Pluripotent stem cell-derived vagal competent cells

Abstract

The presently disclosed subject matter provides for in vitro methods of inducing differentiation of stem cells into hindbrain progenitor cells, enteric neural crest lineage cells, as well as differentiated hindbrain lineage or enteric neural crest lineage cells obtained by such methods. The presently disclosed subject matter also provides for uses of such cells for preventing and / or treating enteric nervous system disorders (e.g, Hirschsprung's disease), and for screening compounds suitable for preventing and / or treating enteric nervous system disorders (e.g., Hirschsprung's disease).

Description

[0001] 072734.1887 PATENT

[0002] PLURIPOTENT STEM CELL-DERIVED VAGAL COMPETENT CELLS

[0003] 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 735,043, filed December 17, 2024, the content of which is hereby incorporated by reference in its entirety, and to which priority is claimed.

[0005] 2. GRANT FUNDING

[0006] This invention was made with government support under HD112917 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] 3. TECHNICAL FIELD

[0008] The presently disclosed subject matter relates to vagal competent cells derived from stem cells (e.g., human stem cells) and uses thereof for cell-based treatment and drug discovery in diseases of the enteric nervous system, such as Hirschsprung’s disease.

[0009] 4. BACKGROUND

[0010] Vagal neural crest are the major cell of origin for the enteric nervous system. Vagal neural crest are capable of differentiating into enteric neuron-like cells and rescuing mouse models of Hirschsprung’s disease. Current methods of producing vagal neural crest and enteric neuron-like derivatives yield large numbers of off-target ectomesenchyme-like cells. Therefore, there remains a need for improved in vitro methods and protocols of generating ENS precursors directly from human stem cells.

[0011] 5. SUMMARY

[0012] The present disclosure provides in vitro methods for inducing differentiation of stem cells. In certain embodiments, the methods comprise contacting a population of stem cells with a first concentration of at least one activator of Wnt signaling for between about 12 and about 24 hours to produce a population of cells expressing at least one hindbrain lineage marker. In certain embodiments, the methods further comprise contacting the population of cells expressing at least one hindbrain lineage marker with at least one inhibitor of SMAD signaling, a second concentration of the at least one activator of Wnt signaling, and at least one molecule that induces vagal neural crest patterning to produce a population of vagal 072734.1887

[0013] PATENT competent cells expressing at least one enteric neural crest lineage marker; wherein the second concentration of the at least one activator of Wnt signaling is decreased from the first concentration of the at least one activator of Wnt signaling. In certain embodiments, the population of stem cells are contacted with the first concentration of the at least one activator of Wnt signaling for between about 18 hours and about 24 hours. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the second concentration of the at least one activator of Wnt signaling for at least about 6 days. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the second concentration of the at least one activator of Wnt signaling for about 6 days. In certain embodiments, the second concentration of the activator of Wnt signaling is decreased from the first concentration of the activator of Wnt signaling by between about 50 and about 95%. In certain embodiments, the second concentration of the activator of Wnt signaling is decreased from the first concentration of the activator of Wnt signaling by between about 70 and about 90%. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling for at least about 6 days. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling for about 6 days. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling and the second concentration of the at least one activator of Wnt signaling for at least about 6 days. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one molecule that induces vagal neural crest patterning for at least about 2 days. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one molecule that induces vagal neural crest patterning for about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days. In certain embodiments, said population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning within an about 8 day period from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said at least one inhibitor of SMAD signaling. In certain embodiments, said population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning at least about 3 days from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said 072734.1887

[0014] PATENT at least one inhibitor of SMAD signaling. In certain embodiments, said population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning about 3 days from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said at least one inhibitor of SMAD signaling. In certain embodiments, said population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning about 3 days from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said at least one inhibitor of SMAD signaling, and said population of cells expressing at least one hindbrain lineage marker are contacted with said at least one molecule that induces vagal neural crest patterning for at least about 3 days. In certain embodiments, said population of stem cells have been differentiated into a population of differentiated cells that express at least one said enteric neural crest lineage marker on or after about 6 days from their initial contact with said at least one inhibitor of SMAD signaling. 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, said at least one inhibitor of TGFp / Activin-Nodal signaling is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. In certain embodiments, said at least one inhibitor of BMP signaling is a small molecule selected from the group consisting of LDN193189, derivatives thereof, and mixtures thereof. In certain embodiments, said at least one activator of Wnt signaling lowers glycogen synthase kinase 3P (GSK3P) for activation of Wnt signaling. In certain embodiments, said at least one activator of Wnt signaling is a small molecule selected from In certain embodiments, the group consisting of CHIR99021, derivatives thereof, and mixtures thereof. In certain embodiments, said at least one molecule that induces vagal neural crest patterning is selected from the group consisting of retinoic acid, retinol, retinal, tretinoin, isotretinoin, alitretinoin, etretinate, acitretin, tazarotene, bexarotene, adapalene, activators of FGF signaling, Wnt activators, and combinations thereof. In certain embodiments, said at least one molecule that induces vagal neural crest patterning is selected from the group consisting of activators of FGF signaling, activators of Wnt signaling, and combinations thereof. In certain embodiments, said at least one molecule that induces vagal neural crest patterning comprises retinoic acid. In certain embodiments, said activators of FGF signaling are selected from the 072734.1887

[0015] PATENT group consisting of FGF2, FGF4, FGF7, and FGF8. In certain embodiments, said activators of Wnt signaling are selected from the group consisting of CHIR99021 and WNT3A. In certain embodiments, said vagal neural crest patterning is characterized by expression of at least one regional specific homoebox (HOX) gene. In certain embodiments, said at least one regional specific HOX gene is selected from the group consisting of HOXB2, HOXB3, HOXB4, and HOXB5. In certain embodiments, said at least one enteric neural crest lineage marker is selected from the group consisting of PAX3, EDRB, RET, PHOX2A, PHOX2B, NTRK-3, HA D2, HOXB3, HOXB5 and ASCL1. In certain embodiments, said at least one hindbrain lineage marker is GBX2. In certain embodiments, the differentiated cells expressing said at least one hindbrain lineage marker do not express OTX2 or TBXT (brachyury). In certain embodiments, said population of differentiated cells expressing at least one enteric neural crest lineage marker further express at least one SOX 10+neural crest lineage marker. In certain embodiments, said SOX10+neural crest lineage marker is CD49D. In certain embodiments, said stem cells are human stem cells. In certain embodiments, said human stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, and F-class pluripotent stem cells. In certain embodiments, the methods further comprise subjecting said population of differentiated cells to conditions favoring maturation of said differentiated cells into a population of cells that express at least one enteric neuron marker. In certain embodiments, said conditions favoring maturation of said differentiated cells into said population of enteric neurons comprise culturing said differentiated cells in a suitable cell culture medium. In certain embodiments, said suitable cell culture medium comprises at least one molecule that enhances maturation of enteric neural crest (ENC) precursors to enteric neurons. In certain embodiments, said at least one molecule that enhances maturation of ENC precursors to enteric neurons is selected from the group consisting of growth factors and Wnt activators. In certain embodiments, said growth factors are selected from the group consisting of activators of FGF signaling, glial cell line derived neurotrophic factor (GDNF), and ascorbic acid. In certain embodiments, said suitable cell culture medium comprises at least one activator of FGF signaling and at least one activator of Wnt signaling. In certain embodiments, said differentiated cells are cultured in said suitable cell culture medium comprising said at least one activator of FGF signaling and said at least one activator of Wnt signaling for about 4 days. In certain embodiments, the at least one activator of FGF signaling is selected from the 072734.1887

[0016] PATENT group consisting of FGF2, FGF4, FGF8, and FGF7. In certain embodiments, said at least one enteric neuron marker is selected from the group consisting of Tuj 1 , MAP2, PHOX2A, PHOX2B, TRKC, ASCL1, HAND2, EDNRB, 5HT, GAB A, NOS, SST, TH, CHAT, DBH, Substance P, VIP, NPY, GnRH, and CGRP.

[0017] The present disclosure further provides cell populations of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by methods disclosed herein.

[0018] The present disclosure further provides compositions comprising cell populations disclosed herein. In certain embodiments, the compositions are pharmaceutical compositions further comprising a pharmaceutically acceptable carrier.

[0019] The present disclosure further provides kits for inducing differentiation of stem cells to vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors), comprising: 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 molecule that induces vagal neural crest patterning. In certain embodiments, the kits further comprise instructions for inducing differentiation of the stem cells into a population of vagal competent cells expressing at least one enteric neural crest lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with a decreased concentration of the at least one activator of Wnt signaling about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours after the cells are initially contacted with the Wnt activator.

[0020] The present disclosure further provides methods of preventing and / or treating an enteric nervous system disorder in a subject, comprising administering to a subject suffering from an enteric nervous system disorder an effective amount of one of the followings: the population of in vitro differentiated cells disclosed herein, or a composition disclosed herein. In certain embodiments, the enteric nervous system disorder is Hirschsprung's disease.

[0021] The present disclosure further provides cell populations or compositions disclosed herein for use in preventing, modeling, and / or treating at least one symptom in a subject having an enteric nervous system disorder in a subject. In certain embodiments, the enteric nervous system disorder is Hirschsprung's disease.

[0022] 6. BRIEF DESCRIPTION OF THE FIGURES

[0023] FIG. 1 shows schematic for “bump” protocol. 072734.1887

[0024] PATENT

[0025] FIG. 2 shows schematic for generic approaches to differentiation of human pluripotent stem cells (hPSCs) into vagal neural crest cells.

[0026] FIGS. 3A-3B show expression of markers in response to retinoic acid. FIG 3A shows schematic for SOXIO reporter line strategy. FIG. 3B shows flow cytometry using the SOXIO reporter and p75 demonstrating no loss of NC efficiency in the presence of retinoic acid.

[0027] FIGS 4A-4E show expression of vagal markers in response to retinoic acid. FIG. 4A shows day 9 immunocytochemistry (ICC) in the presence of retinoic acid showing low proportion of HOXB5 and / or PHOX2B positive neural crest cells. FIGS. 4B-4E provides scRNAseq showing culture-wide transcriptional changes in response to retinoic acid but only a subset of cells respond with the upregulation of vagal markers. Expression is shown for SOXIO (FIG. 4B), PHOX2B (FIG. 4C), H0XA5 (FIG. 4D), and HOXB5 (FIG. 4E).

[0028] FIG. 5 shows schematic for protocol incorporating a long exposure to retinoic acid.

[0029] FIGS. 6A-6N show expression of vagal markers in response to retinoic acid. FIG. 6 A shows schematic showing CellTag experiment. FIG. 6B shows UMAP for CellTag experiment. FIG. 6C shows gene expression overlays for scRNAseq data from CellTag experiment. FIGS. 6D-6E show sub-clustering of clusters 7 and 8 (FIG. 6D) and subclustering of vagal clusters showing both neural crest and CNS lineages (FIG. 6E). FIG. 6F shows network diagram showing a vagal-biased clone and a non-vagal biased clone. FIG. 6G shows location of cells belonging to clones biased toward or against vagal identity. 92% of clones showed 100% commitment. FIG. 6H shows location of cells belonging to biased clones (D4 only). FIG. 61 shows location of cells belonging to biased clones (D4 only). FIG. 6J shows Volcano plot showing significantly differentially expressed genes between vagal biased and non-vagal biased clones at day 4 (before application of retinoic acid). FIG. 6K provides chart showing enrichment of hindbrain-associated genes, stem cell-associated genes, or cell adhesion-associated genes. FIG. 6L shows gene overlays for day 4 showing hindbrain- associated markers that were hits for the vagal-biased clones. FIG. 6M depicts spatial commitment. FIG. 6N shows location of D4 and D9 cells.

[0030] FIG. 7 provides a schematic representing the “bump” protocol.

[0031] FIG. 8 shows ICC demonstrating upregulation of vagal markers HOXB5, PHOX2B and HOXB4 at day 7 of NC induction using the bump protocol.

[0032] FIGS. 9A-9C show expression of markers following “bump” protocol. FIG. 9 A shows schematic depicting replacement of the Wnt “bump” with an inhibitor of Wnt signaling 072734.1887

[0033] PATENT

[0034] (XAV939). Fig. 9B shows ICC demonstrating expression of HOXB5 and SOXIO at day 7. FIG. 9C shows quantification of FIG. 9B.

[0035] FIGS 10A-10D show expression of markers. Fig. 10A shows schematic depicting the “bump” protocol for promoting differentiation of hPSC to vagal neural crest cells, and subsequent differentiation of enteric neurons at day 40 (D40). FIGS. 10B compares expression of VIP, CHAT, and NOS1 at D40 obtained using “bump” protocol or “no bump” protocols. FIG. 10C shows ICC depicting expression of VIP, CHAT, and NOS1 at D40 following “bump” protocol. FIG. 10D provides synthesis signature adapted from Drokhylyanksy et al., 2020.

[0036] FIGS. 11 A-l IB show ENS D40 scRNAseq data. FIGS. 11 A-l IB show expression of TUBB3, SOXIO, and MKI67 (FIG. 11 A) and NOSl, VIP, CHAT, SLC5A7, TAC1, SST, and ADCYAP1 (FIG. 11B).

[0037] FIG. 12 shows scRNAseq data with CellTypist annotation.

[0038] FIG. 13 shows schematic depicting homeobox transcription factor expression.

[0039] FIGS. 14A-14B show gene expression following the “bump” protocol. FIG. 14A shows schematic depicting GBX2 reporter line strategy. FIG. 14B shows gain of GBX2 with bump protocol.

[0040] FIGS. 15A-15B show expression of markers. FIG. 15A shows schematic depicting the “bump” protocol. FIG. 15B provides ICC of DI cells for expression of OCT4, GBX2, and OTX2.

[0041] FIGS. 16A-16B show expression of markers. FIG. 16A shows schematic depicting the “bump” protocol, including extension of the “bump” for up to 48 hours. FIG. 16B provides ICC of D2 cells for expression of GBX2 and TBXT (brachyury).

[0042] FIGS. 17A-17C show expression of markers. FIG. 17A shows schematic depicting the “bump” protocol, including extension of the “bump” for up to 48 hours. FIG. 17B provides ICC of D2 cells for expression of MEIS2, GBX2, and TBXT (brachyury). FIG. 17C shows flow cytometry analysis for expression of SOX2 and TBXT (brachyury).

[0043] FIGS. 18A-18B show expression of markers. FIG. 18A shows schematic depicting the “bump” protocol, including extension of the “bump” for up to 48 hours. FIG. 18B provides ICC of D2 cells for expression of CDX2, GBX2, and OTX2.

[0044] FIGS. 19A-19B show expression of markers. FIG. 19A shows schematic depicting the “bump” protocol, including extension of the “bump” for up to 48 hours. FIG. 19B provides ICC of D7 cells for expression of SOXIO, HOXB5, and CDX2. 072734.1887

[0045] PATENT

[0046] FIGS. 20A-20B show expression of markers. FIG. 20 A shows schematic depicting the “bump” protocol, including variation on the duration of the “bump” for 0, 4, 6, 10, 12, 15, 18, or 24 hours. FIG. 20B provides flow cytometry analysis of DI cells for expression of GBX2 following “bump”.

[0047] FIGS. 21A-21B show expression of markers. FIG. 21A shows schematic depicting the “bump” protocol, including variation on the duration of the “bump” for 0, 4, 6, 10, 12, 15, 18, or 24 hours. FIG. 21B provides ICC of D7 cells for expression of HOXB5, GBX2, and PHOX2B.

[0048] FIGS. 22A-22B show expression of markers. FIG. 22 A shows schematic depicting the “bump” protocol, including delay of Wnt “bump” for 12, 24, or 36 hours. FIG. 22B provides ICC of D7 cells for expression of HOXB5, GBX2, and PHOX2B.

[0049] FIG. 23 shows schematic depicting role of early Wnt activation on regional specification and mutual repression of OTX2 and GBX2.

[0050] FIGS. 24A-24J show expression of vagal markers in response to retinoic acid. FIG. 24A shows day 9 immunocytochemistry (ICC) in the presence of retinoic acid showing low proportion of HOXB5 and / or PHOX2B positive neural crest cells. FIG. 24B shows flow cytometry using the SOX10 reporter and p75 demonstrating no loss of NC efficiency in the presence of retinoic acid. scRNAseq shows that a lot of cells have switched off

[0051] SOX10 after retinoic acid treatment, which supports that they were expressing it at one time. FIG. 24C provides scRNAseq showing culture-wide transcriptional changes in response to retinoic acid but only a subset of cells respond with the upregulation of vagal markers. FIG. 24D shows schematic showing CellTag experiment. FIG. 24E shows UMAP for CellTag experiment. FIG. 24F shows gene expression overlays for scRNAseq data from CellTag experiment. FIG. 24G shows location of cells belonging to clones biased toward or against vagal identity. 92% of clones showed 100% commitment. FIG. 24H shows network diagram showing a vagal -biased clone. FIG. 241 shows network diagram showing a non-vagal biased clone. FIG. 24J shows Volcano plot showing significantly differentially expressed genes between vagal biased and non-vagal biased clones at day 4 (before application of retinoic acid).

[0052] FIGS. 25A-25G shows expression of vagal markers following the “bump” protocol. FIG. 25 A provides a schematic representing the “bump” protocol. FIG. 25B shows ICC demonstrating upregulation of vagal markers HOXB5, PHOX2B and HOXB4 at day 7 of NC induction using the bump protocol. CNS marker PAX6 identifies rare contaminating off- 072734.1887

[0053] PATENT target cells in the “no bump” protocol. These are largely lost in the bump protocol. FIGS. 25C-25D shows integration of bump and no bump scRNAseq data at D4 (FIG. 25C) and D7 (FIG. 25D) with that of the CellTag experiment at D4. Location of cells belonging to vagal- biased clones in the CellTag experiment is shown with the density overlay. FIG. 25E shows violin plots depicting expression of HOX genes. FIG. 25F shows ENS D40 ICC showing VIP, CHAT and NOS. Cells can be identified that correspond to excitatory motor neurons (CHAT+), inhibitor motor neurons (NOS1+, VIP+ and secreto-vasodilator neurons (VIP+). FIG. 25G shows D40 scRNAseq with CellTypist annotation.

[0054] FIGS. 26A-26E show gene expression following the “bump” protocol. FIG. 26 A shows gain of GBX2 with bump protocol. FIGS. 26B-27C provide ICC at D2 (FIG. 26B) and D7 (FIG. 26C) showing that extended CHIR treatment results in loss of vagal competency - no HOXB5 at D7. CDX2 is gained with 48hr bump, although low levels can be detected with 24 hr bump condition. Also shown is gain of TBXT (Brachyury) only with 48hr bump. FIG. 26D provides ICC showing a reduction in bump duration from 24hr to 18hr results in loss of vagal competency. FIG. 26E provides ICC showing a delay in bump application of 24hr or more results in loss of vagal competency.

[0055] FIGS. 27A-27I show molecular characterization of the early progenitor state. FIG. 27A shows DO-2 scRNAseq UMAP showing transcriptionally distinct cultures from bump, no bump and long bump conditions. FIG. 27B provides bar chart showing proportion of differentially expressed (solid) or accessible (dashed) genes across conditions. FIG. 27C shows GO analysis for differentially expressed genes. FIG. 27D shows GO analysis for differentially accessible region-linked genes. FIG. 27E provides heatmaps showing accessibility for enriched differentially accessible regions in bump vs. no bump analysis. Also shown is CUT data RUN data for the same regions for OTX2, GBX2, H3K4me3 and H3K27me3. FIG. 27F shows histone state analysis for bump vs no bump differentially accessible regions with OTX2 and GBX2 linked regions highlighted. FIG. 27G shows GBX2 locus with tracks showing accessibility and CUT and RUN data. FIG. 27H shows top transcription factor motifs identified in bump vs. no bump differentially accessible regions. FIG. 271 provides Venn diagram showing overlap in identified motifs between bump and no bump enriched differentially accessible regions.

[0056] FIGS. 28A-28D shows impact of early progenitor specification on CNS differentiation. FIG. 28A provides schematic showing the location of regional markers within the hindbrain. FIG. 28B provides schematic showing experimental conditions. FIG. 28C. 072734.1887

[0057] PATENT provides D4 ICC data showing GBX2 and OTX2 expression in response to different “bump” durations with and without subsequent FGF8 treatment. FIG. 28D provides D4 ICC data showing GBX2, EGR2 and MAFB expression across different conditions in “bump” and “no bump” cultures.

[0058] FIGS. 29A-29G show CRISPRa experiments and results. FIG. 29 A provides schematic showing format of CRISPRa experiments with Dual SMAD inhibition conditions. FIG. 29B shows D2 ICC from CRISPRa Dual SMAD inhibition experiments. FIG. 29C shows quantification of FIG. 29B. FIG. 29D shows D4 ICC from CRISPRa Dual SMAD inhibition experiments. FIG. 29E shows quantification of FIG. 29D. FIG. 29F provides schematic showing format of CRISPRa experiments with neural crest induction conditions. FIG. 29G shows D7 ICC from CRISPRa NC experiments.

[0059] FIGS. 30A-30D show 3D co-culture experiments. FIG. 30A provides schematic showing format of experiments. FIG. 30B provides ICC showing expression of axial markers in assembloids derived from “no bump” cultures (GFP), “bump” cultures (tdTOM) and “long bump” cultures (3xFLAG).

[0060] FIGS. 31A-31E show results from CellTag experiments. FIG. 31A shows schematic of CellTag transgene. FIG. 3 IB provides Violin plot showing expression of markers from CellTAg scRNAseq data. FIG. 31C shows sub-clustering of vagal clusters shows both neural crest and CNS lineages. FIG. 3 ID shows location of cells belonging to biased clones (D4 only). FIG. 3 IE shows gene overlays for day 4 showing hindbrain-associated markers that were hits for the vagal-biased clones.

[0061] FIGS. 32A-32B show differentiation of hPSC cell lines using the “bump” protocol. FIG. 32A shows ICC demonstrating upregulation of vagal markers HOXB5, PHOX2B and HOXB4 at day 7 of NC induction using the bump protocol for secondary cell lines KOLF and Hl. CNS marker PAX6 identifies rare contaminating off-target cells in the “no bump” protocol. These are largely lost in the bump protocol. FIG. 32B shows ENS D40 (KOLF and Hl) ICC showing VIP, CHAT, NOS, PENK, CGRP and NMU. Cells can be identified that correspond to excitatory motor neurons (CHAT+), inhibitor motor neurons (NOS1+, VIP+ and secreto-vasodilator neurons (VIP+).

[0062] FIGS 33A-33D show characterization of day 40 differentiated cells. FIG. 33A shows ENS D40 scRNAseq showing bump-derived data (regular as well as GDNF and EDN3 treated) integrated with no bump-derived data. FIG. 33B shows ENS D40 flow cytometry showing p75 and SOX10::GFP detection post-dissociation. FIG. 33C shows ENS D40 brightfield 072734.1887

[0063] PATENT imaging of single cell suspension of dissociated ENS cultures prior to flow cytometry and sequencing. FIG. 33D shows D40 ENS ICC using H9-SOX10::GFP and staining for NOS1 and CHAT.

[0064] FIGS. 34A-34D show characterization of cells obtained following manipulation of early signals. FIG. 34A shows D9 NC qPCR after inhibition of WNT signaling DO-1 using XAV939. FIG. 34B shows D7 NC ICC after activation of RA signaling DO-1. FIG. 34C shows D9 NC ICC after treatment with FGF8 DO-1. FIG 34D shows D7 NC ICC after FGF8 treatment D 1 -4 or D 1 -7.

[0065] FIGS. 35A-35F show molecular characterization of the early progenitor state. FIG. 35 A provides Volcano plot showing significantly upregulated / downregulated genes expressed in bump vs. no bump conditions. FIG. 35B shows gene overlay UMAPS for selection of genes of interest from FIG. 35 A. FIG. 35C shows AT AC and CUT and RUN heatmaps for hPSC and long bump conditions. FIG. 35D provides dot plot showing expression of genes (proportion and mean) annotated with an “active” histone signature at DI in the bump condition. Gene names are colored by OTX2 / GBX2 binding status. FIG. 35E shows degree of overlap in motifs identified in bump vs. hPSC differentially accessible regions. FIG. 35F shows accessibility of HOXA and HOXB regions across samples.

[0066] FIGS. 36A-36B show impact of early progenitor specification on CNS differentiation. FIG. 36A show expression of GBX2 and HOXB1 across different experimental conditions described in FIGS. 28A-28D.

[0067] FIG. 37 shows BMP patterning after variable bump duration. D7 ICC is provided for experiments varying BMP activity conditions in “bump”, “no bump” and “long bump” cultures.

[0068] FIGS. 38A-38B show CRISPRa experiments and results. FIG. 38A show D4 ICC from CRISPRa Dual SMAD inhibition experiments outlined in FIGS. 29A-29G (HOXB1). FIG. 38B shows quantification of FIG. 38 A.

[0069] FIG. 39 shows integration and differentiation of enteric neural crest post-graft into NSG mouse colon.

[0070] 7. DETAILED DESCRIPTION OF THE SUBJECT MATTER

[0071] The presently disclosed subject matter relates to in vitro methods for inducing differentiation of stem cells (e.g., human stem cells) to cells expressing at least one hindbrain lineage marker. The presently disclosed subject matter further relates to in vitro methods for 072734.1887 PATENT inducing differentiation of stem cells to vagal competent cells that express at least one enteric neural crest lineage marker (enteric nervous system (ENS) precursors). The cells expressing at least one enteric neural crest lineage marker can further induced in vitro to enteric neurons, cells (ENS precursors and enteric neurons) produced by such methods and compositions comprising such cells. Also provided are uses of such cells for preventing and / or treating Hirschsprung’s disease and for screening compounds suitable for preventing and / or treating Hirschsprung’s disease.

[0072] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0073] 7.1. Definitions

[0074] 7.2. Method of Differentiating Stem Cells

[0075] 7.3. Compositions Comprising Differentiated Cell Populations

[0076] 7.4. Method of Preventing and / or Treating Enteric Nervous System Disorders

[0077] 7.5. Method of Screening Therapeutic Compounds; and

[0078] 7.6. Kits

[0079] 7.1 Definitions

[0080] The terms used in this specification generally have their ordinary meanings in the art, within the context of this 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 disclosure and how to make and use them.

[0081] 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.

[0082] 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, 072734.1887

[0083] PATENT

[0084] NOTCH, transforming growth factor beta (TGFP), Activin, Nodal and glycogen synthase kinase 3p (GSK3P) proteins. 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 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.

[0085] 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.

[0086] 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.

[0087] As used herein, the term “inhibitor”, 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. 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 glycogen synthase kinase 3p (GSK3P)) (e.g., including, but not limited to, the signaling molecules described herein), for one example, via directly contacting SMAD signaling, contacting SMAD mRNA, causing conformational changes of SMAD, decreasing SMAD protein levels, or interfering with SMAD interactions with signaling partners (e.g., including those described herein), and affecting the expression of SMAD target genes (e.g. those described herein). Inhibitors also include molecules that indirectly regulate 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 activins, nodal, TGFP, and BMPs are contemplated for use to neutralize extracellular activators of SMAD signaling, and the like. Inhibitors are described in terms of competitive 072734.1887

[0088] PATENT 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 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.

[0089] As used herein, the term “activator”, refers to compounds that increase, induce, stimulate, activate, facilitate, or enhance activation the signaling function of the molecule or pathway, e.g., Wnt signaling

[0090] As used herein, the term “derivative” refers to a chemical compound with a similar core structure.

[0091] 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 can be a pure population comprising one cell type, such as a population of enteric nervous system precursors, or a population of undifferentiated stem cells. Alternatively, the population can comprise more than one cell type, for example a mixed cell population.

[0092] 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. A human stem cell refers to a stem cell that is from a human.

[0093] As used herein, the term “vagal competent cell” refers to cells which are capable of forming vagal lineages.

[0094] As used herein, the term “enteric nervous system precursor”, “ENS precursor”, “enteric neural crest precursor, “enteric NC precursor” or “ENC precursor” refers to a cell expressing at least one enteric neural crest lineage marker. An ENS precursor is a cell with the ability to mature into an enteric neuron. A human ENS precursor refers to an ENS precursor that is from a human. Non-limiting examples of enteric neural crest lineage markers include PAX3, EDNRB, RET, PHOX2A, PHOX2B, NTRK-3, HAND2, HOXB3, HOXB5 and ASCL1. 072734.1887

[0095] PATENT

[0096] As used herein, the term “enteric neuron” refers to a cell expressing at least one enteric neuron marker. A human enteric neuron refers to an enteric neuron that is from a human. Non-limiting examples of enteric neuron markers include Tuj 1, MAP2, PHOX2A, PHOX2B, TRKC, ASCL1, HAND2, EDNRB, 5HT, GABA, NOS, SST, TH, CHAT, DBH, Substance P, VIP, NPY, GnRH, and CGRP.

[0097] As used herein, the term “embryonic stem cell” refers 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. As used herein, the term “human embryonic stem cell” or “hESC” refers to a type of pluripotent stem cells derived from early stage human 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.

[0098] 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.

[0099] 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.

[0100] As used herein, the term “multipotent” refers to an ability to develop into more than one cell type of the body.

[0101] 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.

[0102] As used herein, the term “induced pluripotent stem cell” or “iPSC” refers to a type of pluripotent stem cell, similar to an embryonic stem cell, formed by the introduction of certain embryonic genes (such as a OCT4, SOX2, and KLF4 transgenes) (see, for example, Takahashi and Yamanaka Cell 126, 663-676 (2006), herein incorporated by reference) into a somatic cell, for examples, CI 4, C72, and the like.

[0103] 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.

[0104] 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 072734.1887

[0105] PATENT for both self renewal (in the laboratory) and differentiation. Such cells vary in their differentiation capacity, but it is usually limited to cell types in the organ of origin.

[0106] 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 dendrites. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

[0107] As used herein, the term “proliferation” refers to an increase in cell number.

[0108] As used herein, the term “undifferentiated” refers to a cell that has not yet developed into a specialized cell type.

[0109] 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 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.

[0110] 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 enteric neuron precursors.

[0111] As used herein, the term “directed differentiation” in reference to a stem cell 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 (e.g. enteric neuron precursors, enteric neurons, etc.).

[0112] 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, such as SOXIO, and CD49D).

[0113] As used herein, the term “cell culture” refers to a growth of cells in vitro in an artificial medium for research or medical treatment.

[0114] 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 072734.1887

[0115] PATENT nourish and support the cells. Culture medium can also include growth factors added to produce desired changes in the cells.

[0116] As used herein, the term “contacting” cells with a compound (e.g., at least one inhibitor, activator, and / or inducer) refers to placing the compound in a location that will allow it to touch the cell. The contacting can be accomplished using any suitable methods. For example, contacting can be accomplished by adding the compound to a tube of cells. Contacting can also be accomplished by adding the compound to a culture medium comprising the cells. Each of the compounds (e.g., the inhibitors, activators, and molecules that induce vagal neural crest patterning disclosed herein) can be added to a culture medium comprising the cells as a solution (e.g., a concentrated solution). Alternatively or additionally, the compounds (e.g., the inhibitors, activators, and molecules that induce vagal neural crest patterning disclosed herein) as well as the cells can be present in a formulated cell culture medium.

[0117] As used herein, the term “zzz 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.

[0118] As used herein, the term “zzz vzvo” 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.

[0119] 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.

[0120] 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 can refer to a “pattern” of markers such that a designated group of markers can identity a cell or cell type from another cell or cell type.

[0121] 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.) a parent cell in a cell line, tissue (such as a dissociated embryo, or fluids using any manipulation, such as, without limitation, single cell isolation, cultured 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 072734.1887

[0122] PATENT 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.

[0123] 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, 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.

[0124] 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.

[0125] 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 can prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0126] 7.2 Method of Differentiating Stem Cells

[0127] The present disclosure provides 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 and at least one activator of Wnt signaling (referred to as “Wnt activator”), to obtain a cell population comprising differentiated cells expressing at least one hindbrain lineage marker. The present disclosure further provides methods for inducing differentiation of stem cells, comprising contacting stem cells with at least one inhibitor SMAD signaling, at least one activator of Wnt signaling, and at least one molecule that induces vagal neural crest patterning to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker.

[0128] 7.2.1. Stem Cells 072734.1887

[0129] PATENT

[0130] The presently disclosed subject matter provides in vitro methods for inducing differentiation of stem cells to cells expressing at least one hindbrain lineage marker. The presently disclosed subject matter further provides in vitro methods for inducing differentiation of stem cells to cells expressing at least one enteric neural crest lineage marker. The presently disclosed subject matter further provides for in vitro methods for inducing differentiation of stem cells to cells expressing at least one enteric neural marker. 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 human stem cell is a human embryonic stem cell (hESC). In certain embodiments, the human stem cell is a human induced pluripotent stem cell (hiPSC). In certain embodiments, the stem cells are non- human stem cells. Non-limiting examples of non-human stem cells non-human primate stem cells, rodent stem cells, dog stem cells, cat stem cells. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the stem cells are embryonic stem cells. In certain embodiments, the stem cells are induced pluripotent stem cells.

[0131] 7.2.2. SMAD Inhibitors

[0132] 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 methods disclosed herein comprise contacting a population of stem cells (e.g., human stem cells) with an effective amount(s) of at least one inhibitor of transforming growth factor beta (TGFP) / Activin-Nodal signaling. In certain embodiments, the inhibitor of TGFp / Activin- Nodal signaling neutralizes the ligands including TGFPs, bone morphogenetic proteins (BMPs), Nodal, and activins, orblocking 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 072734.1887

[0133] PATENT inhibitor is selected from inhibitors of ALK5, inhibitors of ALKA, 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 stimulates SMAD phosphorylation of Smad2 and Smad3. In certain embodiments, the inhibitor of TGFp / Activin-Nodal signaling 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:

[0134] In certain embodiments, the at least one inhibitor of SMAD signaling comprises at least one inhibitor of TGFp / Activin-Nodal signaling. 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.

[0135] The stem cells can be contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling for 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, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, or at least about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin- Nodal signaling for 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 072734.1887

[0136] PATENT to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, up to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling for between 10 days and about 15 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin- Nodal signaling for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling for about 11 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker.

[0137] In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling in a concentration of from about 1 nM to about 300 nM, from about 5 nM to about 250 nM, from about 10 nM to about 200 nM, from about 10 nM to about 50 nM, from about 50 nM to about 150 nM, from about 80 nM to about 120 nM, from about 072734.1887

[0138] PATENT

[0139] 90 nM to about 110 nM, from about 50 nM to about 100 nM, from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, or from about 250 nM to about 300 nM, to produce vagal competent cells. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling in a concentration of from about 80 nM to about 120 nM, to produce vagal competent cells. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling in a concentration of about 100 nM, to produce vagal competent cells. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling in any one of the above-described concentrations daily. In certain embodiments, the stem cells are contacted with the at least one inhibitor of TGFp / Activin-Nodal signaling in a concentration of about 100 nM daily, to produce vagal competent cells.

[0140] 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 those disclosed in WO2011 / 149762, 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 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.

[0141] 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, 072734.1887

[0142] PATENT 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).

[0143] In certain embodiments, the BMP inhibitor comprises LDN193189. In certain embodiments, the BMP inhibitor comprises Noggin.

[0144] 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.

[0145] 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. 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.

[0146] In certain embodiments, the stem cells can be contacted with the at least one inhibitor of SMAD signaling for 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, 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, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, or at least about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling for 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, for 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, up 072734.1887

[0147] PATENT to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling for between 10 days and about 15 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling for about 11 days.

[0148] In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling in a concentration of from about 1 pM to 100 pM, from about 1 pM to 20 pM, from about 1 pM to 15 pM, from about 1 pM to 10 pM, from about 1 pM to 5 pM, from about 5 pM to 10 pM, from about 5 pM to 15 pM, from about 15 pM to 20 pM, from about 20 pM to 30 pM, from about 30 pM to 40 pM, from about 40 pM to 50 pM, from about 50 pM to 60 pM, from about 60 pM to 70 pM, from about 70 pM to 80 pM, from about 80 pM to 90 pM, or from about 90 pM to 100 pM, to produce a population of vagal competent cells 072734.1887

[0149] PATENT expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling in a concentration of from about from about 5 pM to 15 pM. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling in a concentration of about 10 pM. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling in any one of the abovedescribed concentrations daily. In certain embodiments, the stem cells are contacted with the at least one inhibitor of SMAD signaling in a concentration of about 10 pM daily.

[0150] 7.2.3. Wnt A ctivators

[0151] In certain embodiments, the method of in vitro inducing differentiation of stem cells to vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker comprises contacting the cells with an effective amount(s) of at least one activator of wingless (Wnt) signaling (also referred to as “Wnt activators”). As used herein, the term “WNT” or “wingless” in reference to a ligand refers to a group of secreted proteins (i.e. Inti (integration 1) in humans) capable of interacting with a WNT receptor, such as a receptor in the Frizzled and LRPDerailed / RYK receptor family. As used herein, the term “WNT” or “wingless” in reference to a signaling pathway refers to a signal pathway composed of Wnt family ligands and Wnt family receptors, such as Frizzled and LRPDerailed / RYK receptors, mediated with or without P-catenin.

[0152] In certain embodiments, the at least one activator of Wnt signaling lowers glycogen synthase kinase 3P (GSK3P) for activation of Wnt signaling. Thus, the activator of Wnt signaling can be a GSK3P inhibitor. A GSK3P inhibitor is capable of activating a WNT signaling pathway, see e.g., Cadigan, et al., J Cell Sci. 2006; 119:395-402; Kikuchi, et al., Cell Signalling. 2007;19:659-671, which are incorporated by reference herein in their entireties. As used herein, the term “glycogen synthase kinase 3p 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 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 WO201 1 / 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. 072734.1887

[0153] PATENT

[0154] 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 a!., 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.

[0155] In certain embodiments, the Wnt activator is CHIR99021. “CHIR99021” (also known as or “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.

[0156] 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.

[0157] The stem cells can be contacted with the at least one activator of Wnt signaling for 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, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, or at least about 29 days, at least about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem 072734.1887

[0158] PATENT cells are contacted with the at least one activator of Wnt signaling for 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, up to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for between 5 days and about 15 days. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for about 3 days, about 4 days, about 5 days, about 6 days, about

[0159] 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for about 11 days. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for about 10 days. In certain embodiments, the stem cells are contacted with the at least one activator of Wnt signaling for about 9 days. 072734.1887

[0160] PATENT

[0161] In certain non-limiting embodiments, the population of cells described herein are contacted with an initial concentration of CHIR99021 at a concentration of between about 2 and 15 pM, or between abut 3 and 14 pM, or between about 4 and 13 pM, or between about 3 and 5 pM, or about 3 pM, or about 4.5 pM, wherein the concentration of CHIR99021 is decreased, as described herein, for example, about 24 hours after the initial contact of the cells to CHIR99021, to between 0.001 and 2 pM, or between about 0.01 and 1.5 pM, or between about 0.1 and 1 pM, or about 0.9 pM. In certain embodiments, the at least one activator of Wnt signaling is contacted to the cells at an initial concentration of between about 2 and 15 pM, or between abut 3 and 14 pM, or between about 4 and 13 pM, or between about 5 and 12 pM, or between about 6 and 11 pM, or between about 7 and 10 pM, or between about 8 and 9 pM. In certain embodiments, the at least one activator of Wnt signaling is contacted to the cells at an initial concentration of about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 or about 10 pM. In certain embodiments, the at least one activator of Wnt signaling is contacted to the cells at an initial concentration of about 4.5 pM.

[0162] In certain embodiments, the concentration of the activator of Wnt signaling is decreased at least about 12, about 18, about 24, about 30, about 36, about 42, or about 48 hours after the cells are initially contacted with the activator of Wnt signaling, for example, between about 12 and about 48 hours, or between about 12 and about 42 hours, or between about 12 and about 36 hours, or between about 12 and about 30 hours, or between about 12 and about 24 hours, or between about 18 and about 48 hours, or between about 18 and about 42 hours, or between about 18 and about 36 hours, or between about 18 and 30 hours, or between about 18 and about 24 hours, or between about 24 and about 48 hours, or between about 24 and about 42 hours, or between about 24 and about 36 hours, or between about 24 and about 30 hours. In certain embodiments, the stem cells are contacted with the decreased concentration of the Wnt activator for at least about 4, 5, 6, 7, 8, 9, or 10 days or more, for example, between about 4 and 20 days, or between about 5 and 19 days, or between about 6 and 18 days, or between about 7 and 17 days, or between about 8 and 16 days, or between about 9 and 15 days, or between about 8 and 14 days, or between about 9 and 13 days, or between about 10 and 12 days. In certain embodiments, the stem cells are contacted with the decreased concentration of the Wnt activator for up to about 4, 5, 6, 7, 8, 9, or 10 days or more. In certain embodiments, the stem cells are contacted with the decreased concentration 072734.1887

[0163] PATENT of the Wnt activator for about 5, 6, 7, 8, 9, 10, or 11 days. In certain embodiments, the stem cells are contacted with the decreased concentration of the Wnt activator for about 8 days.

[0164] In certain embodiments, the concentration of the activator of Wnt signaling is decreased to a concentration of between about 0.01 pM and about 1 pM, or between about 0.01 pM and about 0.75 pM, or between about 0.01 pM and about 0.5 pM, or between about 0.01 pM and about 0.25 pM, or between about 0.25 pM and about 1 pM, or between about 0.25 pM and about 0.75 pM, or between about 0.25 pM and about 0.5 pM, or between about 0.5 pM and about 1 pM, or between about 0.5 pM and about 0.75 pM. In certain embodiments, the concentration of the activator of Wnt signaling is decreased to a concentration of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1 pM. In certain embodiments, the at least one activator of Wnt signaling is contacted to the cells at a concentration of about 0.9 pM. In certain embodiments, the concentration of the activator of Wnt signaling is decreased from the initial concentration contacted to the cells by between about 50% and about 95%, or between about 50% and about 90%, or between about 50% and about 80%, or between about 50% and about 70%, or between about 50% and about 60%, or between about 60% and about 95%, or between about 60% and about 90%, or between about 60% and about 80%, or between about 60% and about 70%, between about 70% and about 95%, or between about 70% and about 90%, or between about 70% and about 80%, or between about 80% and about 95%, or between about 80% and about 90%, or between about 90% and about 95%, and values in between. In certain embodiments, the concentration of the activator of Wnt signaling is decreased from the initial concentration contacted to the cells by about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 99%, or more.

[0165] 7.2.4. Molecules that Induce Vagal Neural Crest Patterning

[0166] In certain embodiments, the method of in vitro inducing differentiation of stem cells to vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker comprises contacting the cells with an effective amount(s) of at least one molecule that induces vagal neural crest patterning to produce a population of differentiated cells that express at least one enteric neural crest lineage marker or at least one hindbrain lineage marker. Vagal NC patterning can be characterized by the expression of at least one regional specific homeobox (HOX) gene, 072734.1887

[0167] PATENT including, but not limited to, homeobox B2 (HOXB2), homeobox B3 (HOXB3), homeobox B4 (HOXB4), and homeobox B5 (HOXB5).

[0168] In certain embodiments, the at least one molecule that induces vagal neural crest patterning is selected from the group consisting of retinoic acid (RA), retinol, retinal, tretinoin, isotretinoin, alitretinoin, etretinate, acitretin, tazarotene, bexarotene, adapalene, and combinations thereof. In certain embodiments, the at least one molecule that induces vagal neural crest patterning is selected from the group consisting of activators of FGF signaling, activators of Wnt signaling, and combinations thereof.

[0169] In certain embodiments, the molecule that induces vagal neural crest patterning is retinoic acid (RA). RA has been previously used as an extrinsic factor to shift the regional identity of CNS precursors from anterior to more caudal fates such as during motoneuron specification. The inventors discovered that RA not only directs the regional identity in neural crest lineages, but also induces the expression of vagal markers.

[0170] The cells can be contacted with the at least one molecule that induces vagal neural crest patterning for 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, or at least about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for at least about 2 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for up to about 2 days, up to 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, or up to about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one at least one molecule that induces vagal neural crest patterning for between about 2 days and about 21 days, between about 2 days and about 20 days, between about 2 days to about 10 days, 072734.1887

[0171] PATENT between about 10 days and about 15 days, between about 15 days and about 21 days, between about 2 days and about 6 days, between about 2 days and about 5 days, or between about 5 days and about 10 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for between about 5 days and about 10 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for between 5 days and about 20 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for between about 2 days and about 6 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for between about 2 days and about 10 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for up to about 20 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for about 6 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for about 5 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for about 4 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for about 3 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for about 2 days.

[0172] In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning in a concentration of from about 1 pM to 100 pM, from about 1 pM to 20 pM, from about 1 pM to 15 pM, from about 1 pM to 10 pM, from about 1 pM to 5 pM, from about 5 pM to 10 pM, from about 5 pM to 15 pM, from about 15 pM to 20 pM, from about 20 pM to 30 pM, from about 30 pM to 40 pM, from about 40 pM 072734.1887

[0173] PATENT to 50 pM, from about 50 pM to 60 pM, from about 60 pM to 70 pM, from about 70 pM to 80 pM, from about 80 pM to 90 pM, or from about 90 pM to 100 pM, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning in a concentration of from about from about 5 pM to 15 pM. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning in a concentration of about 10 pM. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning in any one of the abovedescribed concentrations daily. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning in a concentration of about 10 pM daily.

[0174] In certain embodiments, the cells are contacted with or exposed to the at least one molecule that induces vagal neural crest patterning 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, or at least about 6 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the cells are contacted with or exposed to the at least one molecule that induces vagal neural crest patterning about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days from the initial contact of the cells with the at least one SMAD inhibitor. In certain embodiments, the cells are contacted with or exposed to the at least one molecule that induces vagal neural crest patterning about 4 days from the initial contact of the cells with the at least one SMAD inhibitor.

[0175] In certain embodiments, the cells are contacted with or exposed to the at least one molecule that induces vagal neural crest patterning at least about 1 day, or at least about 2 days, or at least about 3 days, at least about 4 days, at least about 5 days, or at least about 6 days from the initial contact of the cells with the at least one SMAD inhibitor, and the cells are contacted with or exposed to the at least one molecule that induces vagal neural rest patterning for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days.

[0176] Vagal competent cells can be differentiated from stem cells in less than about 20 days, less than about 19 days, less than about 18 days, less than about 17 days, less than about 16 days, less than about 15 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than 072734.1887

[0177] PATENT about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, or less than about 4 days after their initial contact with at least one of the inhibitor(s) of TGFp / Activin-Nodal signaling, inhibitor(s) of SMAD signaling, activator(s) of Wnt signaling, and molecule(s) inducing vagal neural crest patterning. In certain embodiments, vagal competent cells are differentiated from stem cells on or after about 11 days after their initial contact with at least one of the inhibitor(s) of TGFp / Activin-Nodal signaling, inhibitor(s) of SMAD signaling, activator(s) of Wnt signaling, and molecule(s) inducing vagal neural crest patterning. In certain embodiments, vagal competent cells are differentiated from stem cells on about 11 days after their initial contact with at least one of the inhibitor(s) of TGFp / Activin-Nodal signaling, inhibitor(s) of SMAD signaling, activator(s) of Wnt signaling, and molecule(s) inducing vagal neural crest patterning.

[0178] In certain embodiments, the two or molecules that induce vagal neural crest patterning are at least one activator of FGF signaling and at least one Wnt activator. Non-limiting examples of activators of FGF signaling include FGF2, FGF4, and FGF8. Non-limiting examples of Wnt activators include CHIR99021 and WNT3A.

[0179] 7.2.5. GDNF and EDN3

[0180] In certain embodiments, the differentiated ENS precursors are contacted with GDNF and EDN3. In certain embodiments, the suitable cell culture medium comprises GDNF and EDN3.

[0181] In certain embodiments, the stem cells are contacted with GDNF and EDN3 for 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, or at least about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for at least about 2 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for up to about 2 days, up to 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, 072734.1887

[0182] PATENT up to about 20 days, or up to about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for between about 2 days and about 21 days, between about 2 days and about 20 days, between about 2 days to about 10 days, between about 10 days and about 15 days, between about 15 days and about 21 days, between about 2 days and about 6 days, between about 2 days and about 5 days, or between about 5 days and about 10 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for between about 5 days and about 10 days. In certain embodiments, the stem cells are contacted with the at least one molecule that induces vagal neural crest patterning for between 5 days and about 20 days. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for between about 2 days and about 6 days. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for between about 2 days and about 10 days. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for up to about 20 days. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for about 6 days. In certain embodiments, the stem cells are contacted with GDNF and EDN3 for about 5 days.

[0183] In certain embodiments, the stem cells are contacted with GDNF in a concentration of from about 1 nM to 100 nM, from about 1 ng / mL to 100 ng / mL, from about 1 ng / mL to 20 ng / mL, from about 20 ng / mL to 30 ng / mL, from about 30 ng / mL to 40 ng / mL, from about 40 ng / mL to 50 ng / mL, from about 50 ng / mL to 60 ng / mL, from about 60 ng / mL to 70 ng / mL, from about 70 ng / mL to 80 ng / mL, from about 80 ng / mL to 90 ng / mL, or from about 90 ng / mL to 100 ng / mL. In certain embodiments, the stem cells are contacted with GDNF in a concentration of from about from about 20 ng / mL to 30 ng / mL. In certain embodiments, the stem cells are contacted with GDNF in a concentration of about 25 ng / mL. In certain embodiments, the stem cells are contacted with GDNF signaling in any one of the above- 072734.1887

[0184] PATENT described concentrations daily, every other day or every two days to produce enteric neurons. In certain embodiments, the stem cells are contacted with EDN3 in a concentration of from about 1 nM to 100 nM, from about 1 ng / mL to 100 ng / mL, from about 1 ng / mL to 20 ng / mL, from about 20 ng / mL to 30 ng / mL, from about 30 ng / mL to 40 ng / mL, from about 40 ng / mL to 50 ng / mL, from about 50 ng / mL to 60 ng / mL, from about 60 ng / mL to 70 ng / mL, from about 70 ng / mL to 80 ng / mL, from about 80 ng / mL to 90 ng / mL, or from about 90 ng / mL to 100 ng / mL. In certain embodiments, the stem cells are contacted with EDN3 in a concentration of from about from about 20 ng / mL to 30 ng / mL. In certain embodiments, the stem cells are contacted with EDN3 in a concentration of about 25 ng / mL. In certain embodiments, the stem cells are contacted with EDN3 signaling in any one of the abovedescribed concentrations daily, every other day or every two days to produce enteric neurons. In certain embodiments, the stem cells are contacted with GDNF and EDN3 concurrently. In certain embodiments, the stem cells are contacted with GDNF and / or EDN3 concurrently with the at least one molecule that induces vagal neural crest patterning.

[0185] 7.2.6. BMP Activators

[0186] In certain embodiments, the method of in vitro inducing differentiation of stem cells to vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker comprises contacting a population of human stem cells with effective amounts of at least one inhibitor of TGFp / Activin-Nodal signaling and effective amounts of at least one activator of BMP signaling.

[0187] In certain embodiments, the BMP activator is contacted to the cells for at least 2 days, at least 3 days, at least 4 days, or at lest 5 days, or for up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days or more. In a specific embodiment, the BMP active agent is contacted to the cells for at least about 2 days.

[0188] In certain embodiments, the activator of BMP signaling is selected from the group consisting of BMP2, BMP4, BMP6, BMP7, derivatives thereof, and mixtures thereof. In certain embodiments, the activator of BMP signaling is contacted to the cells at a concentration of between about 0.01 and 5 ng / ml, between about 0.1 and 2 ng / mL, or between about 1 and 1.5 ng / mL. In a specific embodiment the activator of BMP signaling is contacted to the cells at a concentration of about 1 ng / mL.

[0189] 7.2.7. Exemplary Methods

[0190] The present disclosure provides methods for inducing differentiation of stem cells. In certain embodiments, the stem cells are contacted with or exposed to a first concentration of 072734.1887

[0191] PATENT at least one activator of Wnt signaling to produce a population of cells expressing at least one hindbrain lineage marker. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with at least one inhibitor of SMAD signaling. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with a second concentration of the at least one activator of Wnt signaling. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with at least one molecule that induces vagal neural crest patterning to produce a population of cells expressing at least one enteric neural crest lineage marker.

[0192] In certain embodiments, the stem cells are contacted with or exposed to a first concentration of at least one activator of Wnt signaling to produce a population of cells expressing at least one hindbrain lineage marker. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with at least one inhibitor of SMAD signaling and a second concentration of the at least one activator of Wnt signaling. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with at least one molecule that induces vagal neural crest patterning to produce a population of cells expressing at least one enteric neural crest lineage marker.

[0193] In certain embodiments, the stem cells are contacted with or exposed to a first concentration of at least one activator of Wnt signaling to produce a population of cells expressing at least one hindbrain lineage marker. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with at least one inhibitor of SMAD signaling, a second concentration of the at least one activator of Wnt signaling, and at least one molecule that induces vagal neural crest patterning to produce a population of cells expressing at least one enteric neural crest lineage marker.

[0194] In certain embodiments, the stem cells are contacted with or exposed to at least one inhibitor of SMAD signaling and a first concentration of at least one activator of Wnt signaling to produce a population of cells expressing at least one hindbrain lineage marker. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with the at least one inhibitor of SMAD signaling. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with a second concentration of the at least one activator of Wnt signaling. In certain embodiments, the population of cells expressing at least one hindbrain lineage 072734.1887

[0195] PATENT marker are further contacted with at least one molecule that induces vagal neural crest patterning to produce a population of cells expressing at least one enteric neural crest lineage marker.

[0196] In certain embodiments, the stem cells are contacted with or exposed to at least one inhibitor of SMAD signaling and a first concentration of at least one activator of Wnt signaling to produce a population of cells expressing at least one hindbrain lineage marker. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with at least one inhibitor of SMAD signaling and a second concentration of the at least one activator of Wnt signaling. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with at least one molecule that induces vagal neural crest patterning to produce a population of cells expressing at least one enteric neural crest lineage marker.

[0197] In certain embodiments, the stem cells are contacted with or exposed to a first concentration of at least one activator of Wnt signaling to produce a population of cells expressing at least one hindbrain lineage marker. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with at least one inhibitor of SMAD signaling, a second concentration of the at least one activator of Wnt signaling, and at least one molecule that induces vagal neural crest patterning to produce a population of cells expressing at least one enteric neural crest lineage marker.

[0198] In certain embodiments, the stem cells are contacted with or exposed to at least one inhibitor of SMAD signaling and a first concentration of at least one activator of Wnt signaling to produce a population of cells expressing at least one hindbrain lineage marker. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling, a second concentration of the at least one activator of Wnt signaling, and at least one molecule that induces vagal neural crest patterning to produce a population of cells expressing at least one enteric neural crest lineage marker.

[0199] In certain embodiments, the stem cells are contacted with a first concentration of the at least one activator of Wnt signaling for between about 12 hours and about 48 hours, between about 12 hours and about 24 hours, between about 18 hours and about 24 hours, about 12 hours, about 18 hours, about 24 hours, or about 48 hours, to produce a population of cells expressing at least one hindbrain lineage marker. 072734.1887

[0200] PATENT

[0201] In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with a second concentration of the at least one activator of Wnt signaling. In certain embodiments, the second concentration of the at least one activator of Wnt signaling is decreased with respect to the first concentration of the at least one activator of Wnt signaling. In certain embodiments, the cells are contacted with the second concentration of the at least one activator of Wnt signaling (i.e., the decreased concentration of the at least one activator of Wnt signaling) for at least about 3 days or at least about 4 days. In certain embodiments, the stem cells are contacted with the second concentration of the at least one activator of Wnt signaling for about 3 days, about 4 days, about 5 days, or about 6 days. In certain embodiments, the second concentration of the activator of Wnt signaling is decreased from the first concentration of the activator of Wnt signaling contacted to the cells by between about 50 and about 95%, or between about 70 and 90%. In certain embodiments, said at least one activator of Wnt signaling lowers glycogen synthase kinase 3p (GSK3P) for activation of Wnt signaling. In certain embodiments, said at least one activator of Wnt signaling is a small molecule selected from the group consisting of CHIR99021, derivatives thereof, and mixtures thereof.

[0202] In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling concurrently with the first concentration of the at least one activator of Wnt signaling. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling concurrently with the second concentration of the at least one activator of Wnt signaling (i.e., the decreased concentration of the at least one activator of Wnt signaling). In certain embodiments, the cells are contacted with the at least one inhibitor of SMAD signaling for at least about 3 days. In certain embodiments, the cells are contacted with the at least one inhibitor of SMAD signaling for about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In certain embodiments, the at least one inhibitor of SMAD signaling comprises at least one inhibitor of TGFp / Activin-Nodal signaling. 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 SMAD signaling is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof. In certain embodiments, 072734.1887

[0203] PATENT said at least one inhibitor of BMP signaling is a small molecule selected from the group consisting of LDN193189, derivatives thereof, and mixtures thereof.

[0204] In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are further contacted with or exposed to at least one molecule that induces vagal neural crest patterning to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors). In certain embodiments, the cells are contacted with the at least one molecule that induces vagal neural crest patterning for at least about 2 days. In certain embodiments, the cells are contacted with the at least one molecule that induces vagal neural crest patterning for about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days. In certain embodiments, the cells are initially contacted with said at least one molecule that induces vagal neural crest patterning within an about 8 day period from the initial contact of said population of cells with said at least one inhibitor of SMAD signaling. In certain embodiments, the cells are initially contacted with said at least one molecule that induces vagal neural crest patterning at least about 3 days from the initial contact of said population of cells with said at least one inhibitor of SMAD signaling. In certain embodiments, the cells are initially contacted with said at least one molecule that induces vagal neural crest patterning about 3 days from the initial contact of said population of cells with said at least one inhibitor of SMAD signaling. In certain embodiments, the population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning about 3 days from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said at least one inhibitor of SMAD signaling, and the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one molecule that induces vagal neural crest patterning for at least about 3 days. In certain embodiments, the cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling, the at least one activator of Wnt signaling, and the at least one molecule that induces vagal neural crest patterning concurrently for at least about 3 days. In certain embodiments, the cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling, the at least one activator of Wnt signaling, and the at least one molecule that induces vagal neural crest patterning concurrently for at least about 3 days.

[0205] In certain embodiments, said at least one molecule that induces vagal neural crest patterning is selected from the group consisting of retinoic acid, retinol, retinal, tretinoin, 072734.1887 PATENT isotretinoin, alitretinoin, etretinate, acitretin, tazarotene, bexarotene, adapalene, activators of FGF signaling (e.g., FGF2, FGF4, FGF7, and / or FGF8), Wnt activators (e.g., CHIR99021 and / or WNT3 A), and combinations thereof.

[0206] In certain embodiments, population of stem cells have been differentiated into a population of differentiated cells that express at least one said enteric neural crest lineage marker or said at least one hindbrain lineage marker on or after about 6 days from their initial contact with said at least one inhibitor of SMAD signaling. In certain embodiments, said vagal neural crest patterning is characterized by expression of at least one regional specific homoebox (HOX) gene, e.g., at least one regional specific HOX gene selected from the group consisting of HOXB2, HOXB3, HOXB4, and HOXB5. In certain embodiments, said at least one enteric neural crest lineage marker is selected from the group consisting of PAX3, EDRB, RET, PHOX2A, PHOX2B, NTRK-3, HA D2, HOXB3, HOXB5 and ASCL1. In certain embodiments, said at least one hindbrain lineage marker is GBX2. In certain embodiments, the differentiated cells expressing said at least one hindbrain lineage marker do not express OTX2 or TBXT (brachyury). In certain embodiments, said population of differentiated cells further express at least one SOX10+neural crest lineage marker. In certain embodiments, said SOX10+neural crest lineage marker is CD49D.

[0207] In certain embodiments, said stem cells are human stem cells. In certain embodiments, said human stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, and F-class pluripotent stem cells.

[0208] In certain embodiments, the methods further comprise subjecting said population of differentiated cells to conditions favoring maturation of said differentiated cells into a population of cells that express at least one enteric neuron marker. In certain embodiments, said conditions favoring maturation of said differentiated cells into said population of enteric neurons comprise culturing said differentiated cells in a suitable cell culture medium. In certain embodiments, said suitable cell culture medium comprises at least one molecule that enhances maturation of enteric neural crest (ENC) precursors to enteric neurons. In certain embodiments, said at least one molecule that enhances maturation of ENC precursors to enteric neurons is selected from the group consisting of growth factors and Wnt activators. In certain embodiments, said growth factors are selected from the group consisting of activators of FGF signaling, glial cell line derived neurotrophic factor (GDNF), and ascorbic acid. In certain embodiments, said suitable cell culture medium comprises at least one activator of 072734.1887

[0209] PATENT

[0210] FGF signaling and at least one activator of Wnt signaling. In certain embodiments, said differentiated cells are cultured in said suitable cell culture medium comprising said at least one activator of FGF signaling (e.g., FGF2, FGF4, FGF8, and / or FGF7) and said at least one activator of Wnt signaling for about 4 days. In certain embodiments, said at least one enteric neuron marker is selected from the group consisting of Tuj 1 , MAP2, PHOX2A, PHOX2B, TRKC, ASCL1, HAND2, EDNRB, 5HT, GAB A, NOS, SST, TH, CHAT, DBH, Substance P, VIP, NPY, GnRH, and CGRP.

[0211] In certain embodiments, the stem cells are contacted with at least one inhibitor of TGFp / Activin-Nodal signaling and at least one activator of Wnt signaling for about 7 days, wherein the concentration of the at least one activator of Wnt signaling is decreased about 18 hours or about 24 hours after the cells are initially contacted with the Wnt activator; and with the at least one molecule that induces vagal neural crest patterning for about 3 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker.

[0212] In certain embodiments, the stem cells are contacted with at least one inhibitor of TGFp / Activin-Nodal signaling, at least one inhibitor of SMAD signaling, and at least one activator of Wnt signaling for about 7 days, wherein the concentration of the at least one activator of Wnt signaling is decreased about 18 hours or about 24 hours after the cells are initially contacted with the Wnt activator; and with the at least one molecule that induces vagal neural crest patterning for about 3 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker.

[0213] In certain embodiments, the stem cells are contacted with at least one inhibitor of TGFp / Activin-Nodal signaling, at least one activator of BMP signaling, and at least one activator of Wnt signaling for about 7 days, wherein the concentration of the at least one activator of Wnt signaling is decreased about 18 hours or about 24 hours after the cells are initially contacted with the Wnt activator; and with the at least one molecule that induces vagal neural crest patterning for about 3 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker.

[0214] In certain embodiments, the stem cells are contacted with at least one inhibitor of TGFp / Activin-Nodal signaling and the at least one activator of Wnt signaling for about 7 days, wherein the concentration of the at least one activator of Wnt signaling is decreased about 18 hours or about 24 hours after the cells are initially contacted with the Wnt activator; and with the at least one molecule that induces vagal neural crest patterning for about 3 days, 072734.1887

[0215] PATENT to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker.

[0216] In certain embodiments, the stem cells are contacted with at least one inhibitor of TGFp / Activin-Nodal signaling, at least one inhibitor of SMAD signaling, and at least one activator of Wnt signaling for about 7 days, wherein the concentration of the at least one activator of Wnt signaling is decreased about 18 hours or about 24 hours after the cells are initially contacted with the Wnt activator; and with the at least one molecule that induces vagal neural crest patterning for about 3 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker.

[0217] In certain embodiments, the stem cells are contacted with at least one inhibitor of TGFp / Activin-Nodal signaling, at least one activator of BMP signaling, and at least one activator of Wnt signaling for about 7 days, wherein the concentration of the at least one activator of Wnt signaling is decreased about 18 hours or about 24 hours after the cells are initially contacted with the Wnt activator; and with the at least one molecule that induces vagal neural crest patterning for about 3 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker.

[0218] In certain embodiments, the stem cells are contacted with GDNF and EDN3 concurrently. In certain embodiments, the stem cells are contacted with GDNF and / or EDN3 concurrently with the at least one molecule that induces vagal neural crest patterning.

[0219] 7.2.8. In Vitro Induction of ENS Precursors to Enteric Neurons

[0220] Enteric nervous system (ENS) precursors are unique in their ability to give rise to diverse neuron subsets producing dozens of distinct neurotransmitters and hormones. The ENS precursors can be further induced / matured in vitro to enteric neurons. Enteric neurons can be immature enteric neurons, mature enteric neurons, or a combination thereof. The differentiated ENS precursors can be subjected to conditions favoring maturation of ENS precursors into a population of enteric neurons.

[0221] In certain embodiments, the conditions favoring maturation comprises culturing the differentiated ENS precursors in a suitable cell culture medium. In certain embodiments, the suitable cell culture medium is an NB medium. In certain embodiments, the suitable cell culture medium is an NB medium supplemented with L-Glutamine (e.g., from Gibco, 25030- 164), N2 (e.g., from Stem Cell Technologies, 07156), and B27 (e.g., from Life Technologies, 17504044). The differentiated ENS precursors can be cultured in the suitable cell culture medium for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least 072734.1887

[0222] PATENT about 4 days, for at least about 5 days, for at least about 6 days, for at least about 7 days, for at least about 8 days, for at least about 9 days, for at least about 10 days, for at least about 11 days, for at least about 12 days, for at least about 13 days, for at least about 14 days, for at least about 15 days, for at least about 16 days, for at least about 17 days, for at least about 18 days, for at least about 19 days, for at least about 20 days, for at least about 25 days, for at least about 30 days, for at least about 35 days, for at least about 40 days, for at least about 45 days, or for at least about 50 days, to produce enteric neurons.

[0223] In certain embodiments, the suitable cell culture medium comprises at least one molecule that enhances maturation of ENS precursors to enteric neurons. In certain embodiments, the conditions favoring maturation comprises contacting the differentiated ENS precursors with at least one molecule that enhances maturation of ENS precursors to enteric neurons. In certain embodiments, the at least one molecule that enhances maturation of ENS precursors to enteric neurons is selected from the group consisting of growth factors and Wnt activators described herein. Non-limiting examples of growth factors include activators of FGF signaling (FGF activators), glial cell line derived neurotrophic factor (GDNF), and ascorbic acid. In certain embodiments, the differentiated ENS precursors are contacted with at least one activator of FGF signaling and at least one Wnt activator to produce a population of enteric neurons. In certain embodiments, the suitable cell culture medium comprises at least one FGF activator and at least one WNT activator. Non-limiting examples of activators of FGF signaling include FGF2, FGF4, FGF8, and FGF7. In certain embodiments, the at least one FGF activator is FGF2. In certain embodiments, the at least one WNT activator is CHIR99021.

[0224] In certain embodiments, the ENS precursors are contacted with the at least one FGF activator and at least one Wnt 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, or at least about 10 days, to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one FGF activator and at least one Wnt activator for between about 1 day and about 10 days, between about 1 day and about 5 days, between about 5 days and about 10 days, to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one FGF activator and at least one Wnt activator for between about 1 day and about 5 days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one FGF activator and at least one Wnt activator for about 4 days to produce enteric neurons. 072734.1887

[0225] PATENT

[0226] In certain embodiments, the ENS precursors are contacted with the at least one activator of FGF signaling in a concentration of from about 1 nM to 100 nM, from about 1 nM to 20 nM, from about 1 nM to 15 nM, from about 1 nM to 10 nM, from about 1 nM to 5 nM, from about 5 nM to 10 nM, from about 5 nM to 15 nM, from about 15 nM to 20 nM, from about 20 nM to 30 nM, from about 30 nM to 40 nM, from about 40 nM to 50 nM, from about 50 nM to 60 nM, from about 60 nM to 70 nM, from about 70 nM to 80 nM, from about 80 nM to 90 nM, or from about 90 nM to 100 nM, to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one activator of FGF signaling in a concentration of from about from about 5 nM to 15 nM to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one activator of FGF signaling in a concentration of about 10 nM to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one activator of FGF signaling in any one of the above-described concentrations daily, every other day or every two days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one activator of FGF signaling in a concentration of about 10 nM daily to produce enteric neurons.

[0227] In certain embodiments, the ENS precursors are contacted with the at least one Wnt activator in a concentration of from about 1 pM to 100 pM, from about 1 pM to 20 pM, from about 1 pM to 15 pM, from about 1 pM to 10 pM, from about 1 pM to 5 pM, from about 5 pM to 10 pM, from about 5 pM to 15 pM, from about 15 pM to 20 pM, from about 20 pM to 30 pM, from about 30 pM to 40 pM, from about 40 pM to 50 pM, from about 50 pM to 60 pM, from about 60 pM to 70 pM, from about 70 pM to 80 pM, from about 80 pM to 90 pM, or from about 90 pM to 100 pM, to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one Wnt activator in a concentration of from about from about 1 pM to 5 pM to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one Wnt activator in a concentration of about 3 pM to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one Wnt activator in any one of the above-described concentrations daily, every other day or every two days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with the at least one Wnt activator in a concentration of about 3 pM daily to produce enteric neurons.

[0228] In certain embodiments, the ENS precursors are contacted with the at least one FGF activator and at least one Wnt activator in a cell culture medium to produce enteric neurons. 072734.1887

[0229] PATENT

[0230] In certain embodiments, the cell culture medium is an NB medium. In certain embodiments, the cell culture medium is an NB medium supplemented with L-Glutamine (e.g., from Gibco, 25030-164), N2 (e.g., from Stem Cell Technologies, 07156), and B27 (e.g., from Life Technologies, 17504044).

[0231] In certain embodiments, the differentiated ENS precursors are contacted with GDNF and ascorbic acid to produce a population of enteric neurons. In certain embodiments, the suitable cell culture medium comprises GDNF and ascorbic acid.

[0232] In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid 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 15 days, at least about 20 days, at least about 25 days, at least about 30 days, at least about 35 days, at least about 40 days, at least about 45 days, or at least about 50 days, to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid for between about 1 day and about 50 days, between about 1 day and about 10 days, between about 20 days and about 30 days, between about 30 days and about 40 days, or between about 40 days and about 50 days, to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid for between about 10 day and about 20 days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid for between about 20 day and about 30 days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid for between about 40 day and about 50 days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid for about 10 days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid for about 25 days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid for about 45 days to produce enteric neurons.

[0233] In certain embodiments, the ENS precursors are contacted with GDNF in a concentration of from about 1 nM to 100 nM, from about 1 ng / mL to 100 ng / mL, from about 1 ng / mL to 20 ng / mL, from about 20 ng / mL to 30 ng / mL, from about 30 ng / mL to 40 ng / mL, from about 40 ng / mL to 50 ng / mL, from about 50 ng / mL to 60 ng / mL, from about 60 ng / mL to 70 ng / mL, from about 70 ng / mL to 80 ng / mL, from about 80 ng / mL to 90 ng / mL, or from about 90 ng / mL to 100 ng / mL, to produce enteric neurons. In certain embodiments, the ENS 072734.1887

[0234] PATENT precursors are contacted with GDNF in a concentration of from about from about 20 ng / mL to 30 ng / mL to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF in a concentration of about 25 ng / mL to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF signaling in any one of the above-described concentrations daily, every other day or every two days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF of FGF signaling in a concentration of about 25 ng / mL daily to produce enteric neurons.

[0235] In certain embodiments, the ENS precursors are contacted with ascorbic acid in a concentration of from about 50 pM to 200 pM, from about 50 pM to 100 pM, or from about 100 pM to 200 pM, to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with ascorbic acid in a concentration of from about from about 50 pM to 200 pM to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with ascorbic acid in a concentration of about 100 pM to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with ascorbic acid in any one of the abovedescribed concentrations daily, every other day or every two days to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with ascorbic acid in a concentration of about 100 pM daily to produce enteric neurons.

[0236] In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid in a cell culture medium to produce enteric neurons. In certain embodiments, the cell culture medium is an NB medium. In certain embodiments, the cell culture medium is an NB medium supplemented with L-Glutamine (e.g., from Gibco, 25030-164), N2 (e.g., from Stem Cell Technologies, 07156), and B27 (e.g., from Life Technologies, 17504044).

[0237] In certain embodiments, the ENS precursors are contacted with at least one FGF activator and at least one WNT activator, and are subsequently contacted with GDNF and ascorbic acid. In certain embodiments, the ENS precursors are contacted with FGF2 and CHIR990214, and are subsequently contacted with GDNF and ascorbic acid. In certain embodiments, the enteric neurons are immature enteric neurons. In certain embodiments, the immature enteric neurons express at least one enteric neuron marker, including, but not limited to, beta 3 class III tubulin (Tuj l), paired-like homeobox 2A (PHOX2A), paired-like homeobox 2B (PHOX2B), neurotrophic tyrosine kinase receptor type 3 (TRKC), ASCL1, heart and neural crest derivatives expressed 2 (HAND2), and EDNRB.

[0238] The immature enteric neurons can further differentiate to mature enteric neurons. In certain embodiments, the enteric neurons are mature enteric neurons. In certain embodiments, 072734.1887

[0239] PATENT the mature enteric neurons express at least one enteric neuron marker, including, but not limited to, 5-hydroxytryptamine (5HT), gamma-aminobutyric acid (GABA), nitric oxide synthase (NOS), somatostatin (SST), tyrosine hydroxylase (TH), and choline O- acetyltransferase (CHAT).

[0240] In certain embodiments, the enteric neurons are a mixture or combination of immature enteric neurons and mature enteric neurons.

[0241] In certain embodiments, the conditions favoring maturation further comprises aggregating the differentiated ENS precursors into 3D spheroids, and culturing the 3D spheroids in suspension culture. In certain embodiments, the 3D spheroids are cultured in suspension culture for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In certain embodiments, the 3D spheroids are cultured in suspension for about 4 days. In certain embodiments, the suspension culture medium is a Neurobasal medium supplemented with N2 supplement, and B27® supplement comprising CHIR99021 and fibroblast growth factor 2 (FGF2).

[0242] In certain embodiments, the conditions favoring maturation further comprises culturing the 3D spheroids in adherent culture in the presence of ascorbic acid (AA) and GDNF for spontaneous differentiation following culturing the 3D spheroids in suspension culture.

[0243] The 3D spheroids can be cultured in adherent culture for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In certain embodiments, the 3D spheroids are cultured in adherent culture for about 3 weeks, e.g., about 20 days. In certain embodiments, the 3D spheroids are cultured in adherent culture for about 6 weeks, e.g., about 40 days. In certain embodiments, the adherent culture medium is a Neurobasal medium supplemented with N2 supplement, and B27® supplement comprising GDNF and ascorbic acid. In certain non-limiting embodiment, the adherent culture is performed on a surface with a suitable coating, for example, poly ornithine, laminin, fibronectin, or a combination thereof (e.g., the methods described in Zeltner, et al., (2014), , which is incorporated herein by reference in its entirety). In certain embodiments, the cells aggregated to the 3D spheroids first differentiate to a population of immature neurons in the adherent culture and migrate out of the 3D spheroids.

[0244] 7.2.9. Cell Culture Media 072734.1887

[0245] PATENT

[0246] In certain embodiments, the above-described inhibitors, activators and molecules are added to a cell culture medium comprising the stem cells. Suitable cell culture media include, but are not limited to, Knockout® Serum Replacement (“KSR”) medium, N2 medium, and an Essential 8® / Essential 6® (“E8 / E6”) medium, and a Neurobasal (NB) medium (e.g., a NB medium supplemented with N2 and B-27® Supplement). KSR medium, N2 medium, E8ZE6 medium and NB medium are commercially available.

[0247] In certain embodiments, a medium for in vitro differentiation of stem cells to cells expressing at least one enteric neural crest lineage marker (ENS precursors) or cells expressing at least one hindbrain lineage marker is a medium selected from the group consisting of a KSR medium, a N2 medium, and a combination thereof. In certain embodiments, a medium for in vitro differentiation of stem cells to cells expressing at least one enteric neural crest lineage marker (ENS precursors) is an E8ZE6 medium. In certain embodiments, a medium for in vitro induction of cells expressing at least one enteric neural crest lineage marker (ENS precursors) to cells expressing at least one enteric neuron marker (enteric neurons) is an NB medium.

[0248] KSR medium is a defined, serum-free formulation optimized to grow and maintain undifferentiated hESC cells in culture. The components of a KSR medium are disclosed in WO201 1 / 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 can comprise 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.

[0249] In certain embodiments, the stem cells are initially cultured in a KSR medium, which is gradually replaced with increasing amount of a N2 medium from about 1, about 2, about 3, about 4, about 5, about 6, about 7 about 8 days after the initial contact of the stem cells with at least one of the above-described inhibitors, activators, and molecules that induce vagal neural crest patterning. In certain embodiments, the stem cells are initially cultured in a KSR medium, which is gradually replaced with increasing amount of aN2 medium from about day 4 after the initial contact of the stem cells with at least one of the above-described inhibitors, activators, and molecules that induce vagal neural crest patterning (e.g., 4 days after the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling).

[0250] In certain embodiments, the stem cells are contacted with at least one inhibitor of TGFp / Activin-Nodal signaling, at least one inhibitor of SMAD signaling, at least one 072734.1887

[0251] PATENT activator of Wnt signaling, and at least one molecule that induces vagal patterning, wherein the concentration of the at least one activator of Wnt signaling is decreased about 18 hours or about 24 hours after the cells are initially contacted with the Wnt activator. In certain embodiments, at least one inhibitor of TGFp / Activin-Nodal signaling, at least one inhibitor of SMAD signaling, at least one activator of Wnt signaling, and at least one molecule that induces vagal patterning are added to a cell culture medium comprising the stem cells. In certain embodiments, the cell culture medium is a KSR medium.

[0252] 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, selenum, insulin, NaHCOs, transferrin, FGF2 and TGFp. The E8ZE6 medium differs from a KSR medium in that E8ZE6 medium does not include an active BMP or Wnt ingredient. Thus, in certain embodiments, when an E8ZE6 medium is used to culture the presently disclosed population of stem cells to differentiate into a population of enteric neural crest precursors (ENS precursors), at least one inhibitor of SMAD signaling (e.g., those inhibiting BMP) is not required to be added to the E8ZE6 medium. In certain embodiments, the stem cells are contacted with at least one inhibitor of TGFp / Activin-Nodal signaling, at least one activator of Wnt signaling, and at least one molecule that induces vagal patterning, wherein the concentration of the at least one activator of Wnt signaling is decreased about 18 hours or about 24 hours after the cells are initially contacted with the Wnt activator. In certain embodiments, at least one inhibitor of TGFp / Activin-Nodal signaling, at least one activator of Wnt signaling, and at least one molecule that induces vagal patterning are added to a cell culture medium comprising the stem cells. In certain embodiments, the cell culture medium is an E8ZE6 medium.

[0253] N2 supplement is a chemically defined, animal-free, supplement used for expansion of undifferentiated neural stem and progenitor cells in culture. N2 Supplement is intended for use with DMEM / F12 medium. The components of a N2 medium are disclosed in WO201 1 / 149762. In certain embodiments, a N2 medium comprises a DMEM / F12 medium supplemented with glucose, sodium bicarbonate, putrescine, progesterone, sodium selenite, 072734.1887

[0254] PATENT transferrin, and insulin. In certain embodiments, 1 liter of a N2 medium comprises 985 ml dist. H2O with DMEM / F12 powder, 1.55 g of glucose, 2.00 g of sodium bicarbonate, putrescine (100 uL aliquot of 1.61 g dissolved in 100 mL of distilled water), progesterone (20 uL aliquot of 0.032g dissolved in 100 mL 100% ethanol), sodium selenite (60 uL aliquot of 0.5 mM solution in distilled water), 100 mg of transferrin, and 25 mg of insulin in 10 mL of 5 mM NaOH.

[0255] The cell culture medium used for culturing the presently disclosed population of stem cells not only determines the inhibitor(s) to be contacted with the stem cells (e.g., for a KSR medium, at least one inhibitor of TGFp / Activin-Nodal signaling and at least one inhibitor of SMAD signaling are required; and for an E8ZE6 medium, only at least one inhibitor of TGFp / Activin-Nodal signaling is required), but also determines the sequence of contacting the above-described inhibitor(s), activator(s) and molecule(s) with the stem cells.

[0256] In certain embodiments, the initial contact of the cells with an effective amount(s) of the at least one activator of Wnt signaling occurs within about a 4 day period (e.g., concurrently (on the same day), about 1 day, about 2 days, about 3 days, or about 4 days) beginning within the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling. The at least one activator of Wnt signaling can be added the same day as the at least one inhibitor of TGFp / Activin-Nodal signaling or one, or two, or three days later. In certain embodiments, the cells are contacted with at least one activator of Wnt signaling and the at least one inhibitor of TGFp / Activin-Nodal signaling within a 96 hour period.

[0257] In time periods set forth herein, if at least one agents (activators, inhibitors, molecules) are added on the same day (in the same 24 hour period), they can be added in any order unless specified herein to the contrary.

[0258] In certain embodiments, the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling, with the at least one inhibitor of SMAD signaling, and with the at least one activator of Wnt signaling is on the same day, e.g., by initially adding these inhibitors and the Wnt signaling activator(s) to a cell culture medium comprising the stem cells on the same day. In certain embodiments, the cell culture medium is a KSR medium.

[0259] In certain embodiments, the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling is the same day as the initial contact of the stem cells with the at least one inhibitor of SMAD signaling, e.g., by initially adding these 072734.1887

[0260] PATENT inhibitors to a cell culture medium comprising the stem cells on the same day. In certain embodiments, the cell culture medium is a KSR medium. In certain embodiments, the initial contact of the cells with the at least one activator of Wnt signaling is between about 1 and about 4 days (e.g., about 1 day, about 2 days, about 3 days, or about 4 days) from the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling. In certain embodiments, the cell culture medium is a KSR medium. In certain embodiments, the initial contact of the cells with the at least one activator of Wnt signaling is about 2 days from the initial contact with the at least one inhibitor of TGFp / Activin-Nodal signaling.

[0261] In certain embodiments, the cell culture medium for in vitro differentiation of stem cells to cell expressing at least one enteric neural crest lineage marker is an E8ZE6 medium, and the initial contact of the cells with the at least one Wnt activator is the same day as the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling, e.g., by initially adding the Wnt activator(s) and inhibitor(s) of TGFp / Activin- Nodal signaling to a cell culture medium comprising the stem cells on the same day. In certain embodiments, a bone morphogenetic protein (BMP) active agent is added to the E8ZE6 medium. In certain embodiments, the BMP active agent is withdrawn from the medium after about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days of culture. In certain embodiments, the BMP active agent is withdrawn from the medium after about 3 days of culture. In certain embodiments, the BMP active agent is present in the culture medium at a concentration of from between about 0.5 and about 20 ng / mL, or between about 1 and about 15 ng / ml, or between about 2 and about 10 ng / ml, or between about 3 and about 5 ng / ml. In certain embodiments the BMP active agent is present in the culture medium at a concentration of about 5 ng / ml.

[0262] In certain embodiments, the initial contact of the cells with the at least one molecule that induces vagal neural crest patterning is within an about 8 day period (no later than about 8 days) from or beginning with the initial contact of the cells with the at least one activator of Wnt signaling. In certain embodiments, the initial contact of the stem cells with the at least one molecule that induces vagal neural crest patterning is at least about 2 days, or at least about 3 days, or at least about 4 days from the initial contact of the cells with the at least one activator of Wnt signaling. In certain embodiments, initial contact of the cells with the at least one molecule that induces vagal neural crest patterning is about 1 day, about 2 days, 072734.1887

[0263] PATENT about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or about 8 days from the initial contact of stem cells with the at least one activator of Wnt signaling.

[0264] In certain embodiments, the initial contact of the cells with the at least one molecule that induces vagal neural crest patterning is about 4 days from the initial contact of the cells with the at least one activator of Wnt signaling. In certain embodiments, the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling is on day 1, the initial contact of the stem cells with the at least one inhibitor of SMAD signaling is on day 1, the initial contact of the cells with the at least one activator of Wnt signaling on day 0, and the initial contact of the cells with the at least one molecule that induces vagal neural crest patterning is on day 4. In certain embodiments, the cell culture is a KSR medium, a N2 medium, or a combination thereof.

[0265] In certain embodiments, the initial contact of stem cells with the at least one molecule that induces vagal neural crest patterning is about 4 days from the initial contact of the cells with the at least one activator of Wnt signaling. In certain embodiments, the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling is on day 1, the initial contact of the cells with the at least one activator of Wnt signaling is on day 0, and the initial contact of the cells with the at least one molecule that induces vagal neural crest patterning is on day 4. In certain embodiments, the cell culture is an E8ZE6 medium.

[0266] 7.2.10. Differentiated Cells

[0267] In certain embodiments, the differentiated cells express at least one hindbrain lineage marker. Non-limiting examples of hindbrain lineage markers include GBX2. In certain embodiments, the differentiated cells expressing at least one hindbrain lineage marker do not express OTX2 or TBXT (brachyury).

[0268] In certain embodiments, the differentiated cells express at least one enteric neural crest lineage marker. Non-limiting examples of enteric neural crest lineage markers include paired box 3 (PAX3), endothelin receptor type B (EDNRB) and ret proto-oncogene (RET), paired- like homeobox 2A (PHOX2A), paired-like homeobox 2B (PHOX2B), neurotrophic tyrosine kinase receptor type 3 (NTRK-3), achaete-scute complex homolog 1 (ASCL1), heart and neural crest derivatives expressed 2 (HAND2), homeobox B3 (HOXB3), homeobox B5 (HOXB5).

[0269] In certain embodiments, the differentiated ENS precursors further express at least one general neural crest marker. Non-limiting examples of general neural crest marker include forkhead box D3 (FOXD3), transcription factor AP-2 alpha (TFAP2A), T-box 2 (TBX2), 072734.1887

[0270] PATENT

[0271] RP4-792G4.2, RNA, 28S ribosomal 5 (RNA28S5), transcription factor AP-2 beta (TFAP2B), inscuteable homolog (INSC), RP11-200A13.2, cilia and flagella associated protein 126 (Clorfl92), retinoid X receptor gamma (RXRG), complement factor H (CFH), and SOXIO.

[0272] In certain embodiments, the differentiated ENS precursors further express at least one vagal marker. Non-limiting examples of vagal markers include HOXB2, HOXB2, HOXB3, HOXB4, and HOXB5.

[0273] In certain embodiments, the differentiated ENS precursors cells further express at least one SOX10+neural crest lineage marker. In certain embodiments, the SOX10+neural crest lineage marker is CD49D.

[0274] In certain embodiments, the differentiated cells are enteric neurons expressing at least one enteric neuron marker. Non-limiting examples of enteric neuron markers include Tuj 1, MAP2, PHOX2A, PHOX2B, TRKC, ASCL1, HAND2, EDNRB, 5HT, GABA, NOS, SST, TH, CHAT, DBH, Substance P, VIP, NPY, GnRH, and CGRP.

[0275] The differentiated ENS precursors or enteric neurons can further express at least one reporter. Non-limiting examples of reporters 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 promotor of a enteric neural crest lineage marker gene, for example, a SOXIO promoter.

[0276] The differentiated ENS precursors and further matured enteric neurons can be purified after differentiation, e.g., in a cell culture medium. As used herein, the terms “purified,” “purify,” “purification,” “isolated,” “isolate,” and “isolation” refer to the reduction in the amount of at least one contaminant from a sample. For example, a desired cell type is purified by at least 10%, by at least 30%, by at least 50%, by at least 75%, and by at least 90%>, with a corresponding reduction in the amount of undesirable cell types. The term “purify” can refer to the removal of certain cells (e.g., undesirable cells) from a sample. The removal or selection of non-noci ceptor cells results in an increase in the percent of desired nociceptor 072734.1887

[0277] PATENT cells in the sample. In certain embodiments, the cells are purified by sorting a mixed cell population into cells expressing at least one SOX10+neural crest lineage marker, e.g., CD49D. In certain embodiments, the cells are purified by sorting a mixed cell population into cells expressing at least one enteric neural crest lineage marker, e.g., PAX3, EDNRB, RET, PHOX2A, PHOX2B, NTRK-3, HAND2, HOXB3, HOXB5 and / or ASCL1.

[0278] The presently disclosed subject matter also provides a population of in vitro differentiated cells expressing at least one enteric neural crest lineage marker produced by the methods described herein, and compositions comprising such in vitro differentiated cells. The presently disclosed subject matter also provides a population of in vitro differentiated cells expressing at least one enteric neural marker produced by the methods described herein, and compositions comprising such in vitro differentiated cells. The presently disclosed subject matter also provides a population of in vitro differentiated cells expressing at least one hindbrain lineage marker produced by the methods described herein, and compositions comprising such in vitro differentiated cells.

[0279] 7.3 Compositions Comprising Differentiated Cell Populations

[0280] The presently disclosed subject matter provides compositions comprising a population of differentiated enteric neural crest lineage cells (or “ENS precursors”) produced by the in vitro differentiation methods described herewith. The presently disclosed subject matter further provides compositions comprising a population of enteric neurons matured from the in vitro differentiated enteric neural crest lineage cells (or “ENS precursors”) described herewith. The presently disclosed subject matter further provides compositions comprising a population of differentiated hindbrain lineage cells produced by the in vitro differentiation methods described herewith.

[0281] Furthermore, the presently disclosed subject matter provides compositions comprising a population of in vitro differentiated cells, wherein at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.5%) of the population of cells express at least one enteric neural crest lineage markers, and wherein less than about 15% (e.g., 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 population of cells express at least one marker selected from the group consisting of stem cell markers, CNS markers, Cranial Neural Crest (CNC) markers, Melanocyte-competent Neural Crest (MNC) markers, enteric neuron markers, neuronal cell markers, and mesenchymal precursor markers. 072734.1887

[0282] PATENT

[0283] Furthermore, the presently disclosed subject matter provides compositions comprising a population of in vitro differentiated cells, wherein at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.5%)) of the population of cells express at least one enteric neuron markers, and wherein less than about 15% (e.g., 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 population of cells express at least one marker selected from the group consisting of stem cell markers, enteric neural crest lineage markers, CNS markers, Cranial Neural Crest (CNC) markers, Melanocyte-competent Neural Crest (MNC) markers, neuronal cell markers, and mesenchymal precursor markers.

[0284] Non-limiting examples of enteric neural crest lineage markers include PAX3, EDNRB, RET, PHOX2A, PHOX2B, NTRK-3, HAND2, HOXB3, HOXB5 and ASCL1.

[0285] Non-limiting examples of enteric neuron markers include Tuj l, MAP2, PHOX2A, PHOX2B, TRKC, ASCL1, HAND2, EDNRB, 5HT, GABA, NOS, SST, TH, CHAT, DBH, Substance P, VIP, NPY, GnRH, and CGRP.

[0286] Non-limiting examples of stem cell markers include OCT4, NANOG, SOX2, LIN28, SSEA4 and SSEA3.

[0287] Non-limiting examples of CNS markers include PAX6, NESTIN, Vimentin, FOXG1, SOX2, TBR1,TBR2 and SOX1.

[0288] Non-limiting examples of neuronal cell markers include TUJ1, MAP2, NFH, BRN3A, ISL1, TH, ASCL1, CHAT, PHOX2B, PHOX2A, TRKA, TRKB, TRKC, 5HT, GABA, NOS, SST, TH, CHAT, DBH, Substance P, VIP, NPY, GnRH, and CGRP.

[0289] Non-limiting examples of mesenchymal precursor markers are SMA, Vimentin, HLA-ABC, CD 105, CD90 and CD73.

[0290] Non-limiting examples of CNC markers include PAX6, NESTIN, Vimentin, FOXG1, SOX2, TBR1,TBR2 and SOX1.

[0291] Non-limiting examples of MNC markers include PAX6, NESTIN, Vimentin, FOXG1, SOX2, TBR1,TBR2 and SOX1.

[0292] Non-limiting examples of hindbrain lineage markers include GBX2.

[0293] In certain embodiments, the composition comprises a population of from about 1 x 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, 072734.1887

[0294] PATENT from about 1 x 108to about 1 x 109, from about 1 x 108to about 1 x IO10, or from about 1 x 109to about 1 x IO10of the presently disclosed stem-cell-derived enteric neural crest lineage cells or matured enteric neurons. In certain embodiments, the composition comprises a population of from about 1 x 105to about 1 x 107of the presently disclosed stem-cell-derived enteric neural crest lineage cells or matured enteric neurons.

[0295] In certain non-limiting 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 non-limiting 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).

[0296] In certain embodiments, the composition is a pharmaceutical composition that comprises a pharmaceutically acceptable carrier, excipient, diluent or a combination thereof. The compositions can be used for preventing and / or treating enteric neuron related disorders, e.g., Hirschsprung disease (HSCR).

[0297] 7.4 Method of Preventins and / or Treatins Enteric Nervous System Disorders

[0298] The in vitro differentiated cells that express at least one enteric neural crest lineage marker (also referred to as “stem-cell-derived enteric neural crest (NC) precursors” or “ENS precursors”) can be used for preventing and / or treating enteric nervous system disorders (ENSs). The enteric neurons matured from the in vitro differentiated ENS precursors can also be used for preventing and / or treating ENSs. The presently disclosed subject matter provides for methods of preventing and / or treating an ENS disorder comprising administering to a subject suffering from an ENS disorder an effective amount of at least one of the followings:

[0299] (a) a population of differentiated presently disclosed stem-cell-derived enteric NC precursors (ENS precursors) described herein;

[0300] (b) a composition comprising such stem-cell-derived enteric NC precursors (ENS precursors);

[0301] (c) a population of enteric neurons matured from the presently disclosed stem-cell- derived enteric NC precursors (ENS precursors) described herein; and 072734.1887

[0302] PATENT

[0303] (d) a composition comprising such enteric neurons.

[0304] Furthermore, the presently disclosed subject matter provides for uses of the presently disclosed stem-cell-derived enteric NC precursors (ENS precursors) or a composition comprising thereof, or the presently disclosed enteric neurons matured from the stem-cell- derived enteric NC precursors (ENS precursors) for preventing and / or treating an ENS disorder. Non-limiting examples of ENS disorders include Hirschsprung’s disease (HD), toxic megacolon, any intestinal aganglionosis, irritable bowel syndrome, inflammatory bowel disease, gastroparesis, bowel-related drug side effects or other treatment complications. In certain embodiments, the ENS disorder is Hirschsprung’s disease (HD). In certain embodiments, the stem-cell-derived enteric NC precursors express the cell surface marker CD49D.

[0305] The presently disclosed stem-cell-derived enteric NC precursors repopulate the gut and the colon of a subject (e.g., a subject suffering from an ENS disorder (e.g., HD)). In certain embodiments, administration of the presently disclosed stem-cell-derived enteric NC precursors repopulates the colon of a subject suffering from an ENS disorder (e.g., HD) over its entire length from the cecum to the rectum. Widespread engraftment of the presently disclosed stem-cell-derived enteric NC precursors can enable permanent, bona fide repair of the aganglionic portions of the gut. The presently disclosed stem-cell-derived enteric NC precursors can also impact paracrine cytokine release, immunomodulation, and / or changes in barrier function, which can contribute to prevent HD-related death. Therefore, the presently disclosed stem-cell-derived enteric NC precursors provide novel therapeutic opportunities for ENS disorders (e.g., HD).

[0306] The presently disclosed stem-cell-derived enteric NC precursors can be administered or provided systemically or directly to a subject for treating or preventing an ENS disorder. In certain embodiments, the presently disclosed stem-cell-derived enteric NC precursors are directly injected into an organ of interest (e.g., an organ affected by an ENS disorder (e.g., HD)). The presently disclosed stem-cell-derived enteric NC precursors can be administered (injected) directly to a subject’s intestine region, e.g., small intestine, colon, cecum, and / or rectum the. In certain embodiments, the presently disclosed stem-cell-derived enteric NC precursors are administered to the cecum of a subject suffering from an ENS disorder (e.g., HD). In addition, the presently disclosed stem-cell-derived enteric NC precursors can be administered (injected) directly to the wall, smooth muscle, connective issue and / or lymphatic ducts of small intestine, colon, cecum, and / or rectum. In certain embodiments, the presently 072734.1887

[0307] PATENT disclosed stem-cell-derived enteric NC precursors are administered to the wall of the cecum of a subject suffering from an ENS disorder (e.g., HD). The injected cells can migrate to the smooth muscle of small intestine, colon, cecum and / or rectum, and form functional neuromuscular junction.

[0308] The presently disclosed stem-cell-derived enteric NC precursors and / or enteric neurons can be administered in any physiologically acceptable vehicle. Pharmaceutical compositions comprising the presently disclosed stem-cell-derived enteric NC precursors and / or enteric neurons and a pharmaceutically acceptable carrier are also provided. The presently disclosed stem-cell-derived enteric NC precursors and / or enteric neurons and the pharmaceutical compositions comprising thereof can be administered via localized injection, orthotropic (OT) injection, systemic injection, intravenous injection, or parenteral administration. In certain embodiments, the presently disclosed stem-cell-derived enteric NC precursors and / or enteric neurons are administered to a subject suffering from an ENS disorder (e.g., HD) via orthotropic (OT) injection.

[0309] The presently disclosed stem-cell-derived enteric NC precursors and / or enteric neurons and the pharmaceutical compositions comprising thereof can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can 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 enteric NC precursors and / or enteric neurons, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions can 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 072734.1887

[0310] PATENT 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, can be consulted to prepare suitable preparations, without undue experimentation.

[0311] 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 inurn monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the presently disclosed stem-cell-derived enteric NC precursors.

[0312] 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. Obviously, 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).

[0313] 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 enteric NC 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.

[0314] One consideration concerning the therapeutic use of the presently disclosed stem-cell- derived enteric NC precursors is the quantity of cells necessary to achieve an optimal effect. An optimal effect include, but are not limited to, repopulation of gut, repopulation of colon, 072734.1887

[0315] PATENT and repopulation of gut and colon of a subject suffering from an ENS disorder (e.g., HD), and / or improved function of the subject’s intestine.

[0316] 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 ENS disorder (e.g., HD), or otherwise reduce the pathological consequences of the ENS disorder (e.g., HD). 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 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.

[0317] In certain embodiments, an effective amount is an amount that is sufficient to repopulate gut, repopulate colon, or repopulate gut and colon of a subject suffering from an ENS disorder (e.g., HD). In certain embodiments, an effective amount is an amount that is sufficient to improve the function of the intestine of a subject suffering from an ENS disorder (e.g., HD), e.g., the improved function can be about 1%, 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 intestine.

[0318] 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 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 presently disclosed stem-cell- derived enteric NC precursors and / or enteric neurons are administered to a subject. In certain embodiments, from about 1 x 105to about 1 x 107the presently disclosed stem-cell-derived enteric NC precursors and / or enteric neurons are administered to a subject suffering from an ENS disorder (e.g., HD). In certain embodiments, about 2 x 105the presently disclosed stemcell-derived enteric NC precursors and / or enteric neurons are administered to a subject suffering from an ENS disorder (e.g., HD). In certain embodiments, from about 1 x 106to about 1 x 107the presently disclosed stem-cell-derived enteric NC precursors and / or enteric neurons are administered to a subject suffering from an ENS disorder (e.g., HD). In certain 072734.1887

[0319] PATENT embodiments, from about 2 x 106to about 4 x 106the presently disclosed stem-cell-derived enteric NC precursors and / or enteric neurons are administered to a subject suffering from an ENS disorder (e.g., HD). 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.

[0320] In certain embodiments, the cells that are administered to a subject suffering from an ENS disorder (e.g., HD) for preventing and / or treating an ENS disorder are a population of the presently disclosed stem-cell-derived enteric NC precursors. In certain embodiments, the cells that are administered to a subject suffering from an ENS disorder (e.g., HD) for preventing and / or treating an ENS disorder are a population of enteric neurons that are differentiated / matured from the presently disclosed stem-cell-derived enteric NC precursors. In certain embodiments, the cells that are administered to a subject suffering from an ENS disorder (e.g., HD) for treating an ENS disorder are a population of enteric ganglia, sensory neurons and motor neurons differentiated / matured from the presently disclosed stem-cell- derived enteric NC precursors.

[0321] 7.5 Methods of Identifying Therapeutic Compounds

[0322] The presently disclosed stem-cell-derived enteric NC precursors and / or the matured enteric neurons can be used to model an ENS disorder (e.g., HD) and serve as a platform to screen for candidate compounds that can overcome disease related migration defects. The capacity of a candidate compound to alleviate an ENS disorder (e.g., HD) can be determined by assaying the candidate compound’s ability to rescue a physiological or cellular defect caused by a genetic mutation, which causes an ENS disorder (e.g., HD).

[0323] The presently disclosed subject matter provides for in vitro methods of identifying or screening compounds suitable for preventing and / or treating an ENS disorder (e.g., HD and / or a HD-related genetic defect). In certain embodiments, the method comprises identifying a compound that is capable of rescuing at least one migration defect presented by a population of cells comprising homozygous loss-of-function mutations in a gene related to cell mobility, e.g., endothelin receptor type B (EDNRB), wherein the population of cells are selected from the group consisting of presently disclosed enteric NC precursors derived from stem cells (e.g., pluripotent stem cells), presently disclosed enteric neurons derived from the stem-cell derived enteric NC precursors, and a combination or mixture thereof. Loss of function mutations in EDNRB is a well-known genetic cause in a subset of HD patients. 072734.1887

[0324] PATENT

[0325] In certain embodiments, the method comprise: (a) providing (i) a population of cells comprising homozygous loss-of-function mutations in EDNRB, wherein the population of cells are selected from the group consisting of presently disclosed enteric NC precursors derived from stem cells (e.g., pluripotent stem cells), presently disclosed enteric neurons derived from the stem-cell derived enteric NC precursors, and a combination or mixture thereof, and (ii) a test compound; (b) contacting the population of cells with the test compound; and (c) measuring the migration behavior of the population of cells. In certain embodiments, the population of cells (e.g., enteric NC precursors and / or enteric neurons) are contacted with the test compound for at least about 6 hours, about 12 hours, about 24 hours (1 day), e.g., about 24 hours (1 day), about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0326] In certain embodiments, the population of cells are enteric NC precursors derived from stem cells.

[0327] Any suitable migration assays can be used for measuring the migration behavior of the enteric NC precursors include, e.g., scratch assays (e.g., migration to an open space of a cell culture plate), wound healing assays, transmigration assays (e.g., cross through micropores), cell exclusion zone assays, microfluidic assays, and Oris® cell migration assays. In addition, the methods for measuring the migration behavior of the enteric NC precursors include measuring the ability of the enteric NC precursors to migrate in a semi-solid culture medium, e.g.,. a hydrogel culture medium. In certain embodiments, the enteric NC precursors are fixed before being measured for their migration behavior.

[0328] In certain embodiments, the method further comprises determining a causal genetic mutation of an ENS disorder (e.g., HD or toxic megacolon). Methods for determining a causal genetic mutation of HD are known in the art, including, but not limited to, genetic lineage analyses among familial patient groups, forward and reverse genetics using mouse model.

[0329] 7.6. Kits

[0330] The presently disclosed subject matter provides for kits for inducing differentiation of stem cells. In certain embodiments, the kit comprises an effective amount(s) of at least one inhibitor of transforming growth factor beta (TGFP) / Activin-Nodal signaling, an effective amount(s) of at least one activator of wingless (Wnt) signaling, an effective amount(s) of at least one molecule that induces vagal neural crest patterning, and instructions for inducing differentiation of the stem cells into a population of differentiated cells that express at least 072734.1887

[0331] PATENT one enteric neural crest lineage marker. In certain embodiments, the kit further comprises an effective amount of GDNF and an effective amount of EDN3.

[0332] In certain embodiments, the instructions comprise directions to contact the stem cells with the inhibitor(s), activator(s) and molecule(s) in a specific sequence. The sequence of contacting the inhibitor(s), activator(s) and molecule(s) can be determined by the cell culture medium used for culturing the stem cells.

[0333] In certain embodiments, the instructions comprise directions to contact the population of cells with an effective amount(s) of the at least one molecule that induces vagal neural crest patterning for at least about 2 days. In certain embodiments, the instructions comprise directions to contact the population of cells with the at least one molecule that induces vagal neural crest patterning for about 6 days. In certain embodiments, the instructions comprise directions to contact the population of cells with the at least one molecule that induces vagal neural crest patterning for about 5 days. In certain embodiments, the instructions comprise directions to initially contact the cells with an effective amount(s) of the at least one activator of Wnt signaling within an about 4 day period (e.g., concurrently (on the same day), about 1 day, about 2 days, about 3 days, or about 4 days) from or beginning with the initial contact of stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling. In certain embodiments, the instructions comprise directions to contact the cells with a decreased concentration of the at least one activator of Wnt signaling about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours after the cells are initially contacted with the Wnt activator.

[0334] In certain embodiments, the instructions comprise directions to initially contact the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling the same day as the initial contact of the cells with the at least one activator of Wnt signaling.

[0335] In certain embodiments, the kit further comprises an effective amount(s) of at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling and the at least one inhibitor of SMAD signaling concurrently. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling, the at least one inhibitor of SMAD signaling, and the at least one activator of Wnt signaling concurrently. In certain embodiments, the instructions comprise directions to initially contact the cells with at least one activator of Wnt signaling within about 1 to about 4 days from the 072734.1887

[0336] PATENT initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling. In certain embodiments, the instructions comprise directions to initially contact the cells with the at least one activator of Wnt signaling about 2 days from the initial contact of the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling. In certain embodiments, the instructions comprise directions to contact the cells with a decreased concentration of the at least one activator of Wnt signaling about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours after the cells are initially contacted with the Wnt activator.

[0337] In certain embodiments, the instructions comprise directions to initially contact the cells with the at least one molecule that induces vagal neural crest patterning within an about 8 day period from or beginning with the initial contact of the cells with the at least one activator of Wnt signaling. In certain embodiments, the instructions comprise directions to initially contact the cells with the at least one molecule that induces vagal neural crest patterning at least about 2 days, at least about 3 days, or at least about 4 days from the initial contact of the stem cells with the at least one activator of Wnt signaling. In certain embodiments, the instructions comprise directions to initially contact the cells with the at least one molecule that induces vagal neural crest patterning about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or about 8 days from the initial contact of the cells with the at least one activator of Wnt signaling.

[0338] In certain embodiments, the instructions comprise directions to initially contact the cells with the at least one molecule that induces vagal neural crest patterning about 4 days from the initial contact of the cells with the at least one activator of Wnt signaling. In certain embodiments, the instructions comprise directions to initially contact the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling on day 1, directions to initially contact the cells with the at least one activator of Wnt signaling on day 0, directions to contact the cells with a decreased concentration of the at least one activator of Wnt signaling about 18 hours or about 24 hours after the after the cells are initially contacted with the Wnt activator, and directions to initially contact the cells with the at least one molecule that induces vagal neural crest patterning on day 4.

[0339] In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling for 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 072734.1887

[0340] PATENT

[0341] 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, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, or at least about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling for 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, up to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of TGFp / Activin- Nodal signaling for between 10 days and about 15 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of TGFp / Activin-Nodal signaling for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 072734.1887 PATENT days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of TGFp / Activin- Nodal signaling for about 11 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker.

[0342] In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of SMAD signaling for 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, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, or at least about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of SMAD signaling for 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

[0343] 21 days, up to about 22 days, up to about 23 days, up to about 24 days, up to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of SMAD signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days 072734.1887

[0344] PATENT and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of SMAD signaling for between 10 days and about 15 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of SMAD signaling for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with the at least one inhibitor of SMAD signaling for about 11 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker.

[0345] In certain embodiments, the instructions comprise directions to contact the cells with the at least one activator of Wnt signaling for 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, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, or at least about 29 days, at least about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In 072734.1887

[0346] PATENT certain embodiments, the instructions comprise directions to contact the cells with the at least one activator of Wnt signaling for 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, up to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, or up to about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one activator of Wnt signaling for between about 4 days and about 30 days, between about 4 days to about 27 days, between about 4 days and about 26 days, between about 4 days and about 25 days, between about 4 days and about 24 days, between about 4 days and about 20 days, between about 4 days and about 15 days, between about 4 days and about 10 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 15 days, between about 15 days and about 20 days, between about 10 days and about 20 days, between about 20 days and about 25 days, or between about 25 days and about 30 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one activator of Wnt signaling for between 5 days and about 15 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one activator of Wnt signaling for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 day, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one activator of Wnt signaling for about 11 days, to produce a population of vagal competent cells expressing at least one enteric 072734.1887

[0347] PATENT neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one activator of Wnt signaling for about 10 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one activator of Wnt signaling for about 9 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker.

[0348] In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for 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 8 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, or at least about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for at least about 2 days. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for up to about 2 days, up to 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, or up to about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for up to about 20 days. In certain embodiments, the instructions comprise directions to contact the cells with the at least one at least one molecule that induces vagal neural crest patterning for between about 2 days and about 21 days, between about 2 days and about 20 days, between about 2 days to about 10 days, between about 10 days and about 15 days, between about 15 days and about 21 days, between about 2 days and about 6 days, between about 2 days and 072734.1887

[0349] PATENT about 5 days, or between about 5 days and about 10 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprises directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for between about 5 days and about 10 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for between 5 days and about 20 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for between about 2 days and about 6 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for between about 2 days and about 10 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for about 6 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for about 5 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. 072734.1887

[0350] PATENT

[0351] In certain embodiments, the instructions comprise directions to contact the stem cells with at least one inhibitor of TGFp / Activin-Nodal signaling and the at least one inhibitor of SMAD signaling for about 12 days; directions to contact the cells with the at least one activator of Wnt signaling for about 10 days; and directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for about 6 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with at least one inhibitor of TGFp / Activin-Nodal signaling and the at least one inhibitor of SMAD signaling for about 11 days; directions to contact the cells with the at least one activator of Wnt signaling for about 9 days; and directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for about 5 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker.

[0352] In certain embodiments, the instructions comprise directions to contact the stem cells with at least one inhibitor of TGFp / Activin-Nodal signaling and the at least one activator of Wnt signaling for about 12 days; and directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for about 6 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker. In certain embodiments, the instructions comprise directions to contact the stem cells with at least one inhibitor of TGFp / Activin- Nodal signaling and the at least one activator of Wnt signaling for about 11 days; and directions to contact the cells with the at least one molecule that induces vagal neural crest patterning for about 5 days, to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker (ENS precursors) or at least one hindbrain lineage marker.

[0353] In certain embodiments, the kit further comprise direction to contact the stem cells with GDNF. In certain embodiments, the kit further comprise direction to contact the stem cells with EDN3. In certain embodiments, the kit further comprise direction to contact the stem cells with GDNF and EDN3 concurrently. In certain embodiments, the kit further comprise direction to contact the stem cells with GDNF and / or EDN3 concurrently with the at least one molecule that induces vagal neural crest patterning. 072734.1887

[0354] PATENT

[0355] In certain embodiments, the kit further comprise instructions for inducing maturation of ENS precursors to enteric neurons.

[0356] In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise culturing the ENS precursors in a suitable cell culture medium. In certain embodiments, the kit further comprise at least one molecule that enhances maturation of ENS precursors to enteric neurons and the instructions for inducing maturation of ENS precursors to enteric neurons comprise contacting the ENS precursors with the at least one molecule that enhances maturation of ENS precursors to enteric neurons. In certain embodiments, the at least one molecule that enhances maturation of ENS precursors to enteric neurons is selected from the group consisting of growth factors and WNT activators. In certain embodiments, the growth factors are selected from the group consisting of FGF activators, GDNF, and ascorbic acid.

[0357] In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with the at least one FGF activator and the at least one Wnt 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, or at least about 10 days, to produce enteric neurons. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with the at least one FGF activator and the at least one Wnt activator for about 1 day and about 10 days, between about 1 day and about 5 days, between about 5 days and about 10 days, to produce enteric neurons. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with the at least one FGF activator and the at least one Wnt activator for between about 1 day and about 5 days to produce enteric neurons. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with the at least one FGF activator and at least one Wnt activator for about 4 days to produce enteric neurons.

[0358] In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with GDNF and ascorbic acid. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with GDNF and ascorbic acid for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, 072734.1887

[0359] PATENT 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, at least about 20 days, at least about 25 days, at least about 30 days, at least about 35 days, at least about 40 days, at least about 45 days, or at least about 50 days, to produce enteric neurons. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with GDNF and ascorbic acid for between about 1 day and about 50 days, between about 1 day and about 10 days, between about 20 days and about 30 days, between about 30 days and about 40 days, or between about 40 days and about 50 days, to produce enteric neurons. In certain embodiments, the ENS precursors are contacted with GDNF and ascorbic acid for between about 10 day and about 20 days to produce enteric neurons. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with GDNF and ascorbic acid for between about 40 day and about 50 days to produce enteric neurons. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with GDNF and ascorbic acid for about 10 days to produce enteric neurons. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise contacting the ENS precursors with GDNF and ascorbic acid for about 25 days to produce enteric neurons. In certain embodiments, the instructions for inducing maturation of ENS precursors to enteric neurons comprise directions to contact the ENS precursors with GDNF and ascorbic acid for about 45 days to produce enteric neurons. Furthermore, the presently disclosed subject matter provides for kits for preventing and / or treating or preventing an ENS disorder (e.g., HD). In certain embodiments, the kit comprises an effective amount of a population of the presently disclosed stem-cell-derived enteric NC precursors or a composition comprising such precursors 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.

[0360] In certain embodiments, the kit comprises instructions for administering a population of the presently disclosed stem-cell-derived enteric NC precursors or a composition comprising thereof to a subject suffering from an ENS disorder (e.g., HD). The instructions can comprise information about the use of the cells or composition for preventing and / or 072734.1887

[0361] PATENT treating an ENS disorder (e.g., HD). In certain embodiments, the instructions comprise at least one of the following: description of the therapeutic agent; dosage schedule and administration for preventing and / or treating an ENS disorder (e.g., HD) 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.

[0362] 8. EXAMPLES

[0363] Example 1: Early acquisition of vagal competence

[0364] Primary axis patterning in the neural ectoderm results in segregation into discrete domains with phenotypically diverse derivatives. This diversity is particularly true of the neural crest, which exhibits substantial fate restriction depending on the precise axial level at which it is formed. Genetic fate mapping identified a surprisingly early commitment to posterior identity in human pluripotent stem cells (hPSC) differentiating into vagal neural crest-like cells. This early decision enables the cells to respond to retinoic acid with the upregulation of vagal markers, allowing for the generation of pure cultures of neurons that transcriptionally reflect those of the human enteric nervous system. The early, committed posterior progenitor identified in our cultures can be induced with transient WNT activation, shares characteristics with the nascent hindbrain in vivo, and appears to exist in a developmental continuum with neuromesodermal progenitor (NMP)-like cells. This work has significant implications for our understanding of how hPSC differentiating into neural ectoderm can adopt hindbrain-level fate over that of other primary axis identities (such as midbrain or spinal cord), and also provides a platform for the generation of pure cultures of enteric neurons or hindbrain central nervous system fates.

[0365] Early acquisition of vagal competence in hPSC-derived neural crest. In the differentiation of hPSC into neural ectoderm-like derivatives, Retinoic Acid (RA) has long been used to caudalize cultures of cells that would otherwise adopt an anterior identity. This effect of RA in vitro reflects the well characterized role the morphogen plays in primary axis patterning during vertebrate development. The caudalizing action of RA on hPSC-derived neurectoderm has largely been demonstrated through observation of increased expression of vagal markers, as measured using whole-population resolution techniques such as qPCR and bulk RNA sequencing. Assessing vagal identity of hPSC-derived neural crest cells using 072734.1887

[0366] PATENT single-cell resolution approaches revealed that only a subset of cells adopt vagal identity. This is despite transcriptional changes occurring in the whole population in response to RA exposure and without any loss of neural crest induction efficiency (FIGS. 24A-24C). This was found to be the case across different approaches to neural crest differentiation, as well as a variety of RA exposure paradigms.

[0367] To investigate why some cells adopted a vagal fate while others did not, the genetic fate mapping tool “CellTag” was applied. CellTag uses a lentiviral delivery system to sequentially introduce up to 3 libraries of heritable transcribed barcodes that can be sequenced as part of a routine scRNAseq pipeline. This allows for the construction of multilevel cell lineage trees alongside a time course of transcriptional identity and thus identification of developmental commitment or bias in cultures of differentiating cells. To avoid rapid silencing of the CellTag construct the original CMV promoter was switched to EFla (FIG. 31 A). Two CellTag libraries were applied to neural crest differentiation cultures at the point of induction of differentiation (DO; 2hr post-seed; VI) and at the point of RA exposure (D4; V2; FIG. 24D). Samples were taken for scRNAseq at day 4 (D4; immediately before RA exposure) and at day 9 (D9; differentiation endpoint; FIG. 24D). Analysis of the data identified transcriptionally distinct cultures at D4 and D9 (FIG. 24E). At D4 clustering revealed both cells with a developmentally mature signature (SOX10+ / POU5F1-, clusters 2&3) and those with a less mature signature (SOX10- / POU5F1+,- cluster 0; FIGS. 24E-24F, 3 IB). Expression of the ectodermal pioneer factor TFAP2A was widespread at this stage (FIG. 3 IB). At D9, clusters 4-7 exhibited a gene expression signature consistent with neural crest and neural crest derivatives (expression of SOXIO, TFAP2A, TWISTF, FIGS. 24E-24F, 3 IB), while cluster 8 appeared to be a small number of off-target CNS derivatives (PAX6+,- FIG. 3 IB). Notably, expression of vagal markers was entirely restricted to a sub population of cells (clusters 7 and 8; H0XA5+, H0XB5+, PH0X2B+,- FIGS. 24E-24F, 3 IB) as had been seen previously (FIG. 24C) and included both CNS- and neural crest-like cells and derivatives (FIG. 3 IB).

[0368] Lineage reconstruction using the CellTagR package and randomized testing to identify lineage enriched clones revealed that 92% of VI clones (with 3 or more cells) exhibited complete restriction to either vagal (clusters 7-8) or non-vagal (clusters 4-6) fates (FIG. 24G). For the 8% of VI clones where commitment was not seen, branching V2 clones with 3 or more cells often showed commitment toward or against a vagal fate. Together these data indicate that for the vast majority of cells commitment toward or against vagal 072734.1887

[0369] PATENT competence occurs within one cell cycle (~24hr) of the onset of differentiation, and for the remaining cells in culture commitment occurs before the onset of RA exposure. This point is further demonstrated by the cluster location of cells sequenced at D9 that belong to clones which exhibited complete restriction of fate (FIG. 24G). It can also be seen in the provided network diagrams of representative high cell number clones from each group (FIGS. 24H- 241).

[0370] Intriguingly, cells that belonged to committed clones that were sequenced at D4, before the onset of RA exposure, also exhibited a distinct transcriptional profile between the vagal-competent and non-vagal competent groups. Cells belonging to vagal competent lineages were almost entirely located in cluster 0, while cells belonging to non-vagal competent lineages were spread across all 4 clusters with the highest proportion in cluster 2 (FIG. 3 ID). Gene expression analysis between the two groups identified a number of hindbrain-associated genes significantly upregulated in the vagal competent clone members, including FST, OLIG3, EPHA4, MEIS2, STMX2, VGLL3, FZD7 and IRX2 (FIGS. 24J, 3 le). These cells also had a less developmentally mature pattern of gene expression with genes such as POU5F1, SOX2 and PODXL exhibiting a higher level of expression and neural crest markers such as SOX 10, TFAP2B and ETS-1 exhibiting lower levels of expression (FIG. 24J). One possible interpretation could be that the developmentally immature cells in culture retain the ability to adopt vagal fate while those that have committed to a neural crest lineage have lost it. However, this would suggest that earlier treatment with RA would increase the proportion of vagal NC, which is not the case. A second interpretation is that there is an early, spontaneous, acquisition of posterior identity in a subset of cells, and that these cells are able to respond to RA with the upregulation of vagal genes (defined as “vagal competence”).

[0371] Transient WNT induces vagal competence. With the insight that acquisition of vagal competence largely occurs very early during differentiation, culture conditions that would induce this phenotype across the whole population of cells were sought. Early, strong, activation of WNT signaling has been shown to induce an NMP-like identity in differentiating pluripotent stem cells in both the human and mouse. In the case of human PSC, 48hr of activation is required to generate trunk derivatives. Given that vagal competence is established in half this time, 24hr of WNT activation was evaluated for inducing a less caudal, vagal-competent fate.

[0372] Redesigning the protocol with this in mind and using the small molecule CHIR99021 to mimic WNT signaling resulted in a dramatic increase in vagal marker expression in the 072734.1887

[0373] PATENT hPSC-derived neural crest on D7 as measured by ICC (referred to as “WNT bump” protocol; confirmed across 3 separate hPSC lines; FIGS. 25A-25C, 32A). scRNAseq showed expression of vagal HOX genes in the “WNT bump” condition but not “no bump” controls, as well as enriched expression of other caudal genes and WNT signaling targets (FIGS. 25F- 25G). Inhibition of WNT using the tankyrase inhibitor XAV over the same period reduced H0XB5 expression at D7 and also resulted in a loss of neural crest induction at the expense of off-target CNS cell types (FIG. 34A).

[0374] Gene expression in D4 cultures as measured by scRNAseq also showed enrichment for caudal markers in the “WNT bump” condition (FIG. 25D). Some of these markers were also hits in the vagal -committed clones of the CellTag experiment (including MEIS2 and MNP), while others were significantly enriched in cluster 0 from that experiment (such as CRABP1 and DACHP).

[0375] Vagal neural crest are the major cell of origin for the enteric nervous system. Vagal neural crest generated through the application of RA (without a “WNT bump”) are capable of differentiating into enteric neuron-like cells and rescuing mouse models of Hirschsprung’s disease either alone (less severe model) or with sacral neural crest (more severe model). Using these older systems, larger numbers of off-target ectomesenchyme-like cells are obtained, alongside enteric neuron-like derivatives. Using the new “WNT bump” protocol, further differentiation in neurotrophic conditions to day 40 (D40) resulted in pure cultures of neurons with little ectomesenchymal contamination, unlike controls without the “WNT bump” (FIGS. 25H-25I, 32B, 33A, 33D). Using ICC analysis, it is possible to identify putative excitatory motor neurons (CHAT+ / NOSlow / VIP-; white arrow), putative inhibitory motor neurons (CHAT- / NOShigh / VIP+; yellow arrow) and putative secreto-vasodilator neurons (CHAT- / NOSlow / VIP+; blue arrow; FIG. 25H). Accurate quantification of these thick cultures is difficult, but using scRNAseq a more in-depth analysis is possible. Dissociation with Accutase and Papain revealed distinct SOXIO^GFPI^I profiles by flow cytometry, as well as cellular morphology in suspension (FIGS. 33B-33C). D40 ENS cultures were found to contain a majority of neurons (N%; MAP2+ / TUBB3+ / PRPH+ as well as a substantial population of glial cells (N%; S100B+ / SOX10+) and cycling progenitors (N%; MKI67).:some of which showed early signs of differentiation toward either neural or glial fates (Fig 25G, 33A). A high proportion of the neurons exhibit NOS1 expression, potentially indicating an inhibitory motor neuron-like identity, although other branches of the ENS express this factor at lower levels. Markers for cholinergic neurons could also be detected in two otherwise 072734.1887

[0376] PATENT transcriptionally distinct sets of cells (c3,8) indicating the presence of excitatory motor neuron-like cells. The unbiased annotation tool CellTypist was used to annotate the dataset using publicly available human data from both the adult and the fetal enteric nervous system. Both datasets identified the majority of the neurons as corresponding to putative inhibitor motor neurons while also identifying putative excitatory motor neurons, interneurons and sensory neurons. Cells sequenced at D40 derived from the “no bump” control included a large number of fibroblast-like profiles (COL1A1+), although small numbers of neurons and glia were also present (FIG. 33A). It was also possible to generate enteric neuron-like cells after treatment with known ENS morphogens EDN3 or GDNF during the second half of the “WNT bump” protocol (FIG. 33 A).

[0377] As well as WNT activation, alternative posteriorizing factors were attempted during differentiation. Treatment with RA between DO-1 (instead of the WNT “bump”) induced a non-neural ectoderm phenotype, which could be compensated for either with 2.0 pM DMH1 (DO-1) or 4.0 pM DMH1 and 1.2 pM CHIR (Dl-7; FIG. 34B). However, in each case this did not result in expression of vagal markers H0XB5 and PH0X2B in the resulting neural crest. Treatment with FGF8 between DO-1 also did not increase the proportion of vagal neural crest (FIG. 34C). Treatment with FGF8 between Dl-4 or Dl-7 in the absence of the WNT “bump” did not result in induction of vagal fate, and the small number of spontaneous HOXB5 positive cells normally present in this condition were lost (FIG. 34D). Interestingly, treatment with FGF8 (Dl-4 or Dl-7) in conjunction with the WNT “bump” condition appeared to largely block acquisition of vagal identity (FIG. 34D).

[0378] Features of vagal competent cells. The hindbrain-like gene expression signature at D4 suggested that the vagal competent progenitors that exist at DI (either spontaneously or in a manner induced by WNT activation) reflect an early, committed posterior progenitor. In support of this was the small proportion of cells sequenced at D4 in the CellTag experiment in clusters 0-1 that express GBX2 (FIG. 3 IE). GBX2 is not expressed in neural crest cells but is known to be necessary for neural crest formation level with the hindbrain and could have been more widely expressed in vagal competent cells during early stages of the CellTag experiment prior to D4. To study the expression of GBX2 during vagal neural crest differentiation, a reporter cell line was generated using a tdTomato gene fused to a H2B nuclear localization fragment, as has been previously described. Using this reporter, it was possible to demonstrate that early treatment with a “WNT bump” (in excess of ~10hrs duration) results in a broad, high level of GBX2 activation in the culture at DI. Conversely, 072734.1887

[0379] PATENT

[0380] “no bump” controls exhibited only a small proportion of cells with a high level of GBX2 activation (FIG. 26A). This was also detectable at D2 where ICC shows widespread evidence of OTX2 expression in “no bump” controls but OTX2 is not detectable in the “WNT bump” condition (FIG. 26B). Extending the treatment with CHIR99021 to a total of 48hr (“long bump”) resulted in induction of trunk markers TBXT (brachyury) and CDX2 (FIG. 26B). The “long bump” condition does not result in vagal competency as is demonstrated by the absence of HOXB5 expression at D7 in this condition (FIG. 26C). scRNAseq carried out at DI shows segregation between transcriptional signatures of cells in “WNT bump” and “no bump” cultures (FIG. 27A). These populations are also transcriptionally distinct from cultures of hPSC grown in E8 medium, and D2 cultures subjected to 48 hours of strong WNT activation (“long bump”; FIGS. 27A-27B). DI “bump” cultures showed widespread activation of GBX2 in the absence of OTX2 expression (FIG. 27A). They also showed expression of hindbrain associated HOX genes (e.g. H0XA1), without activation of trunk HOX genes, which was only seen in the “long bump” condition (e.g. HOXB8; FIG. 27A). Transcriptional activation of trunk progenitor marker TBXT (brachyury) was detected in DI “WNT bump” cultures, despite the absence of TBXT protein detected at D2 by ICC (FIG. 27A). Differential gene expression analysis also identified a number of hits from the CellTag experiment as being significantly upregulated in the “WNT bump” condition, including FST and STMN2 (FIG. 35A). Gene ontology (GO) analysis identified enriched GO categories associated with WNT signaling and axial patterning (FIG. 27C). ATAC-seq identified enriched accessibility for genes associated with WNT signaling and axial patterning (including HOX gene paralogous groups) in DI “WNT bump” cultures relative to “no bump” or DO hPSC cultures (FIG. 27D, 35E). Accessibility for progressively more posterior HOX genes was identified in the D2 “long bump” cultures (HOX PG 1-10; FIG. 35E).

[0381] During embryonic development GBX2+ neural progenitors give rise to the hindbrain by first forming discrete domains called rhombomeres. This early segregation is characterized by markers that are specifically expressed in one or more rhombomere and highly conserved across species (FIG. 28 A). DI hindbrain progenitor-like cells were challenged by differentiating them further in the presence of dual SMAD inhibition and known modulators of spatial identity in this region (FIG. 28B). In addition to the evidence of rhombomere 7 / 8 (vagal) level identity already seen in the neural crest differentiation (HOXB4+, HOXB5+; FIG. 25B), it was possible to generate neural ectoderm-like cells that were GBX2+ and also 072734.1887

[0382] PATENT either: MAFB+EGR2- (r6-like), MAFB+EGR2+ (r5-like), HOXB1+EGR2- (r4-like), MAFB-EGR2+ (r3-like) and MAFB-EGR2-HOXBl-(r2-like; FIG. 28D). “No bump” cultures were not able to upregulate these markers under these conditions and remained OTX2+ (FIG. 28D).

[0383] A unidirectional continuum of progressively posterior fate can be induced by WNT, The high level of commitment observed in the CellTag experiment indicates that posterior progenitors cannot regress back to an anterior state in this system. It also indicates that capacity for adopting a posterior fate is lost after a certain point. The parameters of this conversion from anterior (OTX2+) to posterior (GBX2+) fate were evaluated by modulating the duration and timing of CHIR99021 exposure (modulating the “WNT bump”; FIG. 26A). About lOhr of 4.5 pM CHIR99021 exposure was sufficient for the upregulation of GBX2 to the levels seen at DI after a 24hr “WNT bump” in the whole population of cells (FIG. 26 A). However, it was only with a full 24hr of CHIR99021 exposure that the vagal competent state was induced (as measured by induction of HOXB5 and PHOX2B at D7 of neural crest induction; FIG. 26D). ICC at D2 under these conditions demonstrated that, although a small number of OTX2 positive cells remain when 12hr of CHIR exposure was used, with 18hr or longer no OTX2 positive cells could be detected suggesting that GBX2 expression at this early stage is necessary but not sufficient for vagal competence (FIG. 26D). Finally, when delaying the onset of the 24hr “WNT bump” by 12 hours vagal competence was still induced.

[0384] However, a delay of 36hr or longer resulted in complete loss of vagal competence (FIG. 26E). Together these results indicated that the duration of strong WNT activation within a certain window of time (~48hr) dictates the primary axis identity of progenitor cells in a progressive, committed manner. Given that others working in similar systems have induced GBX2 expression using lower concentrations of CHIR99021 for longer periods, this likely functions as the definite integral of WNT activation within this 48hr period. After this 48hr period, the capacity for the cells to transition to the next progenitor identity along the primary axis continuum appears to be lost.

[0385] To test the degree of commitment of early progenitors to specific primary axis identities 3D co-culture experiments were conducted, aggregating progenitor cultures at D2 before assembling them into triple-origin organoids and subjecting them to axial patterning factors alongside dual SMAD inhibition (FIG. 30A). This was achieved using H9-derived hPSC cell lines with nucl ear-localized fluorescent tags inserted into the locus of the housekeeping gene GPI, as has been done previously. H2B-GFP was used for rostral “no 072734.1887

[0386] PATENT bump” cultures, H2B-tdTomato was used for posterior (hindbrain-like) “WNT bump” cultures, and H2B-iRFP-3xFLAG was used for posterior (trunk-like) “long bump” cultures. Exposing these organoids to high levels of CHIR99021, either with or without RA, did not result in acquisition of more posterior identity (e.g. OTX2 to GBX2 positive, or GBX2 to TBXT positive; FIG. 30B). Furthermore, treatment with the tankyrase inhibitor XAV (2 pM), either with or without the pan-RAR inhibitor AGN did not result in reversion to more anterior identity (e.g. GBX2 to OTX2 positive or TBXT to GBX2 positive fate; FIG. 30B).

[0387] Whether GBX2 expression alone (in the absence of WNT activity) was sufficient to drive the transition from anterior progenitor identity to posterior progenitor identity was next evaluated using CRISPRa (FIG. 29A). Activation of GBX2 expression at the start of differentiation resulted in an increase in the proportion of cells reporting GBX2 expression with tdTomato from -10% to 50% in “no bump” cultures at D2 (FIGS. 29B-29C). This included an increase in the proportion of OTX2+ GBX2+ double cells, which were only seen at low numbers in control cultures, but it also resulted in a very high level of GBX2 induction in some cells, for which OTX2 was never co-expressed (FIG. 29B). Conversely, activation of OTX2 resulted in a high degree of OTX2 expression in a subset of cells in “bump” cultures, where no OTX2 can be detected in controls (FIG. 29B). These OTX2 positive cells did not show signs of GBX2 induction. Together these results support a mutually repressive role. However, further differentiation under Dual SMAD inhibition conditions did not result in significantly different levels of rhombomere-specific markers EGR2, MAFB or H0XB1 (FIGS. 29D-29E, 38A-38B). Carrying out CRISPRa in the same format in neural crest differentiation conditions (FIG. 29F) did not result in a significant increase in H0XB5 or PHOX2B expression in neural crest cells at D7, either in “WNT bump” or “no bump” cultures (FIGS. 29G-29H).

[0388] An intriguing element of the CellTag experiment was that vagal competent clones could give rise to both neural crest and CNS lineages. This suggests that primary axis identity was determined prior to ectodermal identity in these cultures. To test the ectodermal potential of the axially committed progenitors in the “WNT bump”, “no bump” and “long bump” cultures, the cultures were subjected to either high, low or intermediate levels of BMP activity at D2. This was done either in the presence of dual SMAD inhibition from DO-2 or in E6 medium as used previously (FIG. 37). In the absence of dual SMAD inhibition from DO-2, all three progenitors could generate CNS, neural crest and epidermis-like derivatives, although the latter differed morphologically and in the expression of TFAP2A between 072734.1887 PATENT progenitor types. Interestingly, in the presence of dual SMAD inhibition for DO-2, the flat, epidermal-like cultures obtained in high BMP conditions were lost in favor of neural crestlike derivatives (FIG. 37). This suggests that plasticity between dorsal neural tube and neural crest lineages remains after axial commitment, but that commitment away from non-neural ectoderm lineages can happen in parallel.

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[0421] 31. Yoshida, H. et al. Pepstatin A, an aspartic proteinase inhibitor, suppresses RANKL- induced osteoclast differentiation. J Biochem 139, 583-590, (2006).

[0422] 32. Haas, H. A. Extending the search for folk personality constructs: the dimensionality of the personality-relevant proverb domain. J Pers Soc Psychol 82, 594-609, (2002).

[0423] 33. Gershon, M. D. & Tack, J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 132, 397-414, (2007).

[0424] 34. Vassar, R. et al. Function, therapeutic potential and cell biology of B ACE proteases: current status and future prospects. J Neurochem 130, 4-28, (2014).

[0425] 35. Torii, T. et al. In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration. Biochem Biophys Res Commun 452, 782-788, (2014).

[0426] 36. Wakatsuki, S., Araki, T. & Sehara-Fujisawa, A. Neuregulin-l / glial growth factor stimulates Schwann cell migration by inducing alpha5 betal integrin-ErbB2-focal adhesion kinase complex formation. Genes Cells 19, 66-77, (2014).

[0427] 37. Cully, M. Deal watch: Lilly buys back into the BACE race for Alzheimer's disease. Nature reviews. Drug discovery 13, 804, (2014). 072734.1887

[0428] PATENT

[0429] 38. Zeltner, N., Lafaille, F. G., Fattahi, F. & Studer, L. Feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells. Journal of visualized experiments : JoVE, (2014).

[0430] 39. Hosoda, K. et al. Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice. Cell 79, 1267- 1276, (1994).

[0431] 40. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380-1389, (2013).

[0432] 41. Berthold, M. et al. in Data Analysis, Machine Learning and Applications Studies in Classification, Data Analysis, and Knowledge Organization (eds Christine Preisach, Hans Burkhardt, Lars Schmidt-Thieme, & Reinhold Decker) Ch. 38, 319-326 (Springer Berlin Heidelberg, 2008).

[0433] 42. Dreser, N. et al. Grouping of histone deacetylase inhibitors and other toxicants disturbing neural crest migration by transcriptional profiling. Neurotoxicology 50, 56-70, (2015).

[0434] 43. Frith, T. J. R. et al. Retinoic Acid Accelerates the Specification of Enteric Neural Progenitors from In- Vitro-Derived Neural Crest. Stem Cell Rep 15, 557-565 (2020).

[0435] 44. Fan, Y. et al. hPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung’s disease. Cell Stem Cell 30, 264-282. e9 (2023).

[0436] 45. Fattahi, F. et al. Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease. Nature 531, 105-109 (2016).

[0437] 46. Hackland, J. O. S. et al. Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions. Stem Cell Reports (2017) doi: 10.1016 / j.stemcr.2017.08.008. (note - no retinoic acid treatment / vagal patterning in this one but it is the basis for the low density approach in the schematic)

[0438] 47. Frith, T. J. R. et al. Human axial progenitors generate trunk neural crest cells in vitro. eLife 7, 134 (2018).

[0439] 48. Hackland, J. O. S. et al. FGF Modulates the Axial Identity of Trunk hPSC-Derived Neural Crest but Not the Cranial-Trunk Decision. Stem Cell Reports 12, 920-933 (2019).

[0440] 49. Gouti, M. et al. In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm Identity. PLoS biology 12, el 001937 (2014). 072734.1887

[0441] PATENT

[0442] 50. Kirkeby, A. et al. Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions. Cell Rep. 1, 703-714 (2012).

[0443] 51. Maury, Y. et al. Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nat. Biotechnol. 33, 89-96

[0444] (2015).

[0445] Various patents and other publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.

Claims

072734.1887PATENTWHAT IS CLAIMED IS:

1. An in vitro method for inducing differentiation of stem cells, comprising contacting a population of stem cells with a first concentration of at least one activator of Wnt signaling for between about 12 and about 24 hours to produce a population of cells expressing at least one hindbrain lineage marker.

2. The method of claim 1, further comprising contacting the population of cells expressing at least one hindbrain lineage marker with at least one inhibitor of SMAD signaling, a second concentration of the at least one activator of Wnt signaling, and at least one molecule that induces vagal neural crest patterning to produce a population of vagal competent cells expressing at least one enteric neural crest lineage marker; wherein the second concentration of the at least one activator of Wnt signaling is decreased from the first concentration of the at least one activator of Wnt signaling.

3. The method of claim 1 or 2, wherein the population of stem cells are contacted with the first concentration of the at least one activator of Wnt signaling for between about 18 hours and about 24 hours.

4. The method of claim 2 or 3, wherein the population of cells expressing at least one hindbrain lineage marker are contacted with the second concentration of the at least one activator of Wnt signaling for at least about 6 days.

5. The method of claim 4, wherein the population of cells expressing at least one hindbrain lineage marker are contacted with the second concentration of the at least one activator of Wnt signaling for about 6 days.

6. The method of any one of claims 2-5, wherein the second concentration of the activator of Wnt signaling is decreased from the first concentration of the activator of Wnt signaling by between about 50 and about 95%.

7. The method of claim 6, wherein the second concentration of the activator of Wnt signaling is decreased from the first concentration of the activator of Wnt signaling by between about 70 and about 90%.

8. The method of any one of claims 2-7, wherein the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD072734.1887PATENT signaling for at least about 6 days.

9. The method of claim 8, wherein the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling for about 6 days.

10. The method of any one of claims 2-9, wherein the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one inhibitor of SMAD signaling and the second concentration of the at least one activator of Wnt signaling for at least about 6 days.

11. The method of any one of claims 2-10, wherein the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one molecule that induces vagal neural crest patterning for at least about 2 days.

12. The method of any one of claims 2-11, wherein the population of cells expressing at least one hindbrain lineage marker are contacted with the at least one molecule that induces vagal neural crest patterning for about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days.

13. The method of any one of claims 2-12, wherein said population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning within an about 8 day period from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said at least one inhibitor of SMAD signaling.

14. The method of claim 13, wherein said population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning at least about 3 days from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said at least one inhibitor of SMAD signaling.

15. The method of claim 14, wherein said population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning about 3 days from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said at least one inhibitor of SMAD072734.1887PATENT signaling.

16. The method of claim 14, wherein said population of cells expressing at least one hindbrain lineage marker are initially contacted with said at least one molecule that induces vagal neural crest patterning about 3 days from the initial contact of said population of cells expressing at least one hindbrain lineage marker with said at least one inhibitor of SMAD signaling, and said population of cells expressing at least one hindbrain lineage marker are contacted with said at least one molecule that induces vagal neural crest patterning for at least about 3 days.

17. The method of any one of claims 2-16, wherein said population of stem cells have been differentiated into a population of differentiated cells that express at least one said enteric neural crest lineage marker on or after about 6 days from their initial contact with said at least one inhibitor of SMAD signaling.

18. The method of any one of claims 1-17, 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.

19. The method of claim 18, wherein the at least one inhibitor of TGFp / Activin-Nodal signaling comprises an inhibitor of ALK5.

20. The method of any one of claims 1-18, wherein said at least one inhibitor of TGFp / Activin-Nodal signaling is a small molecule selected from the group consisting of SB431542, derivatives thereof, and mixtures thereof.

21. The method of claim 18, wherein said at least one inhibitor of BMP signaling is a small molecule selected from the group consisting of LDN193189, derivatives thereof, and mixtures thereof.

22. The method of any one of claims 1-21, wherein said at least one activator of Wnt signaling lowers glycogen synthase kinase 3p (GSK3P) for activation of Wnt signaling.

23. The method of any one of claims 1-21, wherein said at least one activator of Wnt signaling is a small molecule selected from the group consisting of CHIR99021, derivatives thereof, and mixtures thereof.072734.1887PATENT24. The method of any one of claims 2-23, wherein said at least one molecule that induces vagal neural crest patterning is selected from the group consisting of retinoic acid, retinol, retinal, tretinoin, isotretinoin, alitretinoin, etretinate, acitretin, tazarotene, bexarotene, adapalene, activators of FGF signaling, Wnt activators, and combinations thereof.

25. The method of any one of claims 2-23, wherein said at least one molecule that induces vagal neural crest patterning is selected from the group consisting of activators of FGF signaling, activators of Wnt signaling, and combinations thereof.

26. The method of claim 24, wherein said at least one molecule that induces vagal neural crest patterning comprises retinoic acid.

27. The method of claim 25, wherein said activators of FGF signaling are selected from the group consisting of FGF2, FGF4, FGF7, and FGF8.

28. The method of claim 25, wherein said activators of Wnt signaling are selected from the group consisting of CHIR99021 and WNT3A.

29. The method of any one of claims 2-28, wherein said vagal neural crest patterning is characterized by expression of at least one regional specific homoebox (HOX) gene.

30. The method of claim 29, wherein said at least one regional specific HOX gene is selected from the group consisting of HOXB2, HOXB3, HOXB4, and HOXB5.

31. The method of any one of claims 2-30, wherein said at least one enteric neural crest lineage marker is selected from the group consisting of PAX3, EDRB, RET, PHOX2A, PHOX2B, NTRK-3, HA D2, HOXB3, HOXB5 and ASCL1.

32. The method of any one of claims 1-31, wherein said at least one hindbrain lineage marker is GBX2.

33. The method of any one of claims 1-32, wherein the differentiated cells expressing said at least one hindbrain lineage marker do not express OTX2 or TBXT (brachyury).

34. The method of any one of claims 2-33, wherein said population of differentiated cells expressing at least one enteric neural crest lineage marker further express at least one SOX10+neural crest lineage marker.072734.1887PATENT35. The method of claim 34, wherein said SOX10+neural crest lineage marker is CD49D.

36. The method of any one of claims 1-35, wherein said stem cells are human stem cells.

37. The method of claim 36, wherein said human stem cells are selected from the group consisting of human embryonic stem cells, human induced pluripotent stem cells, human parthenogenetic stem cells, primordial germ cell-like pluripotent stem cells, epiblast stem cells, and F-class pluripotent stem cells.

38. The method of any one of claims 2-37, further comprising subjecting said population of differentiated cells to conditions favoring maturation of said differentiated cells into a population of cells that express at least one enteric neuron marker.

39. The method of claim 38, wherein said conditions favoring maturation of said differentiated cells into said population of enteric neurons comprise culturing said differentiated cells in a suitable cell culture medium.

40. The method of claim 39, wherein said suitable cell culture medium comprises at least one molecule that enhances maturation of enteric neural crest (ENC) precursors to enteric neurons.

41. The method of claim 40, wherein said at least one molecule that enhances maturation of ENC precursors to enteric neurons is selected from the group consisting of growth factors and Wnt activators.

42. The method of claim 41, wherein said growth factors are selected from the group consisting of activators of FGF signaling, glial cell line derived neurotrophic factor (GDNF), and ascorbic acid.

43. The method of claim 41, wherein said suitable cell culture medium comprises at least one activator of FGF signaling and at least one activator of Wnt signaling.

44. The method of claim 43, wherein said differentiated cells are cultured in said suitable cell culture medium comprising said at least one activator of FGF signaling and said at least one activator of Wnt signaling for about 4 days.

45. The method of any one of claims 42-44, wherein the at least one activator of FGF072734.1887PATENT signaling is selected from the group consisting of FGF2, FGF4, FGF8, and FGF7.

46. The method of any one of claims 38-45, wherein said at least one enteric neuron marker is selected from the group consisting of Tuj 1 , MAP2, PHOX2A, PHOX2B, TRKC, ASCL1, HAND2, EDNRB, 5HT, GAB A, NOS, SST, TH, CHAT, DBH, Substance P, VIP, NPY, GnRH, and CGRP.

47. A cell population of in vitro differentiated cells, wherein the in vitro differentiated cells are obtained by a method of any one of claims 1-46.

48. A composition comprising the cell population of claim 47.

49. The composition of claim 48, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

50. A kit for inducing differentiation of stem cells to vagal competent cells expressing at least one enteric neural crest lineage marker, 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; and(d) at least one molecule that induces vagal neural crest patterning.

51. The kit of claim 50, further comprising (f) instructions for inducing differentiation of the stem cells into a population of vagal competent cells expressing at least one enteric neural crest lineage marker.

52. The kit of claim 50 or 51, wherein the instructions comprise directions to contact the cells with a decreased concentration of the at least one activator of Wnt signaling about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours after the cells are initially contacted with the Wnt activator.

53. A method of preventing and / or treating an enteric nervous system disorder in a subj ect, comprising administering to a subject suffering from an enteric nervous system disorder an effective amount of one of the followings:(a) the population of in vitro differentiated cells of claim 47; or(b) a composition of claim 48 or 49.072734.1887PATENT54. The method of claim 53, wherein the enteric nervous system disorder is Hirschsprung's disease.

55. The cell population of claim 47 or the composition of claim 48 or 49 for use in preventing, modeling, and / or treating at least one symptom in a subject having an enteric nervous system disorder in a subject.

56. The cell population or composition for use of claim 55, wherein the enteric nervous system disorder is Hirschsprung's disease.

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