derived precursors, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON’S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. Attorney Docket No. : 072734.1842

[0254] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, alum inum monostearate and gelatin.

[0255] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0256] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the presently disclosed stem-cell-derived precursors. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0257] One consideration concerning the therapeutic use of the cells is the quantity of cells necessary to achieve an optimal effect. An optimal effect includes, but is not limited to, repopulation of CNS regions of a subject suffering from a neurodegenerative disorder, and / or improved function of the subject’s CNS.

[0258] An “effective amount” (or “therapeutically effective amount”) is an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in at least one doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the neurodegenerative disorder, or otherwise reduce the pathological consequences of the neurodegenerative disorder. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These Attorney Docket No. : 072734.1842 factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the cells administered.

[0259] In certain embodiments, an effective amount of the cells is an amount that is sufficient to repopulate a CNS region of a subject suffering from a neurological disorder. In certain embodiments, an effective amount of the cells is an amount that is sufficient to improve the function of the CNS of a subject suffering from a neurodegenerative disorder, e.g., the improved function can be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99% or about 100% of the function of a normal person’s CNS.

[0260] The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, from about 1 x 104to about 1 x 1010, from about 1 x 104to about 1 x 105, from about 1x105to about 1xio9, from about 1x105to about 1xio6, from about 1x105to about 1x107, from about 1 x 106to about 1 x io7, from about 1 x 106to about 1 x 108, from about 1 x 107to about 1 x io8, from about 1 x 108to about 1 x io9, from about 1 x io8to about 1 x io10, or from about 1 x 109to about 1 x io10of the cells are administered to a subject. In certain embodiments, from about 1 x io5to about 1 x io7of the cells are administered to a subject suffering from a neurological disorder. In certain embodiments, from about 1 x io6to about 1 x 107of the cells are administered to a subject suffering from a neurological disorder. In certain embodiments, from about 1 x 106to about 4 x 106of the cells are administered to a subject suffering from a neurological disorder. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

[0261] 5.5. Kits

[0262] In certain embodiments, the presently disclosed subject matter provides kits for inducing differentiation of stem cells to mDA neurons or precursors thereof. In certain embodiments, the kit comprises (a) at least one inhibitor of SMAD signaling, (b) at least one activator of Wnt signaling, (c) at least one activator of SHH signaling, (d) at least one activator of FGF signaling, and (e) at least one inhibitor of Wnt signaling. In certain embodiments, the kit further comprises (f) instructions for inducing differentiation of the stem cells into a population of differentiated cells that express at least one marker indicating a mDA neuron or a precursor thereof. Attorney Docket No. : 072734.1842

[0263] In certain embodiments, the instructions comprise contacting the stem cells with the inhibitor(s) and activator(s) in a specific sequence. The sequence of contacting the inhibitor(s) and activator(s) can be determined by the cell culture medium used for culturing the stem cells.

[0264] In certain embodiments, the instructions comprise contacting the stem cells with the inhibitor(s) and activator(s) as described by the methods of the present disclosure (see Section 5.2).

[0265] In certain embodiments, the present disclosure provides kits comprising an effective amount of a cell population or a composition disclosed herein in unit dosage form. In certain embodiments, the kit comprises a sterile container which contains the therapeutic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0266] In certain embodiments, the kit comprises instructions for administering the cell population or composition to a subject suffering from a neurological disorder. The instructions can comprise information about the use of the cells or composition for preventing, modeling, and / or treating at least a symptom in a subject having a neurological disorder. In certain embodiments, the instructions comprise at least one of the following: description of the therapeutic agent; dosage schedule and administration for preventing, modeling, and / or treating at least a symptom in a subject having a neurological disorder or symptoms thereof; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0267] 7. EXAMPLES

[0268] The presently disclosed subject matter will be better understood by reference to the following Example, which is provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

[0269] This invention presents a novel method for differentiating human pluripotent stem cells (hPSCs) into A9 midbrain dopaminergic neurons (mDA), the subtype most affected by degeneration in Parkinson’s Disease (PD). Utilizing single-cell-RNA sequencing, the developmental trajectory of hPSC-mDA neuron differentiation was mapped, identifying both progenitors and neuroblast, as well as A9 and A10 neuronal subtypes. Further analysis revealed TGFp / Activin-Nodal and BMP signaling pathways as critical regulators of mDA Attorney Docket No. : 072734.1842 progenitor differentiation. Activation of these pathways through Activin A and BMP7 leads to the preferential expression of A9 markers and a reduction of A10 markers in vitro, indicative of A9 mDA neuron generation.

[0270] Example 1: Exemplary Midbrain DA Neuron Differentiation Protocol incorporating

[0271] Activin A / BMP7 treatment

[0272] The following is an exemplary protocol of the presently disclosed method in accordance with certain embodiments.

[0273] Day 0: Cells were fed with Accutase from hPSC / hiPSC at single cells and plate at a density of 400,000 cells / cm2on Geltrex-coated plated in Medium 1 with Y-drug.

[0274] Day 1 - Day 2: cells should have reached 100% confluence. Cells were double fed with Medium 1.

[0275] Day 3 : Cells were fed with Medium 1.

[0276] Day 4: Cells were fed with Medium 2. For CHIR-Boost protocol, CHIR concentration was changed from 1 pM to 6 pM for WA-09 hESC line-mediated differentiation (this can be slightly vary depending on hPSC / hiPSC lines).

[0277] Day 5 - Day 6: Cells were double fed with Medium 2.

[0278] Day 7: Cells were fed with Medium 3.

[0279] Day 8 - Day 9: Cells were fed daily with Medium 3.

[0280] Day 10: Cells were fed with Medium 4.

[0281] Day 11 : Cells were incubated with Accutase for 30 minutes at 37 C; plate cells at a density of 800,000 cells / cm2in Medium 4.

[0282] Day 12: Cells should have reached 100% confluence. Cells were fed with Medium 5.

[0283] Day 12 - 16: Cells were fed daily with Medium 5; at day 16, more than 90% of cells were FOXA2+ / EN+, as measured by FACS analysis.

[0284] Day 16 - 25: Cells were fed daily with Medium 6.

[0285] Day 25 - Day 100: Cells were fed daily with Medium 7.

[0286] Medium 1 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, 10 pM SB, 250 nM LDN, 500 ng / ml SHH C5II, and 1 pM CHIR.

[0287] Medium 2 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C5II, and 6 pM CHIR.

[0288] Medium 3 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, and 6 pM CHIR. Attorney Docket No. : 072734.1842

[0289] Medium 4 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 3 pM CHIR, 20 ng / ml BDNF, 0.2 pM ascorbic acid (AA), 20 ng / ml GSNF, 0.5 mM dcAMP, and 1 ng / ml TGF-P3.

[0290] Medium 5 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 3 pM CHIR, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF- 33, 1 pM IWP2, and 100 ng / ml FGF18.

[0291] Medium 6 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, 10 nM DAPT, 10 ng / ml Activin A (ActA), and 20 ng / ml BMP7.

[0292] Medium 7 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, and 10 nM DAPT.

[0293] The protocol described in this Example is referred to as “Wnt-boost + ActA (day 16- day 25) + BMP7 (day 16-day 25)” in Example 2.

[0294] Example 2: Exemplary Midbrain DA Neuron Differentiation Protocol

[0295] The “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” protocol is described in Example 1.

[0296] The “Wnt-boost” protocol referred to in this Example is provided below and in Figure 1A.

[0297] Day 0: Cells were fed with Accutase from hPSC / hiPSC at single cells and plate at a density of 400,000 cells / cm2on Geltrex-coated plated in Medium 1 with Y-drug.

[0298] Day 1 - Day 2: cells should have reached 100% confluence. Cells were double fed with Medium 1.

[0299] Day 3 : Cells were fed with Medium 1.

[0300] Day 4: Cells were fed with Medium 2. For CHIR-Boost protocol, CHIR concentration was changed from 1 pM to 6 pM for WA-09 hESC line-mediated differentiation (this can be slightly vary depending on hPSC / hiPSC lines).

[0301] Day 5 - Day 6: Cells were double fed with Medium 2.

[0302] Day 7: Cells were fed with Medium 3.

[0303] Day 8 - Day 9: Cells were fed daily with Medium 3.

[0304] Day 10: Cells were fed with Medium 4.

[0305] Day 11 : Cells were incubated with Accutase for 30 minutes at 37 C; plate cells at a density of 800,000 cells / cm2in Medium 4.

[0306] Day 12: Cells should have reached 100% confluence. Cells were fed with Medium 5. Attorney Docket No. : 072734.1842

[0307] Day 12-16: Cells were fed daily with Medium 5; at day 16, more than 90% of cells were FOXA2+, as measured by FACS analysis.

[0308] Day 16- DaylOO: Cells were fed daily with Medium 6.

[0309] Medium 1 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, 10 pM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 1 pM CHIR.

[0310] Medium 2 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C25II, and 6 pM CHIR.

[0311] Medium 3 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, and 6 pM CHIR.

[0312] Medium 4 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 3 pM CHIR, 20 ng / ml BDNF, 0.2 pM ascorbic acid (AA), 20 ng / ml GDNF, 0.5 mM dcAMP, and 1 ng / ml TGF-P3.

[0313] Medium 5 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3.

[0314] Medium 6 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, and 10 nM DAPT.

[0315] Single cell RNA-sequencing experiments were conducted at Day 16, Day 25, and Day 40 of differentiation to fully characterize the heterogeneity of the cells following the Wnt-boost protocol (Figure IB). The resulting cells included populations that expressed markers consistent with progenitor cells (WNT1+ / SHH+ / TOP2A+ / MKI67+), neuroblast cells (NR4A2+ / LMX1A+ / FOXA2+ / EBF1+), A9-like midbrain dopamine neurons (mDA) (LMO3+ / KCNJ6+ / TH+ / NR4A2+), and AlO-like mDA (CALB1+ / CALB2+ / TH+ / NR4A2+). A9-like mDA and AlO-like mDA populations were observed at Day 40 of differentiation (Figure 2).

[0316] The single cell RNA-sequencing data were used to identify active signaling networks in the cell populations. BMP (Figure 3A) and TGFP (Figure 3B) signaling networks were found to be active in neuronal progenitors, which are evident at Day 16 following the Wnt- boost differentiation protocol.

[0317] Following these results, modified differentiation protocols were tested comprising modulation of BMP and / or TGFP signaling from Day 16 to Day 25 (Figure 4) using human pluripotent stem cells. Single cell RNA-sequencing experiments were conducted and marker expression was evaluated at Day 25. The proportion of CD99+ cells at Day 25 following the Wnt-boost protocol was 31.1% (Figure 5C). This proportion was increased following exposure Attorney Docket No. : 072734.1842 of the cells to ActA and / or BMP7 from Day 16 to Day 25. The proportion was decreased following dual SMAD inhibition (SB431542 and LDN193189) from Day 16 to Day 25. A9- like mDA neurons were identified from the Day 25 populations as cells exhibiting PITX3 expression (PITX3::GFP, Figure 5B) and ALDH activity. The proportion of A9-like mDA neurons at Day 25 of the Wnt-boost protocol was 1.17% among CD99+ cells and 0.39% among CD99- cells. These proportions increased to 2.59%-5.31% following addition of ActA or BMP7; however, the proportions greatly increased following addition of both ActA and BMP7 to 17% among CD99+ populations and to 18.8% among CD99- populations (Figure 5C). Addition of both ActA and BMP7 additionally reduced expression of markers for AlO-like mDA neurons at Day 25, specifically, CALB1 and NR4A2 (Figures 6A-6D).

[0318] Induced pluripotent stem cells (KOLF2.1) were differentiated using the “Wnt-boost” or “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” protocols and evaluated for markers of neuroblasts (CD99), mDA neurons (TH), and A9 subtype mDA neurons (ALDH activity) by flow cytometry. CD99 expression was increased following the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” protocol at Day 25 (Figure 7). For both differentiation protocols, the CD99- population was enriched for expression of TH versus the CD99+ population. The proportion of cells exhibiting both TH expression and ALDH activity was increased following the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” protocol versus the Wnt-boost protocol; although the proportion within the CD99- population (18.8%) was larger than the proportion within the CD99+ population (7.61%).

[0319] Single cell RNA-sequencing experiments were conducted at Day 25 of differentiation to fully characterize the heterogeneity of the cells following the “Wnt-boost + ActA (day 16- day 25) + BMP7 (day 16-day 25)” (Figure 8 A). Marker expression was evaluated for mDA neurons (Figure 8B), A9 subtype mDA neurons (Figure 8C), and A10 subtype mDA (Figure 8D). Twelve clusters were identified, of which cluster #6 was enriched for markers for A9 subtype mDA neurons (ALDH1A1 and LM03). Transcription factor motifs enriched in cluster #6 were evaluated (Figures 8E-8G).

[0320] A FACS sorting strategy was tested for isolating A9-like mDA neurons and neuroblasts. Cells were sorted based on CD99 expression and / or ALDH activity (Figures 9A-9B). Spheroids produced from the sorted CD99+ / ALDH+ population yielded A9 subtype mDA neurons at Day 40 (Figures 10A-10B).

[0321] Cells differentiated according to the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” protocol were evaluated for mDA neuron marker expression at Day 40 in comparison to cells differentiated according to the Wnt-boost protocol. Cells differentiated Attorney Docket No. : 072734.1842 according to the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” protocol exhibited increased expression of ALDH1A1, a marker of A9 subtype mDA neurons, and decreased expression of CALB1, a marker of A10 subtype mDA neurons (Figures 11 A-l IB).

[0322] Example 3: Exemplary Midbrain DA Neuron Differentiation Protocol incorporating

[0323] Activin A / BMP7 andActivin A / BMP4 treatments

[0324] The “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25) and ActA (day 25- day 40) + BMP4 (day 25-day 40)” protocol is provided below and in Figure 12A.

[0325] The “Wnt-boost” protocol referred to in this Example is described in Example 2 and in Figure 1A.

[0326] Day 0: Cells were fed with Accutase from hPSC / hiPSC at single cells and plate at a density of 400,000 cells / cm2on Geltrex-coated plated in Medium 1 with Y-drug.

[0327] Day 1 - Day 2: cells should have reached 100% confluence. Cells were double fed with Medium 1.

[0328] Day 3 : Cells were fed with Medium 1.

[0329] Day 4: Cells were fed with Medium 2. For CHIR-Boost protocol, CHIR concentration was changed from 1 pM to 6 pM for WA-09 hESC line-mediated differentiation (this can be slightly vary depending on hPSC / hiPSC lines).

[0330] Day 5 - Day 6: Cells were double fed with Medium 2.

[0331] Day 7: Cells were fed with Medium 3.

[0332] Day 8 - Day 9: Cells were fed daily with Medium 3.

[0333] Day 10: Cells were fed with Medium 4.

[0334] Day 11 : Cells were incubated with Accutase for 30 minutes at 37 C; plate cells at a density of 800,000 cells / cm2in Medium 4.

[0335] Day 12: Cells should have reached 100% confluence. Cells were fed with Medium 5.

[0336] Day 12-16: Cells were fed daily with Medium 5; at day 16, more than 90% of cells were FOXA2+, as measured by FACS analysis.

[0337] Day 16 - 25: Cells were fed daily with Medium 6.

[0338] Day 25 - 40: Cells were fed daily with Medium 7.

[0339] Day 40 - Day 100: Cells were fed daily with Medium 8.

[0340] Medium 1 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, 10 pM SB, 250 nM LDN, 500 ng / ml SHH C5II, and 1 pM CHIR.

[0341] Medium 2 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C5II, and 6 pM CHIR. Attorney Docket No. : 072734.1842

[0342] Medium 3 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, and 6 pM CHIR.

[0343] Medium 4 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 3 pM CHIR, 20 ng / ml BDNF, 0.2 pM ascorbic acid (AA), 20 ng / ml GSNF, 0.5 mM dcAMP, and 1 ng / ml TGF-P3.

[0344] Medium 5 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 3 pM CHIR, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF- 33, 1 pM IWP2, and 100 ng / ml FGF18.

[0345] Medium 6 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, 10 nM DAPT, 10 ng / ml Activin A (ActA), and 20 ng / ml BMP7.

[0346] Medium 7 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, 10 nM DAPT, 10 ng / ml Activin A (ActA), and 20 ng / ml BMP4.

[0347] Medium 8 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, and 10 nM DAPT.

[0348] Induced pluripotent stem cells were differentiated using the “Wnt-boost”, “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” or “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25) and ActA (day 25-day 40) + BMP4 (day 25-day 40)” protocols and evaluated for markers of neuroblasts (CD99), mDA neurons (TH), and A9 subtype mDA neurons (ALDH activity) by flow cytometry.

[0349] At Day 36, the proportion of cells exhibiting both CD99 and CD56 decreased following the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” versus the Wnt-boost protocol from 90.5% to 71.8%, respectively. This proportion was further decreased to 52.2% following the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25) and ActA (day 25-day 40) + BMP4 (day 25-day 40) protocol (Figure 12B). In addition, the proportion of CC99- cells exhibiting ALDH activity was increased from 0.13% following the Wnt-bump protocol to 2.61% and 35.8% following the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” or “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25) and ActA (day 25-day 40) + BMP4 (day 25-day 40)” protocols, respectively.

[0350] At Day 45, expression of CALB1 was reduced following the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25)” or “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25) or ActA (day 25-day 40) + BMP4 (day 25-day 40)” protocols (Figure 12C). Attorney Docket No. : 072734.1842

[0351] Expression of ALDH1 Al was increased in cells following the “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25) or ActA (day 25-day 40) + BMP4 (day 25-day 40)” protocol.

[0352] Example 4: Exemplary Midbrain DA Neuron Differentiation Protocol incorporating ESRRB agonist

[0353] Positive regulators of A9 development were identified following integrative analyses of RNA expression. Gene expression, ATAC peaks, and transcription factor motifs (Figure 13A-13D) were evaluated. These analysis identified ESRRB as a candidate positive regulator of A9 development.

[0354] The “Wnt-boost + ActA (day 16-day 25) + BMP7 (day 16-day 25) and ActA (day 25- day 40) + BMP4 (day 25-day 40) + DY-13 l(day 25-day 40)” protocol is provided below and in Figure 14 A.

[0355] Day 0: Cells were fed with Accutase from hPSC / hiPSC at single cells and plate at a density of 400,000 cells / cm2on Geltrex-coated plated in Medium 1 with Y-drug.

[0356] Day 1 - Day 2: cells should have reached 100% confluence. Cells were double fed with Medium 1.

[0357] Day 3 : Cells were fed with Medium 1.

[0358] Day 4: Cells were fed with Medium 2. For CHIR-Boost protocol, CHIR concentration was changed from 1 pM to 6 pM for WA-09 hESC line-mediated differentiation (this can be slightly vary depending on hPSC / hiPSC lines).

[0359] Day 5 - Day 6: Cells were double fed with Medium 2.

[0360] Day 7: Cells were fed with Medium 3.

[0361] Day 8 - Day 9: Cells were fed daily with Medium 3.

[0362] Day 10: Cells were fed with Medium 4.

[0363] Day 11 : Cells were incubated with Accutase for 30 minutes at 37 C; plate cells at a density of 800,000 cells / cm2in Medium 4.

[0364] Day 12: Cells should have reached 100% confluence. Cells were fed with Medium 5.

[0365] Day 12-16: Cells were fed daily with Medium 5; at day 16, more than 90% of cells were FOXA2+, as measured by FACS analysis.

[0366] Day 16 - 25: Cells were fed daily with Medium 6.

[0367] Day 25 - 35: Cells were fed daily with Medium 7.

[0368] Day 35 - Day 100: Cells were fed daily with Medium 8.

[0369] Medium 1 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, 10 pM SB, 250 nM LDN, 500 ng / ml SHH C5II, and 1 pM CHIR. Attorney Docket No. : 072734.1842

[0370] Medium 2 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, 10 mM SB, 250 nM LDN, 500 ng / ml SHH C5II, and 6 pM CHIR.

[0371] Medium 3 composition: neurobasal medium, N2 supplement, B27 supplement, Pen / Strep, L-Glutamine, and 6 pM CHIR.

[0372] Medium 4 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 3 pM CHIR, 20 ng / ml BDNF, 0.2 pM ascorbic acid (AA), 20 ng / ml GSNF, 0.5 mM dcAMP, and 1 ng / ml TGF-P3.

[0373] Medium 5 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 3 pM CHIR, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF- 33, 1 pM IWP2, and 100 ng / ml FGF18.

[0374] Medium 6 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, 10 nM DAPT, 10 ng / ml Activin A (ActA), and 20 ng / ml BMP7.

[0375] Medium 7 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, 10 nM DAPT, 10 ng / ml Activin A (ActA), 20 ng / ml BMP4, and DY-131.

[0376] Medium 8 composition: neurobasal medium, B27 supplement, Pen / Strep, L-Glutamine, 20 ng / ml BDNF, 0.2 pM AA, 20 ng / ml GDNF, 0.5 mM dcAMP, 1 ng / ml TGF-P3, and 10 nM DAPT.

[0377] The impact of ESRRB agonists on A9 development was evaluated. ALDH+and ALDH' populations were isolated from Day 25 A9-enriched cultures and treated with ActA, BMP4, and ESRRB agonist DY-131 (Figure 14A). AlO-encriched cultures (LDN-treated) were additionally tested. ALDH activity was sustained through Day 35 and Day 44 in A9 populations (Figures 14B-14D).

[0378] Example 5: Integration analysis of A9 andAlO midbrain dopaminergic neurons

[0379] Single-cell RNA sequencing profiling was conducted for Day 36 A9 and A10 midbrain dopaminergic neurons. Analysis of mDA markers revealed expression of general mDA markers (LMX1A, NR4A2, FOXA2, EN1, TH) A9 subtype mDA neurons (ALDH1A1, LM03), and A10 subtype mDA neurons (CALB1) (Figures 15A-15D). Integration analysis identified biological pathways (Figure 15E) and transcriptional activity observed in A9 and A10 subtype mDA neurons. Attorney Docket No. : 072734.1842

[0380] Integration analysis was additionally used to compare mDA subtypes generated herein through in vitro differentiation to those identified in human fetal midbrain samples (Figures 16A-16C).

[0381] The functional properties of in vitro differentiated A9 and A10 subtype mDA neurons were compared following treatment with Levodopa (L-DOPA). First, differences in pigmentation were observed. L-DOPA induced pigmentation in A9 mDA neurons but not A10 mDA neurons (Figures 17A-17B). Second, dopamine metabolism was evaluated. L-DOPA is converted by mDA neurons to dopamine which is then released for synaptic signaling. Excess dopamine can be degraded to 3,4-dihydroxyphenylacetic acid (DOPAC). In vitro differentiated A9 mDA neurons exhibited significantly greater intracellular and extracellular concentrations of dopamine in comparison to A10 mDA neurons (Figure 17C). Intracellular concentrations of DOPAC were not statistically different. Following treatment with L-DOPA, the concentrations for dopamine and DOPAC increased for both A9 and A10 mDA neurons. However, L-DOPA- treated A9 mDNA neurons exhibited significantly greater intracellular and extracellular concentrations of dopamine in comparison to L-DOPA-treated A10 mDA neurons (Figure 17D). In addition, L-DOPA-treated A9 mDNA neurons exhibited significantly greater intracellular concentration of DOPAC in comparison to L-DOPA-treated A10 mDA neurons.

[0382] * * *

[0383] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. Attorney Docket No. : 072734.1842

[0384] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the present disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

Attorney Docket No. : 072734.1842WHAT IS CLAIMED IS:

1. An in vitro method for inducing differentiation of stem cells, comprising:(a) contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and(b) contacting the cells with at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling and at least one activator of bone morphogenetic protein (BMP) signaling to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof.

2. The method of claim 1, wherein the contact of the cells with the at least one activator of (TGFP) / Activin-Nodal signaling is initiated at least about 15 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.

3. The method of claim 1 or 2, wherein the contact of the cells with the at least one activator of (TGFP) / Activin-Nodal signaling is initiated no later than about 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.

4. The method of any one of claims 1-3, wherein the contact of the cells with the at least one activator of (TGFP) / Activin-Nodal signaling is initiated about 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.

5. The method of any one of claims 1-4, wherein the contact of the cells with the at least one activator of (TGFP) / Activin-Nodal signaling is initiated 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.

6. The method of any one of claims 1-5, wherein the cells are contacted with the at least one activator of (TGFP) / Activin-Nodal signaling for at least about 1 day.

7. The method of any one of claims 1-6, wherein the cells are contacted with the at least one activator of (TGFP) / Activin-Nodal signaling for up to about 9 days, or up to about 10 days, or up to about 25 days, or up to about 30 days.Attorney Docket No. : 072734.18428. The method of any one of claims 1-7, wherein the cells are contacted with the at least one activator of (TGFP) / Activin-Nodal signaling for about 5 days, about 10 days, or about 20 days.

9. The method of any one of claims 1-8, wherein the cells are contacted with the at least one activator of (TGFP) / Activin-Nodal signaling for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, or 20 days.

10. The method of any one of claims 1-9, wherein the contact of the cells with the at least one activator of BMP signaling is initiated at least about 15 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling.

11. The method of any one of claims 1-10, wherein the contact of the cells with the at least one activator of BMP signaling is initiated no later than about 16 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling.

12. The method of any one of claims 1-11, wherein the contact of the cells with the at least one activator of BMP signaling is initiated about 16 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling.

13. The method of any one of claims 1-12, wherein the contact of the cells with the at least one activator of BMP signaling is initiated 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling.

14. The method of any one of claims 1-13, wherein the cells are contacted with the at least one activator of BMP signaling for at least about 1 day.

15. The method of any one of claims 1-14, wherein the cells are contacted with the at least one activator of BMP signaling for up to about 9 days, or up to about 10 days, or up to about 25 days, or up to about 30 days.

16. The method of any one of claims 1-15, wherein the cells are contacted with the at least one activator of BMP signaling for about 5 days, about 10 days, or about 20 days.

17. The method of any one of claims 1-16, wherein the cells are contacted with the at least one activator of BMP signaling for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, or 20 days.Attorney Docket No. : 072734.184218. The method of any one of claims 1-17, wherein the cells are contacted with the at least one inhibitor of SMAD signaling for about 5 days.

19. The method of any one of claims 1-18, wherein the cells are contacted with the at least one inhibitor of SMAD signaling for 6 days or 7 days.

20. The method of any one of claims 1-19, wherein the cells are contacted with the at least one activator of SHH signaling for about 5 days.

21. The method of any one of claims 1-20, wherein the cells are contacted with the at least one activator of SHH signaling for 6 days or 7 days.

22. The method of any one of claims 1-21, wherein the cells are contacted with the at least one activator of Wnt signaling for about 10 days.

23. The method of any one of claims 1-22, wherein the cells are contacted with the at least one activator of Wnt signaling for 10 days or 11 days.

24. The method of any one of claims 1-23, wherein the concentration of the at least one activator of Wnt signaling is increased about 4 days from its initial contact with the stem cells.

25. The method of claim 24, wherein the concentration of the at least one activator of Wnt signaling is increased by between about 200 % and about 1000 % from the initial concentration of the at least one activator of Wnt signaling.

26. The method of claim 24 or 25, wherein the concentration of the at least one activator of Wnt signaling is increased by about 500 % from the initial concentration of the at least one activator of Wnt signaling.

27. The method of any one of claims 24-26, wherein the concentration of the at least one activator of Wnt signaling is increased to from about 1 pM to between about 5 pM and about 10 pM.

28. The method of any one of claims 24-27, wherein the concentration of the at least one activator of Wnt signaling is increased to a concentration of about 6 pM.

29. The method of any one of claims 1-28, wherein the at least one activator of (TGFP) / Activin-Nodal signaling comprises Activin A.Attorney Docket No. : 072734.184230. The method of any one of claims 1-29, wherein the at least one activator of BMP signaling comprises BMP7 or TCS21311.

31. The method of any one of claims 1-20, wherein the at least one inhibitor of SMAD signaling comprises an inhibitor of TGFp / Activin-Nodal signaling, an inhibitor of BMP signaling, or a combination thereof.

32. The method of claim 31, wherein the at least one inhibitor of TGFp / Activin-Nodal signaling comprises an inhibitor of ALK5.

33. The method of claim 31 or 32, wherein the at least one inhibitor of TGFp / Activin-Nodal signaling is selected from the group consisting of SB431542, derivatives of SB431542, and combinations thereof.

34. The method of claim 33, wherein the derivative of SB431542 comprises A83-01.

35. The method of any one of claims 31-34, wherein the at least one inhibitor of TGFp / Activin-Nodal signaling comprises SB431542.

36. The method of claim 31, wherein the at least one inhibitor of BMP signaling is selected from the group consisting of LDN193189, Noggin, dorsomorphin, derivatives of LDN193189, derivatives of Noggin, derivatives of dorsomorphin, and combinations thereof.

37. The method of claim 31 or 36, wherein the at least one inhibitor of BMP comprises LDN-193189.

38. The method of any one of claims 1-37, wherein the at least one activator of Wnt signaling comprises an inhibitor of glycogen synthase kinase 3p (GSK3P) signaling.

39. The method of any one of claims 1-38, wherein the at least one activator of Wnt signaling is selected from the group consisting of CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, deoxycholic acid, BIO, SB-216763, Wnt3A, Wntl, Wnt5a, derivatives thereof, and combinations thereof.

40. The method of any one of claims 1-39, wherein the at least one activator of Wnt signaling comprises CHIR99021.Attorney Docket No. : 072734.184241. The method of any one of claims 1-40, wherein the at least one activator of SHH signaling is selected from the group consisting of SHH proteins, Smoothened agonists (SAG), and combinations thereof.

42. The method of claim 41, wherein the SHH protein is selected from the group consisting of recombinant SHHs, modified N-terminal SHHs, and combinations thereof.

43. The method of claim 42, wherein the modified N-terminal SHH comprises two Isoleucines at the N-terminus.

44. The method of claim 42 or 43, wherein the modified N-terminal SHH has at least about 90% sequence identity to an un-modified N-terminal SHH.

45. The method of claim 44, wherein the un-modified N-terminal SHH is a un-modified mouse N-terminal SHH or a un-modified human N-terminal SHH.

46. The method of any one of claims 42-45, wherein modified N-terminal SHH comprises SHH C25II.

47. The method of claim 41, wherein the SAG comprises purmorphamine.

48. The method of any one of claims 1-47, wherein at least about 80% of the differentiated cells express FOXA2 and EN1 about 15 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.

49. The method of any one of claims 1-48, wherein greater than about 80% or greater than about 90% of the differentiated cells express FOXA2 and EN1 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.

50. The method of any one of claims 1-49, wherein the at least one marker indicating a midbrain dopamine neuron or a precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LM03, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof.

51. The method of any one of claims 1-50, wherein the differentiated cells do no express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, H0XA1, H0XA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.Attorney Docket No. : 072734.184252. The method of any one of claims 1-51, further comprising isolating cells that express at least one positive marker and do not express at least one negative marker.

53. The method of claim 52, wherein the at least one positive marker is selected from the group consisting of PITX3, ALDH1A1, TH, and combinations thereof.

54. The method of claim 52 or 53, wherein the at least one positive marker comprises ALDH1A1.

55. The method of any one of claims 52-54, wherein the at least one negative marker is selected from CD49e, CD340, and combination thereof.

56. The method of any one of claims 1-55, wherein the stem cells are pluripotent stem cells.

57. The method of any one of claims 1-56, wherein the stem cells are selected from the group consisting of nonembryonic stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

58. The method of any one of claims 1-57, wherein the stem cells are human stem cells, non-human primate stem cells, or rodent stem cells.

59. The method of any one of claims 1-58, wherein the stem cells are human stem cells.

60. An in vitro method for inducing differentiation of stem cells, comprising:(a) contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and(b) contacting the cells with at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling and at least one activator of bone morphogenetic protein (BMP) signaling for up to about 9 days; and(c) contacting the cells with at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling and at least one activator of bone morphogenetic protein (BMP) signaling for up to about 15 days to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof.Attorney Docket No. : 072734.184261. The method of claim 60, wherein the at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling of step (b) comprises Activin A.

62. The method of claim 60 or 61, wherein the at least one BMP of step (b) comprises BMP7, TCS21311, or a combination thereof.

63. The method of any one of claims 60-62, wherein the at least one activator of transforming growth factor beta (TGFP) / Activin-Nodal signaling of step (c) comprises Activin A.

64. The method of any one of claims 60-63, wherein the at least one BMP of step (c) comprises BMP4.

65. The method of any one of claims 60-64, wherein step (c) further comprises contacting the cells with at least one activator of estrogen-related receptor p (ESRRB).

66. The method of claim 65, wherein the at least one activator of ESRRB comprises DY- 131.

67. A cell population of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by a method of any one of claims 1-66.

68. A composition comprising the cell population of claim 67.

69. The composition of claim 68, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

70. A kit for inducing differentiation of stem cells to midbrain dopamine neurons or precursors thereof, comprising:(a) at least one inhibitor of SMAD signaling;(b) at least one activator of SHH signaling;(c) at least one activator of Wnt signaling;(d) at least one activator of TGFp / Activin-Nodal signaling; and(e) at least one activator of BMP signaling.

71. The kit of claim 70, further comprising (f) instructions for inducing differentiation of the stem cells into a population of differentiated cells that express at least one marker indicating a midbrain dopamine neuron or a precursor thereof.Attorney Docket No. : 072734.184272. A method of preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder, comprising administering to the subject an effective amount of one of the followings:(a) the cell population of claim 67; or(b) the composition of claim 68 or 69.

73. The method of claim 72, wherein the neurological disorder is characterized by reduction of midbrain dopamine neuron function.

74. The method of claim 73, wherein the reduction of midbrain dopamine neuron function is age related.

75. The method of any one of claims 72-74, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and combinations thereof.

76. The method of any one of claims 72-75, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof.

77. The method of any one of claims 72-76, wherein the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia.

78. The cell population of claim 67 or the composition of claim 68 or 69 for use in preventing, modeling, and / or treating at least one symptom in a subject having a neurological disorder in a subject.

79. The cell population or composition for use of claim 78, wherein the neurological disorder is characterized by reduction of midbrain dopamine neuron function.

80. The cell population or composition for use of claim 79, wherein the reduction of midbrain dopamine neuron function is age related.

81. The cell population or composition for use of any one of claims 78-80, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, multiple sclerosis, and combinations thereof.Attorney Docket No. : 072734.184282. The cell population or composition for use of any one of claims 78-81, wherein the neurological disorder is selected from the group consisting of Parkinsonism, Parkinson’s disease, and combinations thereof.

83. The cell population or composition for use of any one of claims 78-82, wherein the symptom for a neurological disorder is selected from the group consisting of tremor, bradykinesia, flexed posture, postural instability, rigidity, dysphagia, and dementia.

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