Systems, cell lines, and methods for producing and using same - Patents.com

Abstract

The present disclosure generally relates to two-dimensional and three-dimensional cultures of cells, the cultures comprising one or more enteric neurons and / or one or more enteric glial cells. These cells and cultures can be used for screening compounds for neurologically active compounds, transplantation into subjects or animal models, and administration to subjects with intestinal motility disorders. TIFF2025502844000253.tif44128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under U.S. Patent Act § 119(e) to U.S. Provisional Application No. 63 / 296,157, filed January 3, 2022, which is incorporated by reference in its entirety.

[0002] Statement regarding federally funded research This invention was made with Government support under Grant Nos. DP2NS116769 and R01CA240984 awarded by the National Institute of Health (NIH), and Grant No. R01DK121169 awarded by the National Institute of Diabetes and Digestive and Kidney Diseases. The Government has certain rights in this invention.

[0003] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (37944_0027P1.xml, size: 49,238 bytes, and creation date: December 30, 2022) are incorporated by reference in their entirety into this specification.

[0004] Technical Field The present disclosure generally relates to compositions comprising glial cells, methods of culturing pluripotent stem cells under defined conditions and inducing the pluripotent stem cells to differentiate into enteric neuronal cells and glial cells, which are then components of either two-dimensional or three-dimensional cell cultures. The present disclosure also relates to cultured two-dimensional neuronal cell-containing cultures and three-dimensional spheroids, and methods of their use. [Background technology]

[0005] background The enteric nervous system (ENS) is the largest and most complex division of the autonomic nervous system (De Giorgio, 2006). More than 500 million enteric neurons and almost seven times as many enteric glia form interconnected enteric ganglia embedded in two distinct layers within the intestinal wall. The myenteric plexus is located between the longitudinal and circular muscles, and the submucosal plexus is located between the circular muscle and the mucosa (Grubissic and Gulbransen, 2017; Grundmann et al., 2019; Hamnett et al., 2021; Sasselli et al., 2012).

[0006] The ENS, uniquely, does not depend on input from the central nervous system (CNS) to command gastrointestinal function (Furness et al., 2014). This autonomy is exemplified by studies in which gut segments removed from the body continue to generate complex motor patterns ex vivo. The autonomy of the ENS is the result of a large diversity of neuronal and glial cell types with different neurochemical signatures that work together in unison (Brehmer, 2021; Qu et al., 2008; Fung and Vanden Berghe, 2020). Thus, the ENS is equipped to control complex intestinal functions, including motility, secretion, absorption, blood flow regulation, and assisting barrier function. Furthermore, the ENS communicates externally with the CNS, the enteric endocrine system, the immune system, and the gut microbiota to maintain vitality and proper intestinal homeostasis ( Furness et al., 2014 , Long-Smith et al., 2020 , Muller et al., 2014 , Obata and Pachnis, 2016 , Schneider et al., 2019 , Yoo and Mazmanian, 2017 ).

[0007] Although the neurochemical and functional complexity of the ENS is similar to that of the CNS (Gershon, 1999), progress in the field of ENS research has been much slower. Despite the ENS being the largest and most complex division of the peripheral nervous system and playing a central role in the development and progression of enteric neuropathies and gut-brain axis diseases, ENS research has been disproportionately affected by multiple long-standing technical challenges. For example, enteric neurons are diluted throughout the gastrointestinal tract, constituting less than 1% of intestinal tissue (Drokhlyansky et al., 2020). Thus, to access ENS tissue, scientists must rely on samples collected during gastrointestinal resection surgery rather than the more routinely performed gastrointestinal biopsies. Furthermore, it is difficult to isolate the ENS without significant sampling bias associated with harsh tissue dissociation techniques that damage fragile neurites, and there is a lack of reliable surface markers suitable for FACS purification of enteric neurons and glia.

[0008] The complex developmental process and elaborate cellular architecture of the ENS, as well as its remarkable communication with the rest of the body, allow for the development of a wide variety of abnormalities. Enteric neuropathy, also known as disorders of gut-brain interaction (DGBI), including some of the most challenging clinical disorders, result from the loss, degeneration or dysfunction of ENS cell types (De Giorgio et al., 2016; Niesler et al., 2021). Our incomplete understanding of ENS development and function may explain the long-term morbidity and mortality of gastrointestinal disorders, as well as the limited availability of therapeutic interventions. Summary of the Invention

[0009] overview The present disclosure relates to neuronal cell lines and cell cultures comprising the same. The cell cultures comprise enteric neurons as disclosed herein, or glial cells as disclosed herein, or a combination of both enteric neuronal cells and glial cells. In some embodiments, the present disclosure relates to methods of making and culturing the enteric neuronal cells and glial cells as disclosed herein. The resulting cultures are suitable for screening potential therapeutic agents for the treatment of enteric neuropathies such as gastroparesis, esophageal achalasia, chronic intestinal pseudo-obstruction, and hypertrophic pyloric stenosis, as well as for regenerative medicine applications. In some embodiments, the present disclosure relates to compositions comprising cell lines and cultures comprising neuronal cells, in some embodiments, these compositions are for transplantation or administration to a mammalian subject.

[0010] In one aspect, the disclosure relates to a method of inducing nitric oxide-sensitive enteric neurons, comprising exposing one or more enteric neurons to one or a series of cell culture media disclosed herein in combination with a platelet-derived growth hormone receptor inhibitor disclosed herein.

[0011] In another aspect, the disclosure relates to a method of enriching a cell population for a subtype of nitrergic neurons, comprising exposing one or more iPSCs to a PDFGR inhibitor.

[0012] In another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD24.

[0013] In another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD45RA.

[0014] In another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD57.

[0015] In another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD63.

[0016] In yet another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD71.

[0017] In yet another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD121b.

[0018] In yet another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD147.

[0019] In yet another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD164.

[0020] In yet another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD184.

[0021] In yet another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD193.

[0022] In yet another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD243.

[0023] In yet another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and CD275.

[0024] In another aspect, the present disclosure relates to a composition comprising a spheroid comprising enteric neurons, wherein the enteric neurons comprise SOX10 and at least one or a combination of CD24, CD45RA, CD57, CD63, CD71, CD121b, CD147, CD164, CD184, CD193, CD243, CD275.

[0025] In another aspect, the disclosure relates to a method of differentiating a neuronal cell into an enteric neuronal cell, the method comprising exposing a neuronal cell to an effective amount of a platelet-derived growth factor receptor (PDGFR) inhibitor, or a pharma- ceutically acceptable salt thereof, for a period of time sufficient to differentiate the neuronal cell into an enteric neuronal cell.

[0026] In some embodiments, the PDGFR inhibitor is (Z)-orantinib, AC710, AC710 mesylate, AG1295, amuvatinib, amuvatinib hydrochloride, avapritinib, axitinib, AZD2932, cediranib, cediranib maleate, tiauranib, CHIR-124, CP-673451, crenolanib, dovitinib, dovitinib lactate, dovitinib lactate hydrate, dovitinib-D8, ENMD-2076, ENMD-2076 tartrate, flumatinib ... matinib mesylate, GZD856, GZD856 formate, HG-7-85-01, hypothemycrin, ilorasertib, ilorasertib hydrochloride, imatinib, imatinib D4, imatinib D8, imatinib mesylate, JI-101, JNJ-10198409, KG5, Ki20227, lenvatinib, lenvatinib mesylate, linifanib, masitinib, masitinib mesylate, methylnisoline, multikinase inhibitor 1, N-(p-coumaroyl) Serotonin, nintedanib, nintedanib esylate, NVP-ACC789, orantinib, pazopanib, pazopanib hydrochloride, PD-089828, PD-161570, PDGFRα kinase inhibitor 1, ponatinib, ponatinib D8, PP121, PP58, regorafenib, regorafenib D3, regorafenib hydrochloride, regorafenib monohydrate, ripretinib, sennoside B, seraltinib, SU5402, SU14813, SU14813 maleate, SU16f, SU4 312, SU4984, sunitinib, sunitinib D10, sunitinib malate, sunitinib-d4, TAK-593, tandutinib, tandutinib hydrochloride, telatinib, telatinib mesylate, TG100572, TG100572 hydrochloride, TG100801, TG100801 hydrochloride, toceranib, toceranib salt, toceranib-d8, trapidil, tyrosine kinase-IN-1, tyrphostin AG1296, tyrphostin AG1433, and borolanib.

[0027] In some embodiments, the PDGFR inhibitor is TIFF2025502844000002.tif172128TIFF2025502844000003.tif222133TIFF2025502844000004.tif208128TIFF2025502844000005.tif180135TIFF2025502844000006.tif206144TIFF2025502844000007.tif220140TIFF2025502844000008.tif194136TIFF2025502844000009.tif162128, or a pharma- ceutically acceptable salt thereof.

[0028] In some embodiments, the PDGFR inhibitor is a hydrate.

[0029] In some embodiments, the PDGFR inhibitor is TIFF2025502844000010.tif72128 or a pharma- ceutically acceptable salt thereof. In some embodiments, the PDGFR inhibitor is an isotope.

[0030] In some embodiments, the PDGFR inhibitor is deuterated.

[0031] In some embodiments, the PDGFR inhibitor is Selected from TIFF2025502844000011.tif90136TIFF2025502844000012.tif161128.

[0032] In some embodiments, the PDGFR inhibitor is exposed to the stem or neuronal cells as a pharmaceutically acceptable salt. In some embodiments, the PDGFR inhibitor is exposed to the Crestsphere as a pharmaceutically acceptable salt.

[0033] In some embodiments, the pharma- ceutically acceptable salt is a mesylate, hydrochloride, maleate, lactate, tartrate, formate, esylate, phosphate, or malate salt.

[0034] In some embodiments, the pharma- ceutically acceptable salt is TIFF2025502844000013.tif215143TIFF2025502844000014.tif215134TIFF2025502844000015.tif211143TIFF2025502844000016.tif117128, or a pharma- ceutically acceptable salt thereof.

[0035] In another aspect, the disclosure relates to a method for modulating NO neuronal activity in a cell culture, comprising exposing cells in the cell culture to an effective amount of a PDGFR inhibitor, or a pharma- ceutically acceptable salt thereof, for a period of time sufficient to cause differentiation of the cells into enteric neurons.

[0036] In some embodiments, the cell culture comprises neural crest cells in two or three dimensions.

[0037] In some embodiments, modulating NO neuronal activity is nitric oxide responsive.

[0038] In another aspect, the disclosure relates to a method of enriching NO enteric neurons in a cell culture, comprising exposing a composition of neural crest cells or crestspheres to an effective amount of a PDGFR inhibitor, or a pharma- ceutically acceptable salt thereof, for a period of time sufficient to differentiate the cells into enteric neurons.

[0039] In some embodiments, the cell culture comprises neural crest cells in two or three dimensions. In some embodiments, modulating NO neuronal activity is nitric oxide responsive.

[0040] In another aspect, the disclosure relates to a kit comprising a PDGFR inhibitor, or a pharma- ceutically acceptable salt thereof, and one or more selected from a) instructions for treating an intestinal motility disorder, and b) instructions for administering the compound in association with treating an intestinal motility disorder.

[0041] In some embodiments, the kit comprises a cell line comprising neural crest cells or crestspheres. In some embodiments, the kit comprises stem cells or differentiated human stem cells. In some embodiments, the kit further comprises one or more enteric neurons. In some embodiments, the agent is selected from a parasympathomimetic agent, a prokinetic agent, an opioid antagonist, an antidiarrheal agent, and an antibiotic.

[0042] In some embodiments, the drug is selected from neostigmine, bethanechol, metoclopramide, cisapride, and loperamide. In some embodiments, PDGFR and cell line or cell culture are packaged together. In another aspect, the present disclosure relates to a composition comprising one or more enteric glial cells, wherein the enteric glial cells comprise SOX10 and PMP22. In another aspect, the present disclosure relates to a composition comprising one or more enteric glial cells, wherein the enteric glial cells comprise PMP22.

[0043] In another aspect, the present disclosure relates to a composition comprising a spheroid comprising one or more enteric glial cells, wherein the enteric glial cells comprise or express SOX10 and PMP22. In some embodiments, the enteric glial cells further comprise SB100. In some embodiments, the enteric glial cells further comprise PLP1. In some embodiments, the enteric glial cells further comprise AQP4. In some embodiments, the enteric glial cells further comprise GFAP. In some embodiments, the enteric glial cells further comprise MPZ. In some embodiments, the enteric glial cells further comprise MBP. In some embodiments, the one or more enteric glial cells are derived from induced pluripotent stem cells. In some embodiments, the one or more enteric glial cells are derived from human induced pluripotent stem cells.

[0044] In some embodiments, the one or more enteric glial cells are present in a ganglioid or spheroid or substantially spherical cell composition.

[0045] In some embodiments, the one or more enteric glial cells are in cell culture for about 5, 10, 12, or 15 days or more.

[0046] In some embodiments, one or more enteric glial cells and one or more enteric neuronal cells, wherein the enteric glial cells comprise SOX10 and PMP22, and the enteric neuronal cells comprise SOX10 and at least one or a combination of CD24, CD45RA, CD57, CD63, CD71, CD121b, CD147, CD164, CD184, CD193, CD243, CD275.

[0047] In another aspect, the present disclosure relates to a composition comprising one or more enteric neuronal cells, (i) one or more mesenchymal cells, and (ii) one or more epithelial cells, wherein the epithelial cells are disposed within a spheroid or ganglioid.

[0048] In some embodiments, the composition further comprises one or more smooth muscle cells and / or enteric glial cells.

[0049] In some embodiments, the mesenchymal cells express one or a combination of PRRX1, RNX2, TWIST1, COL11A1, COL1A2, COL1A1, COL3A1, COL5A2, FN1, LAMA4, EDNRA, PDGFRA, and PDGFRB. In some embodiments, the epithelial cells express one or a combination of GALR1, CFC1, AC073941.1, TTC6, ARX, AC012405.1, CMTM8, SHH, PLSCR5, and CNTN4-AS2. In some embodiments, the enteric neurons express one or a combination of NRXN3, NRXN1, DCX, MAPT, ELAVL2, NRCAM, RBFOX3, NCAM1, NRG1, SYN1, and SYP.

[0050] In some embodiments, the enteric neuron cells comprise SOX10 and at least one or a combination of CD24, CD45RA, CD57, CD63, CD71, CD121b, CD147, CD164, CD184, CD193, CD243, and CD275.

[0051] In some embodiments, the composition further comprises one or more progenitor or stem-like cells that express one or a combination of the biomarkers in FIG. 8F.

[0052] In some embodiments, the glial cells express one or a combination of GFAP, ERBB4, NTRK2, NTRK3, PAX3, EDNRB, FZD3, and SOX2.

[0053] In some embodiments, the epithelial cells express one or a combination of CDH1, EPCAM, and KRT119. In some embodiments, the smooth muscle cells express ACTA1, PAX7, MYOD1, MYL4, CHRNA1, TNNT2, MYOG, DES, and TBX1.

[0054] In some embodiments, the composition further comprises RPE cells that express one or a combination of the biomarkers in Figure 8H.

[0055] In some embodiments, the glial cells express one or a combination of GFAP, ERBB4, NTRK2, NTRK3, PAX3, EDNRB, FZD3, and SOX2, the epithelial cells express one or a combination of CDH1, EPCAM, KRT119, the enteric neurons express one or a combination of NRXN3, NRXN1, DCX, MAPT, ELAVL2, NRCAM, RBFOX3, NCAM1, NRG1, SYN1, and SYP, and the mesenchymal cells express one or a combination of PRRX1, RNX2, TWIST1, COL11A1, COL1A2, COL1A1, COL3A1, COL5A2, FN1, LAMA4, EDNRA, PDGFRA, and PDGFRB.

[0056] In some embodiments, the mesenchymal cells express KRT119 and one or a combination of PRRX1, RNX2, TWIST1, COL11A1, COL1A2, COL1A1, COL3A1, COL5A2, FN1, LAMA4, EDNRA, PDGFRA, and PDGFRB.

[0057] In some embodiments, the composition is free or substantially free of retinal pigment epithelial (RPE) cells.

[0058] In another aspect, the disclosure relates to a method of enriching cells in crestspheres or spheroids by exposing the crestspheres to a PDFGR inhibitor or a pharma- ceutically acceptable salt thereof and one or a combination of GDNF, ascorbic acid, neurobasal™, n2 and b27.

[0059] In some embodiments, the PDGFR inhibitor is not PP1121, or is free or substantially free of PP1121 or a pharma- ceutically acceptable salt thereof.

[0060] In another aspect, the present disclosure relates to a method of transplanting a spheroid of cells into a subject by administering the spheroid of cells to the gastrointestinal tract of the subject.

[0061] In some embodiments, the spheroids comprise a composition according to any one of claims 50-72.

[0062] In some embodiments, the subject has or is suspected of having a bowel motility disorder.

[0063] In some embodiments, the intestinal motility disorder is selected from achalasia, Hirschsprung's disease, intestinal pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence.

[0064] In some embodiments, the administering step comprises seeding the cells into the small intestine, stomach, or colon of the subject.

[0065] In another aspect, the disclosure provides a method of treating an intestinal motility disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the enteric neuronal or glial cell compositions disclosed herein.

[0066] In some embodiments, the intestinal motility disorder is selected from achalasia, Hirschsprung's disease, intestinal pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence.

[0067] In another aspect, the present disclosure relates to a subject comprising any one of any of the compositions of enteric neurons or glial cells disclosed herein. In some embodiments, the subject is a mouse or a human. In some embodiments, the mouse is a NOS double knockout (NOS- / -).

[0068] The present disclosure also relates to a cell line comprising the enteric neuronal cells disclosed herein. In another aspect, the present disclosure relates to a cell line comprising the enteric glial cells disclosed herein. [Brief description of the drawings]

[0069] [Figure 1A] Schematic of the protocol for in vitro differentiation and maturation of hPSCs into enteric neural crest and enteric crestspheres. [Figure 1B] scRNA-seq UMAP of cell types present in enteric neural crest cells (D10, upper panel) and enteric crestsphere cells (D15, lower panel) of the differentiated cultures shown in Figure 1A. [Figure 1C] UMAP of enteric neural crest (D10, top) and enteric crestsphere (D15, bottom) subtypes in differentiated cultures. [Figure 1D]Violin plot stacks showing expression of classical enteric neural crest markers in enteric neural crest (top) and enteric crestsphere (bottom) subtypes. [Figure 1E] Protocol schematic for in vitro differentiation and maturation of hPSC-derived intestinal crestspheres into 2D ENS cultures and 3D gangliosides. [Figure 1F] snRNA-seq UMAP of cell types present in stage 1 intestinal ganglioids. [Figure 1G] snRNA-seq UMAP of cell types present in stage 2 intestinal ganglioids. [Figure 1H] Immunofluorescence analysis for expression of neuronal TUBB3 and glial GFAP in stage 1 and stage 2 intestinal ganglioids. [Figure 1I] Immunofluorescence analysis for expression of the neuronal activity marker cFOS in stage 1 and stage 2 intestinal ganglioids. [Figure 1J] Flow cytometric quantification of the neuronal activity marker cFOS in intestinal ganglioids at maturity. [Figure 1K] In vivo fluorescence imaging of human hSYN-ChR2-EYFP in intestinal ganglioids at maturity. [Figure 1L] Quantification of multielectrode array (MEA) analysis of baseline and blue light stimulated neuronal activity in stage 1 hSYN-ChR2-EYFP (left) and control (right) intestinal gangliosides. [Figure 1M] Dot plot of the mean module scores of stage 1 enteric ganglioid cell type transcriptional signatures in stage 2 enteric ganglioid cell types. [Figure 1N] Projection of stage 2 cell types (right) onto the SWNE of stage 1 intestinal ganglioid cells, with overlay projections of stage 1 cell type specific transcription factors from Figure 13 . [Figure 2A] snRNA-seq UMAP of neuronal subtypes present in stage 1 intestinal ganglioids. [Figure 2B]snRNA-seq UMAP of neuronal subtypes present in stage 2 intestinal ganglioids. [Figure 2C] Projection of a stage 2 cell type (right) onto the SWNE of a stage 1 enteric ganglioid neuron with overlaid projection rate-limiting neurotransmitter synthesis enzymes. [Figure 2D] Dot plots of mean module scores for stage 1 (bottom) and stage 2 (top) ganglioid cell type transcriptional signature adult colon cell types. [Figure 2E] Dot plots of mean module scores for stage 1 (bottom) and stage 2 (top) ganglioid neuron subtype transcriptional signatures of adult enteric neuron subtypes. [Figure 2F] Immunofluorescence analysis for expression of ENS cell-type markers (serotonin, CHAT, GABA, and NOS1) in stage 1 intestinal ganglioids. [Figure 2G] Quantification of flow cytometry analysis for expression of neuronal subtype markers serotonin, CHAT, GABA and NOS1 in stage 1 2D ENS cultures (left) and 3D intestinal gangliosides (right). [Figure 2H] Flow cytometry validation of stage 1 EN 8 surface markers CCR6 (left) and GYPB (right) that co-label with neurochemical markers showing enrichment of neurochemical identity of marker positive populations normalized to baseline neurochemical population levels. [Figure 2I] Overall percentage of neurotransmitter-synthesizing neurons in stage 1 and 2 enteric gangliosides compared with mouse and human primary enteric neurons. [Figure 2J] Schematic diagram of single and multiple neurotransmitter synthesis in enteric neurons. [Figure 2K] Percentage of neurons exhibiting single and multiple neurotransmitter profiles in stage 1 and 2 enteric ganglioid neurons compared with mouse and human primary enteric neurons. [Figure 2L]Immunostaining of primary human colon with antibodies against NOS1, GABA and TUBB3 (top), and CHAT, GABA and TUBB3 (bottom). White dashed lines indicate the borders of TUBB3+ ganglia. White arrows indicate colocalization. [Figure 2M] Percentage of single (top) and dual neurotransmitter (bottom) producing enteric neurons in stage 1, 2 enteric ganglioid and primary datasets. [Figure 3A] Schematic diagram of snRNA-seq analysis of stage 2 intestinal ganglioids and subsequent glial subclustering. [Figure 3B] UMAP of glial subtypes present in stage 2 enteric ganglioids (snRNA-seq, left) and distribution of glial subtypes in biological replicates of enteric ganglioid cultures (right). [Figure 3C] UMAP of enteric glial subtypes present in the primary adult dataset. [Figure 3D] Distribution of enteric glial subtype expression in individual human tissue samples. [Figure 3E] Violin stack plot of expression of classical glial markers in stage 2 enteric ganglioglioid and adult glial subtypes. [Figure 3F] Immunofluorescence staining of classical glial markers GFAP and S100 in stage 2 intestinal ganglioids and human primary colon tissue. [Figure 3G] Co-staining of GFAP and S100 in stage 2 intestinal ganglioids. [Figure 3H] Dot plot of mean module scores of stage 2 enteric ganglioid glial subtype transcriptional signatures in adult enteric glial subtypes. [Figure 3I] Immunostaining of myelin markers in human colon and intestinal ganglioids. PMP22 expression in stage 2 intestinal ganglioids (top), PMP22 expression (middle) and MPZ expression (bottom) in human colon. [Figure 3J]Feature plots showing module scores of stage 1 enteric ganglioid precursor 1 (top) and 2 (bottom) transcriptional signatures in ganglioid glial cells. [Figure 3K] Heatmap showing normalized enrichment scores for GO pathways enriched in each glial class as determined by hierarchical clustering. [Figure 4A] Schematic of bulk RNA-seq analysis of CD24+ / NOS1:GFP+ FACS-sorted neurons (top), and snRNA-seq analysis of stage 1 intestinal ganglioids and subsequent NO neuron subclustering (bottom). [Figure 4B] snRNA-seq UMAP of NO subtypes present in stage 1 enteric ganglioid neurons. [Figure 4C] snRNA-seq UMAP of subclustered NO neuron subtypes from stage 1 intestinal ganglioids. [Figure 4D] Violin plot of (top) NOS1 expression and (bottom) module scoring for nitric oxide biosynthesis gene ontology (GO) term genes by stage 1 intestinal ganglioid NO subtypes. [Figure 4E] UMAP of pNO subtypes in adult enteric neurons. [Figure 4F] UMAP of subclustered pNO subtypes from adult enteric neurons. [Figure 4G] Dot plots of mean module scores of adult pNO neuron subtype transcriptional signatures in stage 1 enteric ganglioid NO neuron subtypes (left) and stage 1 enteric ganglioid NO neuron subtype transcriptional signatures in adult pNO neuron subtypes (right). [Figure 4H] Heatmap matrix of Spearman correlations based on scaled expression of 3000 anchor features that shared significantly variable genes (or anchor features) between adult (x-axis) and stage 1 enteric ganglioglioid (y-axis) NO neuron subtypes. [Figure 4I]Dot plots of scaled mean expression of NO neuron-specific transcription factors (TFs), neuropeptides (NPs), neurotransmitter receptors (NT-Rs), neuropeptide receptors (NP-Rs), and surface markers (SMs) in stage 1 gut ganglioid (I) and adult (J) NO neuron subtypes relative to non-NO neurons. [Figure 4J] Dot plots of scaled mean expression of NO neuron-specific transcription factors (TFs), neuropeptides (NPs), neurotransmitter receptors (NT-Rs), neuropeptide receptors (NP-Rs), and surface markers (SMs) in stage 1 gut ganglioid (I) and adult (J) NO neuron subtypes relative to non-NO neurons. [Figure 4K] Feature plots of the identity of predicted neurotransmitter-producing neurons in stage 1 gut ganglioid (K) and adult (L) subclustered NO neurons. [Figure 4L] Feature plots of the identity of predicted neurotransmitter-producing neurons in stage 1 gut ganglioid (K) and adult (L) subclustered NO neurons. [Figure 4M] Distribution of neurochemical identity in stage 1 gut ganglioid (left) and adult (right) NO neuron subtypes relative to non-NO neurons. [Figure 4N] Dot plots of mean module scores of myenteric and submucosal neuronal transcriptional signatures relative to non-NO neurons in stage 1 intestinal ganglioid (left) and adult (right) NO neuron subtypes. [Figure 5A] Schematic of a high-throughput flow cytometry-based screen to identify compounds that induce cFOS expression in hESC-derived stage 2 intestinal ganglioid NO neurons. [Figure 5B]Target classes of hits identified in the enteric NO neuron cFOS induction screen (Figure S20D, red dots). Data in Figure S20 are not shown, but we describe the identification of enteric NO neuron modulators by functional high-throughput screening. Data in S20 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted January 3, 2022, and are incorporated by reference in their entirety. [Figure 5C] Schematic of a high-throughput calorimetry-based screen to identify compounds that induce NO release in hESC-derived stage 1 2D ENS cultures. [Figure 5D] Target classes of hits identified in the NO release screen (fig. S20E, red dots). Protein classes in common with (B) are indicated with an asterisk. [Figure 5E] Feature plot showing predicted responsiveness of subclustered stage 1 ganglioid enteric NO neurons to neurotransmitters by module scoring of neurotransmitter receptor gene families. [Figure 5F] B and D. Dot plots of expression of genes belonging to the target classes indicated in hESC-derived stage 1 and primary human intestinal nitrergic neuron subtypes compared to all other neurons. [Figure 5G] Combined protein target analysis for selected screening hits showing shared protein classes. Color code matches target classes in (B) and (D). [Figure 5H] Schematic diagram of testing the effect of selected candidate hits (listed in (G)) on mouse colonic motility ex vivo. [Figure 5I] Representative spatiotemporal map of mouse colonic contractions along the proximal-distal axis over a 10 min period. [Figure 5J]Quantification of colonic migratory motor complex (CMMC) intervals at the 75th percentile of CMMC cumulative percentages for selected hit compounds (data not shown). [Figure 5K] Experimental design to measure the effect of selected candidate hits on mouse colonic motility ex vivo. Representative spatiotemporal maps of mouse colonic contractions along the proximal-distal axis over a 26 min period. Three representative longitudinal muscle contraction events (LCEs) are indicated per condition (arrows). [Figure 5L] Quantification of CMMC cumulative percentiles and CMMC interval (time difference between two consecutive contractions) at the 75th percentile for dexmedetomidine. Mean and SEM error bars are shown for five pairs of colons of untreated and drug-treated mice. [Figure 5M] Total number of colonic longitudinal muscle contraction events (LCEs) within each 6 min treatment condition for five dexmedetomidine-treated mouse colons measured from spatiotemporal maps. *: p-value < 0.05. [Figure 5N] Average LCE duration calculated for three LCEs within each 6 min treatment (one at the beginning, one in the middle, and one at the end of each spatiotemporal map, see (K)). Data are shown for the colons of five dexmedetomidine-treated mice. SEM error bars are shown. [Figure 6A] Schematic of a high-throughput pharmacological screen to identify compounds that enrich for NO neurons in hESC-derived 2D ENS cultures. [Figure 6B] Combined protein target analysis of the top 12 HTS hits showing shared protein classes among structurally similar hits. [Figure 6C] Effect of PP121 treatment window on NOS1::GFP induction efficiency. [Figure 6D] Immunofluorescence staining of NOS1 and neuronal TUBB3 in stage 1 intestinal ganglioids treated or not with PP121 between days 15 and 20. [Figure 6E]Split UMAP of cell types present in stage 1 control (top) and PP121-treated (bottom) intestinal ganglioid cultures. [Figure 6F] Dot plot of mean module scores in PP121-treated ganglioid subtype vs. intestinal ganglioid subtype transcriptional signature only controls. [Figure 6G] Split UMAP of neuronal subtypes present in stage 1 control (top) and PP121-treated (bottom) intestinal ganglioglioid cultures. [Figure 6H] Dot plot of mean module scores of neuronal subtype transcriptional signatures in PP121-treated ganglioside neuronal subtypes vs. control only. [Figure 6I] Distribution of NO neuron subtypes in control versus PP121-treated stage 1 intestinal ganglioside cultures. [Figure 6J] Split UMAP of subclustered NO subtypes present in stage 1 control (top) and PP121-treated (bottom) intestinal ganglioid cultures. [Figure 6K] Dot plot of mean module scores of control-only NO neuron subtype transcriptional signatures in PP121-treated ganglioid NO neuron subtypes. [Figure 6L] Feature plot showing expression of ERBB, PDGFR and VEGFR in sub-clustered intestinal crestspheres at D15. [Figure 6M] Schematic diagram of receptor tyrosine kinase (RTK) natural agonists and selected pharmacological antagonists, including the NO neuron-enriched top hit PP121. [Figure 6N] Effect of RTK ligand treatment on stage 1 intestinal ganglioid NO neuron induction. [Figure 6O] Effect of PDGFRA(O) and PDGFRB(P) knockout in intestinal crestspheres at D15 on stage 1 intestinal ganglioid NO neuron enrichment as measured by flow cytometry. [Figure 6P] Effect of PDGFRA(O) and PDGFRB(P) knockout in intestinal crestspheres at D15 on stage 1 intestinal ganglioid NO neuron enrichment as measured by flow cytometry. [Figure 7A] Schematic showing transplantation of hESC-derived stage 1 intestinal ganglioids into mouse proximal colon. [Figure 7B] Engraftment of hESC-derived stage 1 intestinal ganglioid cells along the entire length of the mouse colon as indicated by expression of the human cytoplasmic marker SC121 in red. [Figure 7C] Immunohistochemical analysis of human cytoplasmic protein SC121 and NO neuronal marker NOS1 in Nos1 − / − mouse colon 8 weeks after transplantation. [Figure 8A] Dot plots of scaled mean expression of cell type annotation genes for enteric neural crest (left) and enteric crestsphere (right) cell types. All data are derived from scRNA-seq analysis. [Figure 8B] Dot plot of the average module scores of enteric neural crest cell type transcriptional signatures in enteric crestsphere cell types. All data are derived from scRNA-seq analysis. [Figure 8C] Dot plot of the mean module scores of the enteric neural crest cell (D10) subtype transcriptional signature in the enteric crestsphere (D15) subtype. [Figure 8D] Dot plots of scaled mean expression of the top 10 differentially expressed genes (unknown cluster showing top 10 differentially expressed genes) for each enteric neural crest (D10, D), enteric crestsphere (D15, E), and stage 1 enteric ganglioside (F) cell type. [Figure 8E] Dot plots of scaled mean expression of the top 10 differentially expressed genes (unknown cluster showing top 10 differentially expressed genes) for each enteric neural crest (D10, D), enteric crestsphere (D15, E), and stage 1 enteric ganglioside (F) cell type. [Figure 8F] Dot plots of scaled mean expression of the top 10 differentially expressed genes (unknown cluster showing top 10 differentially expressed genes) for each enteric neural crest (D10, D), enteric crestsphere (D15, E), and stage 1 enteric ganglioside (F) cell type. [Figure 8G] Dot plot of mean module scores of the intestinal crestsphere (D15) subtype transcriptional signature in stage 1 intestinal ganglioid cell types. [Figure 8H] Dot plot of scaled mean expression of the top 10 differentially expressed genes for stage 2 intestinal ganglioid cell types. [Figure 8I] UMAP of epithelial and mesenchymal subtypes present in stage 2 intestinal ganglioside cultures. [Figure 8J] Dot plot of scaled mean expression of 10 10 differentially expressed genes of stage 2 intestinal ganglioid epithelial and mesenchymal subtypes. [Figure 9A] Representative diagram of spontaneous neuronal firing recorded during stage 1 intestinal ganglioid multielectrode array analysis (MEA). [Figure 9B] MEA analysis of baseline and blue light stimulated neuronal activity in stage 1 hSYN-ChR2-EYFP (left) and control (right) gut gangliosides. [Figure 9C] Flow cytometry analysis of cFOS expression in hSYN-ChR2-EYFP-derived stage 2 intestinal ganglioids in response to blue light stimulation. [Figure 10A] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), secreted ligands (F), ligand receptors (G), and surface markers (H) in stage 1 intestinal ganglioids. [Figure 10B]Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), secreted ligands (F), ligand receptors (G), and surface markers (H) in stage 1 intestinal ganglioids. [Figure 10C] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), secreted ligands (F), ligand receptors (G), and surface markers (H) in stage 1 intestinal ganglioids. [Figure 10D] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), secreted ligands (F), ligand receptors (G), and surface markers (H) in stage 1 intestinal ganglioids. [Figure 10E] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), secreted ligands (F), ligand receptors (G), and surface markers (H) in stage 1 intestinal ganglioids. [Figure 10F] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), secreted ligands (F), ligand receptors (G), and surface markers (H) in stage 1 intestinal ganglioids. [Figure 10G] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), secreted ligands (F), ligand receptors (G), and surface markers (H) in stage 1 intestinal ganglioids. [Figure 10H]Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), secreted ligands (F), ligand receptors (G), and surface markers (H) in stage 1 intestinal ganglioids. [Figure 10I] Violin plot stacks of cell type-specific transcription factors (I), neurotransmitter receptors (J), neuropeptide receptors (K), cytokines (L), cytokine receptors (M), secreted ligands (N), ligand receptors (O), and surface markers (P) in stage 2 intestinal ganglioids. [Figure 10J] Violin plot stacks of cell type-specific transcription factors (I), neurotransmitter receptors (J), neuropeptide receptors (K), cytokines (L), cytokine receptors (M), secreted ligands (N), ligand receptors (O), and surface markers (P) in stage 2 intestinal ganglioids. [Figure 10K] Violin plot stacks of cell type-specific transcription factors (I), neurotransmitter receptors (J), neuropeptide receptors (K), cytokines (L), cytokine receptors (M), secreted ligands (N), ligand receptors (O), and surface markers (P) in stage 2 intestinal ganglioids. [Figure 10L] Violin plot stacks of cell type-specific transcription factors (I), neurotransmitter receptors (J), neuropeptide receptors (K), cytokines (L), cytokine receptors (M), secreted ligands (N), ligand receptors (O), and surface markers (P) in stage 2 intestinal ganglioids. [Figure 10M] Violin plot stacks of cell type-specific transcription factors (I), neurotransmitter receptors (J), neuropeptide receptors (K), cytokines (L), cytokine receptors (M), secreted ligands (N), ligand receptors (O), and surface markers (P) in stage 2 intestinal ganglioids. [Figure 10N]Violin plot stacks of cell type-specific transcription factors (I), neurotransmitter receptors (J), neuropeptide receptors (K), cytokines (L), cytokine receptors (M), secreted ligands (N), ligand receptors (O), and surface markers (P) in stage 2 intestinal ganglioids. [Figure 10O] Violin plot stacks of cell type-specific transcription factors (I), neurotransmitter receptors (J), neuropeptide receptors (K), cytokines (L), cytokine receptors (M), secreted ligands (N), ligand receptors (O), and surface markers (P) in stage 2 intestinal ganglioids. [Figure 10P] Violin plot stacks of cell type-specific transcription factors (I), neurotransmitter receptors (J), neuropeptide receptors (K), cytokines (L), cytokine receptors (M), secreted ligands (N), ligand receptors (O), and surface markers (P) in stage 2 intestinal ganglioids. [Figure 11A] Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 1 intestinal ganglioid cell types. Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 2 intestinal ganglioid cell types. [Figure 11B]Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 1 intestinal ganglioid cell types. Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 2 intestinal ganglioid cell types. [Figure 11C] Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 1 intestinal ganglioid cell types. Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 2 intestinal ganglioid cell types. [Figure 11D] Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 1 intestinal ganglioid cell types. Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 2 intestinal ganglioid cell types. [Figure 11E] Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 1 intestinal ganglioid cell types. Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 2 intestinal ganglioid cell types. [Figure 11F] Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 1 intestinal ganglioid cell types. Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 2 intestinal ganglioid cell types. [Figure 11G] Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 1 intestinal ganglioid cell types. Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 2 intestinal ganglioid cell types. [Figure 11H] Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 1 intestinal ganglioid cell types. Dot plots of scaled average expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in stage 2 intestinal ganglioid cell types. [Figure 12A] Dot plot of mean module scores of stage 1 enteric ganglioid neuron subtype transcriptional signatures in stage 2 enteric ganglioid neuron subtypes. [Figure 12B] UMAP of neuronal subtypes in stage 2 2D ENS cultures. [Figure 12C] Dot plots of scaled mean expression of the top 10 differentially expressed genes for each stage 2 2D ENS culture neuronal subtype. [Figure 12D] Dot plot of mean module scores of stage 2 enteric ganglioneuron subtype transcriptional signatures in stage 2 2D ENS cultures neuronal subtypes. [Figure 12E] Comparison of the distribution of enteric neuron subtypes in two-dimensional versus three-dimensional enteric neuron cultures. [Figure 12F] Heatmap matrix of Spearman correlations based on scaled expression of 3000 anchor features that shared significantly variable genes (or anchor features) between stage 2 2D ENS cultures (x-axis) and gangliosides (y-axis). [Figure 12G] UMAP of cell types (top) and neuronal subtypes (bottom) present in the primary adult colon dataset. [Figure 12H]Heatmap matrix of Spearman correlations based on scaled expression of 3000 anchor features that shared significantly variable genes (or anchor features) between stage 1 and 2 ganglioid neuron subtypes and adult enteric neuron subtypes. [Figure 12I] Dot plots of mean module scores of myenteric and submucosal neuronal transcriptional signatures in adult enteric neuron subtypes (left) and in stage 1 (middle) and stage 2 (right) ganglioside neuron subtypes. [Figure 13A] Feature plots showing expression of rate-limiting enzymes in the neurotransmitter synthesis pathway by stage 1 (A) and stage 2 (B) enteric ganglioid neurons. [Figure 13B] Feature plots showing expression of rate-limiting enzymes in the neurotransmitter synthesis pathway by stage 1 (A) and stage 2 (B) enteric ganglioid neurons. [Figure 13C] Feature plots showing neurotransmitter identity scores by stage 1 (C) and stage 2 (D) enteric ganglioid neurons by modular scoring of genes related to the synthesis, metabolism, and reuptake of each neurotransmitter. [Figure 13D] Feature plots showing neurotransmitter identity scores by stage 1 (C) and stage 2 (D) enteric ganglioid neurons by modular scoring of genes related to the synthesis, metabolism, and reuptake of each neurotransmitter. [Figure 13E] UMAPs of predicted neurotransmitter producing neuronal identity in stage 1 (E) and stage 2 (F) gut gangliosides. [Figure 13F] UMAPs of predicted neurotransmitter producing neuronal identity in stage 1 (E) and stage 2 (F) gut gangliosides. [Figure 13G] Distribution of neurochemical identity among stage 1 (left) and stage 2 (right) enteric ganglioid neuron subtypes. [Figure 13H] Distribution of neurochemical identity in stage 2 2D cultures of enteric neuron subtypes. [Figure 13I] Comparison of the distribution of neurochemical identity in enteric neurons in 2D versus 3D cultures. [Figure 13J] Feature plots showing predicted responsiveness of stage 1 (J) and stage 2 (K) enteric ganglioid neurons to each neurotransmitter by module scoring of neurotransmitter receptor gene families. [Figure 13K] Feature plots showing predicted responsiveness of stage 1 (J) and stage 2 (K) enteric ganglioid neurons to each neurotransmitter by module scoring of neurotransmitter receptor gene families. [Figure 14A] Distribution of neurochemical identity among enteric neuron subtypes in the adult (left) and adult mouse (right). [Figure 14B] Distribution of neurochemical identity in mouse enteric neuron subtypes at E15 (left), E18 (middle), and P21 (right). [Figure 15A] scRNA-seq UMAP (left) and distribution of glial subtypes in biological replicates (right) in stage 2 2D ENS cultures. [Figure 15B] UMAP of enteric glial subtypes present in primary adult mouse datasets. [Figure 15C] UMAP of enteric glial subtypes present at P21 (left) and enteric glial and progenitor subtypes present in the E18 (right) adult mouse dataset. [Figure 15D] Dot plots of scaled mean expression of the top 10 differentially expressed genes for each enteric ganglioid (top left), 2D ENS cultures (top right), adult (middle left), adult mouse (middle right), P21 mouse (bottom left) enteric glial subtypes, and E18 (bottom right) enteric glial and progenitor subtypes. [Figure 15E] Violin plot stacks showing expression of classical glial markers in 2D ENS cultures, adult mice, and P21 and E18 mouse glial (and progenitor) subtypes. [Figure 15F]Dot plots of mean module scores of stage 2 enteric ganglioglioid glial subtype transcriptional signatures (snRNA-seq) in 2D ENS cultures glial subtypes (scRNA-seq). [Figure 15G] Heatmap matrix of Spearman correlations based on scaled expression of 3000 anchor features that shared significantly variable genes (or anchor features) between 2D ENS cultures (x-axis) and intestinal ganglioids (y-axis). [Figure 16A] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 16B] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 16C] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 16D] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 16E]Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 16F] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 16G] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 16H] Violin plot stacks of cell type-specific transcription factors (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 17A] Dot plots of scaled mean expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 17B]Dot plots of scaled mean expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 17C] Dot plots of scaled mean expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 17D] Dot plots of scaled mean expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 17E] Dot plots of scaled mean expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 17F] Dot plots of scaled mean expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 17G]Dot plots of scaled mean expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 17H] Dot plots of scaled mean expression of selected transcription factor families (A), neurotransmitter receptors (B), neuropeptide receptors (C), cytokines (D), cytokine receptors (E), selected secreted ligands (F), selected ligand receptors (G) and surface markers (H) in enteric ganglioid glial subtypes. [Figure 18A] Hierarchical clustering of enteric ganglioid glial subtypes with primary human and mouse glial subtypes based on normalized enrichment scores of biological process Gene Ontology (GO) pathways. [Figure 18B] Dot plot of mean module scores of myenteric and submucosal glial transcriptional signatures in primary human enteric glial subtypes. [Figure 18C] Distribution of enteric glial subtype expression in primary human myenteric versus submucosal tissue samples. [Figure 18D] Dot plot of mean module scores of intermyenteric and submucosal glial transcriptional signatures in stage 2 ganglioid glial subtypes. [Figure 19A] Schematic diagram of the NOS1::GFP reporter construct. [Figure 19B] Representative immunofluorescence images of stage 2 intestinal ganglioids stained for GFP and NOS1. [Figure 19C] Representative flow cytometry analysis for GFP and NOS1 expression in stage 1 intestinal ganglioids derived from NOS1::GFP. [Figure 19D]Top 50 differentially expressed transcripts from bulk RNA-seq in FACS sorted CD24+ / NOS1::GFP+ cells versus CD24+ / NOS1::GFP- cells, p-value <0.05, upregulated in red and downregulated in red. [Figure 19E] Distribution of NO neuron subtypes in biological replicates of stage 1 intestinal ganglioside cultures. [Figure 19F] Dot plots of scaled mean expression of the top 10 differentially expressed genes for each stage 1 enteric ganglioid NO neuron subtype. [Figure 19G] snRNA-seq analysis violin plot of module scoring of the top 100 differentially expressed genes from CD24+ / NOS1+ sorted neurons versus other neurons in stage 1 gut ganglioid NO subtype versus other neurons. [Figure 20A] snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20B] snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20C] snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20D] snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20E] snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20F]snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20G] snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20H] snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20I]snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. [Figure 20J] snRNA-seq dot plots of bulk RNA-seq differentially expressed (Log2FC, p-value < 0.05) genes in NOS1::GFP+ neurons versus other neurons, and mean expression of selected transcription factor families (A), neurotransmitter synthesis genes (B), neurotransmitter receptors (C), neuropeptide receptors (D), neuropeptides (E), cytokines (F), cytokine receptors (G), selected secreted ligands (H), selected ligand receptors (I) and surface markers (J) in stage 1 enteric ganglioid NO neurons. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Detailed Description of the Preferred Embodiments The present disclosure relates to compositions comprising ganglioid cells, spheroids and crestspheres comprising one or more enteric neurons. The present disclosure relates to compositions comprising ganglioid cells, spheroids and crestspheres comprising enteric glial cells. The present disclosure further relates to methods of differentiating human pluripotent stem cells to produce any two-dimensional or three-dimensional cultures comprising enteric neurons and / or glial cells. The present disclosure further relates to methods of implanting these compositions into a subject to produce, in one case, an animal model comprising enteric neurons and / or enteric glial cells as disclosed herein, and in another case, a method of administering spheroids, treated crestspheres comprising enteric neurons and / or enteric glial cells to a subject for the treatment of intestinal motility disorders. In some embodiments, the enteric neurons are NO-responsive or more NO-responsive than enteric neurons from human pluripotent stem cells that have not been exposed to a PDGFR inhibitor. The present disclosure relates to exposing crestspheres from iPSCs to a physiologically effective amount of a PDGFR inhibitor for a period of time sufficient to enrich the number of enteric neurons or enteric glial cells in the culture.

[0071] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. For example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994) provides a general guide to those skilled in the art for many of the terms used in this application. In addition, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry that are within the skill of the art, unless otherwise indicated. Such techniques are fully described, for example, in the following documents: “Molecular Cloning: A Laboratory Manual”, 2nd edition (Sambrook et al., 1989), “Oligonucleotide Synthesis” (MJ Gait, ed., 1984), “Animal Cell Culture” (RIFreshney, ed., 1987), “Methods in Enzymology” (Academic Press, Inc.), “Handbook of Experimental Immunology”, 4th edition (DMWeir & C.C. Blackwell, eds., Blackwell Science Inc., 1987), “Gene Transfer Vectors for Mammalian Cells” (JMMiller & M.P. Calos, eds., 1987), “Current Protocols in Molecular Biology” (FMAusubel et al., eds., 1987), and “PCR: The Polymerase Chain Reaction”, (Mullis et al. al., eds., 1994).

[0072] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0073] When an embodiment is described herein using the language "comprising," it is understood that other similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided. When an embodiment is described herein using the language "consisting essentially of," it is understood that other similar embodiments described using the term "consisting of" are also provided.

[0074] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0075] The term "substantially free" as used herein refers to a composition having only trace or negligible amounts of the substance to which it refers. In some embodiments, substantially free means that the composition contains only about 0.1%, 0.2%, 0.3%, 0.4%, or 0.5% of the substance to which it refers. In some embodiments, substantially free means that the composition contains less than about 1.0% of the substance to which it refers, relative to the number or weight of the substances in the composition, and does not impart a biological effect to the composition.

[0076] The term "culture vessel" as used herein is defined as any vessel suitable for growing, culturing, cultivating, expanding, or otherwise similarly manipulating cells. The culture vessel may also be referred to herein as a "culture insert." In some embodiments, the culture vessel is made of biocompatible plastic and / or glass. In some embodiments, the plastic is a thin layer of plastic containing one or more pores that allow proteins, nucleic acids, nutrients (such as heavy metals and hormones), antibiotics, and other cell culture medium components to diffuse through the pores. In some embodiments, the pores are about 0.1, 0.5, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 microns or less in width. In some embodiments, the culture vessel is a hydrogel matrix and does not include a substrate or any other structure. In some embodiments, the culture vessel is designed to include a hydrogel or hydrogel matrix and various culture media. In some embodiments, the culture vessel consists of, or consists essentially of, a hydrogel or hydrogel matrix. In some embodiments, the only plastic components of the culture vessel are those components of the culture vessel that make up the sidewalls and / or bottom of the culture vessel that separate the volume of the well or cell growth zone from a point outside the culture vessel. In some embodiments, the culture vessel comprises a hydrogel and one or more isolated glial cells. In some embodiments, the culture vessel comprises a hydrogel and one or more isolated glial cells, onto which one or more neuronal cells are seeded.

[0077] The term "expose" as used herein refers to contacting, directly or indirectly, a disclosed compound with a cell, a target receptor, or other biological entity in such a way that the disclosed compound can affect the activity of the cell (e.g., receptor, cell, etc.). This can occur directly, by physical contact between the disclosed compound and the cell, receptor, or other entity, i.e., by contacting the target or cell itself, or it can occur indirectly, by interacting with another molecule, cofactor, factor, or protein on which the activity of the cell depends. In some embodiments, the activity of the cell in response to the compound or molecule is differentiation. In some embodiments, the compound is one or more differentiation factors.

[0078] The "analogs" of the compounds disclosed herein are pharma- ceutically acceptable salts, prodrugs, deuterated forms, radioactively labeled forms, isomers, solvates, and combinations thereof. The "combination" referred to in this context refers to derivatives that fall into at least two of the following groups: pharma- ceutically acceptable salts, prodrugs, deuterated forms, radioactively labeled forms, isomers, and solvates. Examples of radioactively labeled forms include compounds labeled with tritium, phosphorus-32, iodine-129, carbon-11, fluorine-18, and the like. The compounds described herein may be in the form of pharma- ceutically acceptable salts. For use in medicine, the salts of the compounds described herein refer to non-toxic "pharma- ceutically acceptable salts". Pharmaceutically acceptable salt forms include pharma- ceutically acceptable acidic / anionic or basic / cationic salts. Suitable pharma- ceutically acceptable acid addition salts of the compounds described herein include, for example, salts of inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid) and salts of organic acids (such as acetic acid, benzenesulfonic acid, benzoic acid, methanesulfonic acid, and p-toluenesulfonic acid). Examples of pharma- ceutically acceptable base addition salts include, for example, sodium, potassium, calcium, ammonium, organic amino, or magnesium salts. As used herein, the term "salt" refers to an acid salt or a base salt of a compound used in the method of the present disclosure. Illustrative examples of acceptable salts are salts of mineral acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid), salts of organic acids (such as acetic acid, propionic acid, glutamic acid, citric acid), and salts of quaternary ammonium (such as methyl iodide, ethyl iodide).

[0079] The term "progenitor cell" as used herein is defined as a cell that is a pluripotent cell that has been exposed to a cell culture medium that includes a differentiation factor, but remains at least partially undifferentiated and pluripotent. In some embodiments, the progenitor cell comprises WNT2B+. In some embodiments, the progenitor cell comprises PAX6 + Includes.

[0080] The term "pluripotent stem cell" as used herein is defined as a cell capable of self-renewal that can develop into cells and tissues of the three major germ layers. Pluripotent stem cells include embryonic and induced pluripotent cells as defined herein. Pluripotent stem cells contemplated are derived from mammals such as humans, mice, rats, monkeys, horses, goats, sheep, dogs, and cats.

[0081] The term "induced pluripotent stem cells" (iPSCs) refers to a type of pluripotent cell created by reprogramming somatic cells to have the same properties as embryonic stem cells, i.e., the ability to self-renew and differentiate into the three major germ layers. In some embodiments, iPSCs include mammalian cells, such as human, mouse, rat, monkey, horse, goat, sheep, dog, cat, etc., that have been reprogrammed to express Oct4, Nanog, Sox2, and optionally c-Myc. In some embodiments, iPSCs include reprogrammed primary cell lines. In some embodiments, iPSCs are obtained from the Coriell Institute for Medical Research (e.g., catalog IDs GM25256 (WTC-11), GM25430, GM23392, GM23396, GM24666, GM27177, GM24683), the California Institute for Regenerative Medicine: California's Stem Cell Agency (e.g., CW60261, CW60354, CW60359, CW60480, CW60335, CW60280, CW60594, CW60083, CW60086, CW60087, CW60167, CW60186), and the American Type Culture Collection (ATCC®) (e.g., ATCC-DYR0530 Human Induced Pluripotent Stem (IPS) Cells may be obtained from repositories such as ATCC® ACS-1012™, ATCC® ACS-1011™, ATCC® Number: ACS-1024™, ATCC® Number: ACS-1028™, ATCC® Number: ACS-1031™, ATCC® Number: ACS-1004™, ATCC® Number: ACS-1029™, ATCC® Number: ACS-1020™, ATCC® Number: ACS-1007™, ATCC® Number: ACS-1030™).Induced pluripotent stem cells can be derived from cell types such as fibroblasts taken from the skin, lungs, or veins of apparently healthy or diseased subjects.

[0082] As defined herein, the terms "inhibit," "inhibit," "inhibiting," and the like in reference to the interaction of a protein inhibitor (e.g., an antagonist) refer to adversely affecting (e.g., decreasing) the activity or function of a protein relative to the activity or function of the protein in the absence of the inhibitor. In some embodiments, inhibition refers to a reduction in a disease or a symptom of a disease. In some embodiments, inhibition refers to a reduction in a signaling pathway or the activity of a signaling pathway. Thus, inhibition includes at least partially, partially, or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating a signaling or enzymatic activity or the amount of a protein.

[0083] As used herein, the term "embryonic stem cell line" is defined as a cell derived from the inner cell mass of a preimplantation blastocyst that is capable of self-renewal and differentiation into the three primary germ layers. In some embodiments, the NIH Human Embryonic Stem Cell Embryonic stem cell lines listed in the Registry, e.g., CHB-1, CHB-2, CHB-3, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, RUES1, RUES2, HUES1, HUES2, HUES3, HUES4, HUES5, HUES6, HUES7, HUES8, HUES9, HUES10, HUES11, HUES12, HUES13, HUES14, HUES15, HUES16, HUES17, HUES18, HUES19, HUES20, HUES21, HUES22, HUES23, HUES24, HUES26, HUES27, HUES28, CyT49, RUES3, WA01(H1), UCS F4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, MFS5, HUES48, HUES49, HUES53 , HUES65, HUES66, UCLA1, UCLA2, UCLA3, WA07(H7), WA09(H9), WA13(H13), WA14(H14), HUES62, HUES63, HUES64, CT1, CT2, CT3, CT4, MA135, Endeavor-2, WIBR1, WIBR2, HUES45, Shef3, Shef6, WIBR3, WIBR4, WIBR5, WIBR6, BJNhem19, BJNhem20, SA001, SA002, UCLA4, UCLA5, UCLA6, HUES PGD13, HUES PGD3, ESI-014, ESI-017, HUES PGD11, HUES PGD12, WA15, WA16, WA17, WA18, WA19, etc. In some embodiments, the embryonic stem cells comprise a gene or genes associated with a disease or disorder.

[0084] The term "enteric neural crest cells" refers to cells produced by inducing differentiation of pluripotent stem cells, wherein the enteric neural crest cells express SOX10, PHOX2B, EDNRB, TFAP2A, BRN3A, ISL1 and / or ASCL1. In some embodiments, the enteric neural crest cells comprise FOX3D. In some embodiments, the neural crest cells are present in embryoid bodies or neural rosettes. In some embodiments, the neural crest cells express the vagus nerve markers HOXB2, HOXB3, and / or HOXB5. In some embodiments, the neural crest cells express p75 and HNK1. In some embodiments, the neural crest cells express HOXB2, HOXB3, HAND2, and EDNRB.

[0085] The term "enteric neurons" refers to cells that exhibit downregulation of SOX10, persistent expression of EDNRB, ASCL1 and PHOX2B, and upregulation of TUJ1 and TRKC. In some embodiments, the enteric neurons express neural subtype-specific markers including the cholinergic neural marker choline acetyltransferase (CHAT), serotonin (5-HT) receptor, gamma-aminobutyric acid (GABA), and neuronal nitric oxide synthase (nNOS). In some embodiments, CHAT expression indicates the presence of cholinergic neurons. In some embodiments, NOS1 expression indicates the presence of nitrergic neurons. In some embodiments, the enteric neurons include glial cells that express glial fibrillary acidic protein (GFAP) and SOX10. In some embodiments, the enteric neurons are produced by inducing differentiation of enteric neural crest cells. In some embodiments, the enteric neurons express SOX10, persistently express EDNRB, ASCL1 and PHOX2B, and upregulate TUJ1 and TRKC.

[0086] The term "enteric glial cells" refers to cells that exhibit expression of SOX10 and GPAP and / or PMP22. In some embodiments, enteric glial cells exhibit expression of SOX10 and PMP22. In some embodiments, enteric glial cells are produced by inducing differentiation of enteric neural crest cells.

[0087] The term "rho kinase inhibitor" refers to a compound that reduces the activity of rho kinase. In some embodiments, the rho kinase inhibitor is N-[(3-hydroxyphenyl)methyl]-N'-[4-(4-pyridinyl)-2-thiazolyl]urea dihydrochloride (RKI-1447), (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632), fasudil (HA-1077), hydroxyfasudil (HA 1100 hydrochloride), thiazovivin, GSK429286A, narciclasine, and / or (+)-(R)-trans 4-(1-aminoethyl)-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)cyclohexanecarboxamide dihydrochloride (Y-30141).

[0088] The term "hydrogel" as used herein is defined as any water-insoluble, cross-linked, three-dimensional network of polymer chains that is filled with water or has voids between the polymer chains that can be filled with water. The term "hydrogel matrix" as used herein is defined as any three-dimensional hydrogel construct, system, device, or similar structure. In some embodiments, the hydrogel or hydrogel matrix comprises one or more proteins and / or glycoproteins. In some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following proteins: collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, silk fibroin, and any derivative or combination thereof. In some embodiments, the hydrogel or hydrogel matrix comprises Matrigel® or vitronectin. In some embodiments, the hydrogel or hydrogel matrix can be solidified into various shapes, for example, branched shapes designed to mimic the neural tube. In some embodiments, the hydrogel or hydrogel matrix comprises poly(ethylene glycol) dimethacrylate (PEG). In some embodiments, the hydrogel or hydrogel matrix comprises Puramatrix. In some embodiments, the hydrogel or hydrogel matrix comprises glycidyl methacrylate-dextran (MeDex). In some embodiments, two or more hydrogels or hydrogel matrices are used simultaneously in a cell culture vessel. In some embodiments, two or more hydrogels or hydrogel matrices are used simultaneously in the same cell culture vessel, but the hydrogels are separated by walls that create independently addressable microenvironments within the tissue culture vessel, such as wells. In multiple tissue culture vessels, some embodiments can include any number of the aforementioned wells or independently addressable locations within the cell culture vessel, such that the hydrogel matrix in one well or location is different or the same as the hydrogel matrix in another well or location of the cell culture vessel.

[0089] The term "Matrigel®" refers to a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma that contains ECM proteins including laminin, collagen IV, heparin sulfate proteoglycan, entactin / nidogen, and other growth factors. In some embodiments, Cultrex® BME (Trevigen, Inc.) or Geltrex® (Thermo-Fisher Inc.) can be used in place of Matrigel®.

[0090] In some embodiments, the hydrogel or hydrogel matrix can have a variety of thicknesses. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 10 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 150 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 200 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 250 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 300 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 350 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 400 μm to about 3000 μm. In some embodiments, the hydrogel or hydrogel matrix has a thickness of about 450 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 550 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 600 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 650 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 700 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 750 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 800 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 850 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 900 μm to about 3000 μm, In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 950 μm to about 3000 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 1000 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 1500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 2000 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 2500 μm to about 3000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 2500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 2000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 1500 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 1000 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 950 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 900 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 850 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 800 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 750 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 700 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 650 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 550 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 100 μm to about 500 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​from about 100 μm to about 450 μm.In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 400 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 350 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 300 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 250 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 200 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 100 μm to about 150 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 300 μm to about 600 μm. In some embodiments, the thickness of the hydrogel or hydrogel matrix is ​​about 400 μm to about 500 μm.

[0091] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic polymers, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following synthetic polymers: polyethylene glycol (polyethylene oxide), polyvinyl alcohol, poly-2-hydroxyethyl methacrylate, polyacrylamide, silicone, and any derivatives or combinations thereof.

[0092] In some embodiments, the hydrogel or hydrogel matrix comprises one or more synthetic and / or natural polysaccharides, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following polysaccharides: hyaluronic acid, heparin sulfate, heparin, dextran, agarose, chitosan, alginate, and any derivative or combination thereof.

[0093] In some embodiments, the hydrogel or hydrogel matrix comprises one or more proteins and / or glycoproteins, hi some embodiments, the hydrogel or hydrogel matrix comprises one or more of the following proteins: collagen, gelatin, elastin, titin, laminin, fibronectin, fibrin, keratin, silk fibroin, and any derivatives or combinations thereof.

[0094] The term "vitronectin" refers to a protein encoded by the VTN gene. In some embodiments, vitronectin has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a fragment thereof. >sp|P04004|VTNC_Human Vitronectin OS=Homo sapiens OX=9606 GN=VTN PE=1 SV=1 SEQ ID NO:1 MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKPE EEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFRGQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPDY PRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISG MAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRATWLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL >tr|Q3KR94|Q3KR94_Rat Vitronectin OS=Rattus norvegicus OX=10116 GN=Vtn PE=1 SV=1 SEQ ID NO:2 MASLRPFFILALLALVSLADQESCKGRCTQGFMASKKCQCDELCTYYQSCCVDYMEQCKPQVTRGDVFTMPEDEYWSYDYPEETKNSTSTGVQSENTSLHFNLKPRAEETIKPTTPDPQ EQSNTQEPEVGQQGVAPRPDTTDEGTSEFPEEELCSGKPFDAFTDLKNGSLFAFRGEYCYELDETAVRPGYPKLIQDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPDYPR NISEGFSGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYEFQQQPSQEECEGSSLSAVFEHFALLQRDSWENIFELLFWGRSSDGAKGPQFISRDWHGVPGKVDAAMAGRIYITGST FRSVQAKKQKSGRRSRKRYRSRRGRGHSRSRSRSMSSRRPSRSVWFSLLSSEESGLGTYNYDYDMNWRIPATCEPIQSVYFFSGDKYYRVNLRTRRVDSVNPPYPRSIAQYWLGCPTSEK >sp|P29788|VTNC_Mouse Vitronectin OS=Mus musculus OX=10090 GN=Vtn PE=1 SV=2 SEQ ID NO:3 MAPLRPFFILALVAWVSLADQESCKGRCTQGFMASKKCQCDELCTYYQSCCADYMEQCKPQVTRGDVFTMPEDDYWSYDYVEEPKNNTNTGVQPENTSPPGDLNPRTDGTLKPTAFLDP EEQPSTPAPKVEQQEEILRPDTTDQGTPEFPEEELCSGKPFDAFTDLKNGSLFAFRGQYCYELDETAVRPGYPKLIQDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPGYP RNISEGFSGIPDNVDAAFALPAHRYSGRERVYFFKGKQYWEYEFQQQPSQEECEGSSLSAVFEHFALLQRDSWENIFELLFWGRSSDGAREPQFISRNWHGVPGKVDAAMAGRIYVTGS LSHSAQAKKQKSKRRSRKRYRSRRRGHRRSQSSNSRRSSRSIWFSLFSSEESGLGTYNNYDYDMDWLVPATCEPIQSVYFFSGDKYYRVNLRTRRVDSVNPPYPRSIAQYWLGCPTSEK

[0095] The term "biomarker" as used herein refers to a biological molecule present in an individual or on the surface of a call at various concentrations that is useful for determining the phenotype of a cell. Biomarkers can include, but are not limited to, nucleic acids, proteins, and variants and fragments thereof. A biomarker may be DNA that contains all or a portion of a nucleic acid sequence encoding the biomarker, or the complement of such a sequence. Biomarker nucleic acids useful in the present invention are believed to include both DNA and RNA that contain the entire sequence or a partial sequence of any of the nucleic acid sequences of interest.

[0096] Choline acetyltransferase (CHAT) refers to an enzyme that catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline to produce acetylcholine (ACh). In some embodiments, CHAT has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a fragment thereof. >sp|P28329|CLAT_Human choline O-acetyltransferase OS=Homo sapiens OX=9606 GN=CHAT PE=1 SV=4 SEQ ID NO:4 MGLRTAKKRGLGGGGKWKREEGGGTRGREVRPACFLQSGGRGDPGDVGGPAGNPGCSPHPRAATRPPPLAHTPAHTPEWCGAASAEAAEPRRAGPHLCIPAPGLTKPILEKVPRKMAAKTPSEESGLPKLPVPPLQQTLATYLQCMRHLVSEEQFRKSQAIVQQFGAPGGLGETLQQKLLERQ EKTANWVSEYWLNDMYLNNNRLALPVNSSPAVIFARQHFPGTDDQLRFAASLISGVLSYKALLDSHSIPTDCAKGQLSGQPLCMKQYYGLFSSYRLPGHTQDTLVAQNSSIMPEPEPHVIVACCNQFFVLDVVINFRRLSEGDLFTQLRKIVKMASNEDERLPPIGLLTSDGRESWAEARTVLVKDSTN RDSLDMIERCICLVCLDAPGGVELSDTHRALQLLHGGGYSKNGANRWYDKSLQFVVGRDGTCGVVCEHSPFDGIVLVQCTEHLLKHVTQSSRKLIRADSVSELPAPRLRWKCSPEIQGHLASSAEKLQRIVKNLDFIVYKFDNYGKTFIKKQKCSPDAFIQVALQLAFYRLHRRLVPTYESASIRR FQEGRVDNIRSATPEALAFVRAVTDHKAAVPASEKLLLLKDAIRAQTAYTVMAITGMAIDNHLLALRELARAMCKELPEMFMDETYLMSNRFVLSTSQVPTTTEMFCCYGPVVPNGYGACYNPQPETILFCISSFHSCKETSSSKFAKAVEESLIDMRDLCSLLPPTESKPLATKEKATRPSQGHQP >sp|P32738|CLAT_ラットコリンO-アアテルトランスフェラエーOS=Rattus norvegicus OX=10116 GN=Chat PE=1 SV=2 sequence number 5 MPILEKAPQKMPVKASSWEELDLPKLPVPPLQQTLATYLQCMQHLVPEEQFRKSQAIVKRFGAPGGLGETLQEKLLERQEKTANWVSEYWLNDMYLNNRLALPVNSSPAVIFARQHFQDTNDQLRFAACLISGVLSYKTLLDSHSLPTDWAKGQLSGQPL CMKQYYRLFSSYRLPGHTQDTLVAQKSSIMPEPEHVIVACCNQFFVLDVVINFRRLSEGDLFTQLRKIVKMASNEDERLPPIGLLTSDGRSEWAKARTVLLKDSTNRDSLDMIERCICLVCLDGPGTGELSDTHRALQLLHGGGCSLNGANRWYDKSLQF VVGRDGTCGVVCEHSPFDGIVLVQCTEHLLKHMMTSNKKLVRADSVSELPAPRRLRLKCSPETQGHLASSAEKLQRIVKNLDFIVYKFDNYGKTFIKKQKYSPDGFIQVALQLAYYRLYQRLVPTYESASIRRFQEGRVDNIRSATPEALAFVQAMTDHK AAMPASEKLQLLQTAMQAHKQYTVMAITGMAIDNHLLALRELARDLCKEPPEMFMDETYLMSNRFVLSTSQVPTTMEMFCCYGPVVPNGNGACYNPQPEAITFCISSFHSCKETSSVEFAEAVGASLVDMRDLCSSRQPADSKPPAPKEKARGPSQAKQS >sp|Q03059|CLAT_Mouse choline O-acetyltransferase OS=Mus musculus OX=10090 GN=Chat PE=2 SV=2 SEQ ID NO:6 MPILEKVPPKMPVQASSCEEVLDLPKLPVPPLQQTLATYLQCMQHLVPEEQFRKSQAIVKRFGAPGGLGETLQEKLLERQEKTANWVSEYWLNDMYLNNRLALPVNSSPAVIFARQHFQDTNDQLRFAASLISGVLSYKALLDSQSIPTDWAKGQLSGQP LCMKQYYRLFSSYRLPGHTQDTLVAQKSSIMPEPEHVIVACCNQFFVLDVVINFRRLSEGDLFTQLRKIVKMASNEDERLPPIGLLTSDGRSEWAKARTVLLKDSTNRDSLDMIERCICLVCLDGPGTGDLSDTHRALQLLHGGGCSLNGANRWYDKSLQ FVVGRDGTCGVVCEHSPFDGIVLVQCTEHLLKHMMTGNKKLVRVDSVSELPAPRRLRWKCSPETQGHLASSAEKLQRIVKNLDFIVYKFDNYGKTFIKKQKCSPDGFIQVALQLAYYRLYQRLVPTYESASIRRFQEGRVDNIRSATPEALAFVQAMTDH KAAVLASEKLQLLQRAIQAQTEYTVMAITGMAIDNHLLALRELARDLCKEPPEMFMDETYLMSNRFILSTSQVPTTMEMFCCYGPVVPNGYGACYNPHAEAITFCISSFHGCKETSSVEFAEAVGASLVDMRDLCSSRQPADSKPPTAKERARGPSQAKQS

[0097] "Serotonin receptor" or "5-hydroxytryptamine (5-HT) receptor" is a G protein-coupled receptor and ligand-gated ion channel found in the central and peripheral nervous systems. Serotonin activates serotonin receptors, which mediate both excitatory and inhibitory neurotransmission. In some embodiments, the serotonin receptor has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or a fragment thereof. >sp|P08908|5HT1A_Human 5-hydroxytryptamine receptor 1A OS=Homo sapiens OX=9606 GN=HTR1A PE=1 SV=3 SEQ ID NO:7 MDVLSPGQGNNTTSPPAPFETGGNTTGISDVTVSYQVITSLLLGTLIFCAVLGNACVVAAIALERSLQNVANYLIGSLAVTDLMVSVLVLPMAALYQVLNKWTLGQVTCDLFIALDVLCCTSSILHLCAIALDRYWAITDPIDYVNKRTPRRAAAALISLTWLIGFLISIPPMLGWRTPEDRSDPDACTISKDHGYTIYSTFGAFYIPLLLM LVLYGRIFRAARFRIRKTVKKVEKTGADTRHGASPAPQPKKSVNGESGSRNWRLGVESKAGGALCANGAVRQGDDGAALEVIEVHRVGNSKEHLPLPSEAGPTPCAPASFERKNERNAEAKRKMALARERKTVKTLGIIMGTFILCWLPFFIVALVLPFCESSCHMPTLLGAIINWLGYSNSLLNPVIYAYFNKDFQNAFKKIIKCKFCRQ >sp|P19327|5HT1A_Rat 5-hydroxytryptamine receptor 1A OS=Rattus norvegicus OX=10116 GN=Htr1a PE=1 SV=1 SEQ ID NO:8 MDVFSFGQGNNTTASQEPFGTGGNVTSISDVTFSYQVITSLLLGTLIFCAVLGNACVVAAIALERSLQNVANYLIGSLAVTDLMVSVLVLPMAALYQVLNKWTLGQVTCDLFIALDVLCCTSSILHLCAIALDRYWAITDPIDYVNKRTPRRAAALISLTWLIGFLISIPPMLGWRTPEDRSDPDACTISKDHGYTIYSTFGAFYIPLLLMLVLYGRIFRAARFRIRKTVRKVEKKGAGTSLGTSSAPPPKKSLNGQPGSGDWRRCAENRAVGTPCTNGAVRQGDDEATLEVIEVHRVGNSKEHLPLPSESGSNSYAPACLERKNERNAEAKRKMALARERKTVKTLGIIMGTFILCWLPFFIVALVLPFCESSCHMPALLGAIINWLGYSNSLLNPVIYAYFNKDFQNAFKKIIKCKFCRR >sp|Q64264|5HT1A_μαύς5-hydroxyτριプタミン receptor1A OS=Mus musculus OX=10090 GN=Htr1a PE=2 SV=2 sequence number 9 MDMFSLGQGNNTTTSLEPFGTGGNDTGLSNVTFSYQVITSLLLGTLIFCAVLGNACVVAAIALERSLQNVANYLIGSLAVTDLMVSVLVLPMAALYQVLNKWTLGQVTCDLFIALDVLCCTSSILHLCAIALDRYWAITDPIDYVNKRTPRRAAALISLTWLIGFLISIPPMLGWRTPEDRSNPNECTISKDHGYTIYSTFGAFYIPLLLMLVLYGRIFRAARFRIRKTVKKVEKKGAGTSFGTSSAPPPKKSLNGQPGSGDCRRSAENRAVGTPCANGAVRQGEDDATLEVIEVHRVGNSKGHLPLPSESGATSYVPACLERKNERTAEAKRMALARERKTVKTLGIIMGTFILCWLPFFIVALVLPFCESSCHMPELLGAIINWLGYSNSLLNPVIYAYFNKDFQNAFKKIIKCKFCR

[0098] Gamma-aminobutyric acid (GABA) functions as a trophic factor that regulates several important developmental processes, including neuronal proliferation, migration, and differentiation.

[0099] Neuronal nitric oxide synthase (nNOS) produces nitric oxide (NO) in the central and peripheral nervous systems. In some embodiments, nNOS has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a fragment thereof. >sp|P29475|NOS1_Human nitric oxide synthase, brain OS=Homo sapiens OX=9606 GN=NOS1 PE=1 SV=2 SEQ ID NO:10 >sp|P29476|NOS1_Rat nitric oxide synthase, brain OS=Rattus norvegicus OX=10116 GN=Nos1 PE=1 SV=1 SEQ ID NO:11 >sp|Q9Z0J4|NOS1_Mouse Nitric Oxide Synthase, Brain OS=Mus musculus OX=10090 GN=Nos1 PE=1 SV=1 SEQ ID NO: 12 MEEHTFGVQQIQPNVISVRLFKRKVGGLGFLVKERVSKPPVIISDLIRGGAAEQSGLIQAGDIILAVNDRPLVDLSYDSALEVLRGIASETHVVLILRGPEGFTTHLETTFTGDGTPKTIRVTQPLGTPTKAVDLSRQPSASKDQPLAVDRVPGPSNGPQHAQGRGQGAGSVSQANGVAIDPTMKNTKANLQDSGEQDELLKEIEPVLSILTGGGKAVNRGGPAKAEMKDTGIQVDRDLDGKLHKAPPLGGENDRVFNDLWGKGNVPVVLNNPYSENEQSPASGKQSPTKNGSPSRCPRFLKVKNWETDVVLTDTLHLKSTLETGCTEQICMGSIMLPSHHIRKSEDVRTKDQLFPLAKEFLDQYYSSIKRFGSKAHMDRLEEVNKEIESTSTYQLKDTELIYGAKHAWRNASRCVGRIQWSKLQVFDARDCTTAHGMFNYICNHVKYATNKGNLRSAITIFPQRTDGKHDFRVWNSQLIRYAGYKQPDGSTLGDPANVEFTEICIQQGWKPPRGRFDVLPLLLQANGNDPELFQIPPELVLEVPIRHPKFDWFKDLGLKWYGLPAVSNMLLEIGGLEFSACPFSGWYMGTEIGVRDYCDNSRYNILEEVAKKMDLDMRKTSSLWKDQALVEINIAVLYSFQSDKVTIVDHHSATESFIKHMENEYRCRGGCPADWVWIVPPMSGSITPVFHQEMLNYRLTPSFEYQPDPWNTHVWKGTNGTPTKRRAIGFKKLAEAVKFSAKLMGQAMAKRVKATILYATETGKSQAYAKTLCEIFKHAFDAKAMSMEEYDIVHLEHEALVLVVTSTFGNGDPPENGEKFGCALMEMRHPNSVQEERKS YKVRFNSVSSYSDSRKSGDGPDLRDNFESTGPLANVRFSVFGLGSRAYPHFCAFGHAVDTLLEELGGERILKMREGDELCGQEEAFRTWAKKVFKAACDVFCVGDDVNIEKANNSLISNDRSWKRNKFRLTYVAEAPELTQGLSNV HKKRVSAARLLSRQNLQSPKSSRSTIFVRLHTNGNQELQYQPGDHLGVFPGNHEDLVNALIERLEDAPPANHVVKVEMLEERNTALGVISNWKDESRLPPCTIFQAFKYYLDITTPPTPLQLQQFASLATNEKEKQRLLVLSKGLQE YEEWKWGKNPTMVEVLEEFPSIQMPATLLLTQLSLLQPRYYSISSSPDMYPDEVHLTVAIVSYHTRDGEGPVHHGVCSSWLNRIQADDVVPCFVRGAPSFHLPRNPQVPCILVGPGTGIAPFRSFWQQRQFDIQHKGMNPCPMVLVF GCRQSKIDHIYREETLQAKNKGVFRELYTAYSREPDRPKKYVQDVLQEQLAESVYRALKEQGGHIYVCGDVTMAADVLKAIQRIMTQQGKLSEEDAGVFISRLRDDNRYHEDIFGVTLRTYEVTNRLRSESIAFIEEESKKDTDEVFSS

[0100] Glial fibrillary acidic protein (GFAP) is a class III intermediate filament. During central nervous system development, GFAP is a cell-specific marker that distinguishes astrocytes from other glial cells. In some embodiments, GFAP has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or a fragment thereof. >sp|P14136|GFAP_Human glial fibrillary acidic protein OS=Homo sapiens OX=9606 GN=GFAP PE=1 SV=1 SEQ ID NO:13 MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM >sp|P47819|GFAP_Rat glial fibrillary acidic protein OS=Rattus norvegicus OX=10116 GN=Gfap PE=1 SV=2 SEQ ID NO: 14 MERRRITSARRSYASSETMVRGHGPTRHLGTIPRLSLSRMTPPLPARVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTTNSARLEVERDNLTQDLGTLRQKLQDETNLRLEAENNLAVYRQEADEATLARVDLERKVESLEEEIQFLRKIHEEEVRELQEQLAQQQVHVEMDVAKPDLTAALREIRTQYEAVATSNMQETEEWYRSKFADLTDVASRNAELLRQAKHEANDYRRQLQALTCDLESLRGTNESLERQMREQEERHARESASYQEALARLEEEGQSLKEEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM >sp|P03995|GFAP_Mouse glial fibrillary acidic protein OS=Mus musculus OX=10090 GN=Gfap PE=1 SV=4 SEQ ID NO:15 MERRRITSARRSYASETTVVRGLGPSRQLGTMPRFSLSRMTPPLPARVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPT KLADVYQAELRELRLRLDQLTANSARLEVERDNFAQDLGTLRQKLQDETNLRLEAENNLAAYRQEADEATLARVDLERKVESLEEEIQFLRKIYEEEVRELREQLAQQ QVHVEMDVAKPDLTALREIRTQYEAVATSNMQETEEWYRSKFADLTDAASRNAELLRQAKHEANDYRRQLQALTCDLESLRGTNESLERQMREQEERHARESASYQ EALARLEEEGQSLKEEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKDSKQEHKDVVM

[0101] Enteric neural crest cells express SOX10, which signals neural crest cells to direct the activity of other genes to become more specific cell types, including enteric neurons. In some embodiments, SOX10 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or a fragment thereof. >sp|P56693|SOX10_Human transcription factor SOX-10 OS=Homo sapiens OX=9606 GN=SOX10 PE=1 SV=1 SEQ ID NO:16 MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGTAAIQAHYKSAHLDHRHPGEGSPMSDGNPEHPSGQSHGPPTPPTTPKTELQSGKADPKRDGRSMGEGGKPHIDFGNVDIGEISHEVMSNMETFDVAELDQYLPPNGHPGHVSSYSAAGYGLGSALAVASGHSAWISKPPGVALPTVSPPGVDAKAQVKTETAGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSGPYYGHSGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSPTHWEQPVYTTLSRP >sp|O55170|SOX10_Rat transcription factor SOX-10 OS=Rattus norvegicus OX=10116 GN=Sox10 PE=1 SV=1 Sequence number 17 MAEEQDLSEVELSPVGSEEPRCLSPSSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGETDQGGAAAIQAHYKSAHLDHRHPEEGSPMSDGNPEHPSG QSHGPPTPPTTPKTELQSGKADPKRDGRSLGEGGKPHIDFGNVDIGEISHEVMSNMETFDVTELDQYLPPNGHPGHVGSYSAAGYGLSSALAVASGHSAWISKPPGVALPTSPAVDKAQVKTETTGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSGPYYGHAGQASGLYSAFSYMGPSQRPLYTAISDPPSGPQSHSPTHWEQPVYTTLSRP >sp|Q04888|SOX10_μαύς τρόκρανικόνη SOX-10 OS=Mus musculus OX=10090 GN=Sox10 PE=1 SV=2 sequence number 18 MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWAQ AARRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGAAAIQAHYKSAHLDHRHPEEGSPMSDGNPEHPSG QSHGPPTPPTTPKTELQSGKADPKRDGRSLGEGGKPHIDFGNVDIGEISHEVMSNMETFDVTELDQYLPPNGHPGHVGSYSAAGYGLGSALAVASGHSAWISKPPGVALPTVSPPG VDAKAQVKTETTGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSGPYYGHAGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSPTHWEQPVYTTLSRP

[0102] Enteric neural crest cells express CD24. In some embodiments, CD24 has about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 19, or a fragment thereof. SEQ ID NO:19 MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGGALQSTASLFVVSLSLLHLYS

[0103] Enteric neural crest cells express CD45RA, which in some embodiments has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to GENBANK® Accession Nos. NP_002829.3, NP_563578.2, NP_563578.2, and NP_002829.3, all of which are incorporated herein by reference.

[0104] Enteric neural crest cells express CD57, in some embodiments, CD57 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or a fragment thereof. SEQ ID NO:20 MGNEEPWVQPALEMPKRRDILAIVLIVLPWTLLITVWHQSTLAPLLAVHKDEGSDPRRETPPGADPREYCTSDRDIVEVVRTEYVYTRPPPWSDTLPTIHVVTPTYSRPVQKAELTRMANTLHVPNLHWLVVEDAPRRTPLTARLLRDTGLNYTHLHVETPRNYKLRGDARD PRIPRGTMQRNLALRWLRETFPRNSSQPGVVYFADDDNTYSLELFEEMRSTRRVSVWPVAFVGGLRYEAPRVNGAGKVVGWKTVFDPHRPFAIDMAGFAVNLRLILQRSQAYFKLRGVKGGYQESSLLRELVTLNDLEPKAANCTKILVWHTRTEKPVLVNEGKKGFTDPSVEI SEQ ID NO:21 MGNEEPWVQP ALEMPKRRDI LAIVLIVLPW TLLITVWHQS TLAPLLAVHK DEGSDPRRET PPGADPREYC TSDRDIVEVV RTEYVYTRPP PWSDTLPTIH VVTPTYSRPV QKAELTRMANTLLHVPNLHW LVVEDAPRRT PLTARLLRDT GLNYTHLHVE TPRNYKLRGD ARDPRIPRGTMQRNLALRWL RETFPRNSSQ PGVVYFADDD NTYSLELFEE MRSTRRVSVW PVAFVGGLRYEAPRVNGAGK VVGWKTVFDPHRPFAIDMAGFAVNLRLILQRSQAYFKLRGVKGGYQESSLLRELVTLNDL EPKAANCTKI LVWHTRTEKP VLVNEGKKGF TDPSVEI SEQ ID NO:22 MPKRRDILAI VLIVLPWTLL ITVWHQSTLA PLLAVHKDEG SDPRRETPPG ADPREYCTSDRDIVEVVRTE YVYTRPPPWS DTLPTIHVVT PTYSRPVQKA ELTRMANTLL HVPNLHWLVVEDAPRRTPLT ARLLRDTGLN YTHLHVETPR NYKLRGDARD PRIPRGTMQR NLALRWLRETFPRNSSQPGV VYFADDDNTY SLELFEEMRS TRRVSVWPVA FVGGLRYEAP RVNGAGKVVGWKTVFDPHRP FAIDMAGFAV NLRLILQRSQ AYFKLRGVKG GYQESSLLRE LVTLNDLEPKAANCTKILVW HTRTEKPVLV NEGKKGFTDP SVEI SEQ ID NO:23 MPKRRDILAI VLIVLPWTLL ITVWHQSTLA PLLAVHKDEG SDPRRETPPG ADPREYCTSDRDIVEVVRTE YVYTRPPPWS DTLPTIHVVT PTYSRPVQKA ELTRMANTLL HVPNLHWLVVEDAPRRTPLT ARLLRDTGLN YTHLHVETPR NYKLRGDARD PRIPRGTMQR NLALRWLRETFPRNSSQPGV VYFADDDNTY SLELFEEMRS TRRVSVWPVA FVGGLRYEAP RVNGAGKVVGWKTVFDPHRP FAIDMAGFAV NLRLILQRSQ AYFKLRGVKG GYQESSLLRE LVTLNDLEPKAANCTKILVW HTRTEKPVLV NEGKKGFTDP SVEI

[0105] Enteric neural crest cells express CD63. In some embodiments, CD63 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:24, or a fragment thereof. SEQ ID NO:24 MAVEGGMKCV KFLLYVLLLA FCACAVGLIA VGVGAQLVLS QTIIQGATPG SLLPVVIIAVGVFLFLVAFV GCCGACKENY CLMITFAIFL SLIMLVEVAA AIAGYVFRDK VMSEFNNNFRQQMENYPKNN HTASILDRMQ ADFKCCGAAN YTDWEKIPSM SKNRVPDSCC INVTVGCGINFNEKAIHKEG CVEKIGGWLR KNVLVVAAAA LGIAFVEVLG IVFACCLVKS IRSGYEVM

[0106] Enteric neural crest cells express CD71. In some embodiments, CD71 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:25, or a fragment thereof. SEQ ID NO:25 MMDQARSAFS NLFGGEPLSY TRFSLARQVD GDNSHVEMKL AVDEEENADN NTKANVTKPK RCSGSICYGT IAVIVFFLIG FMIGYLGYCK GVEPKTECER LAGTESPVRE EPGEDFPAAR RLYWDDLKRK LSEKLDSTDF TGTIKLLNEN SYVPREAGSQ KDENLALYVE NQFREFKLSK VWRDQHFVKI QVKDSAQNSV IIVDKNGRLV YLVENPGGYV AYSKAATVTG KLVHANFGTK KDFEDLYTPV NGSIVIVRAG KITFAEKVAN AESLNAIGVL IYMDQTKFPI VNAELSFFGH AHLGTGDPYT PGFPSFNHTQ FPPSRSSGLP NIPVQTISRA AAEKLFGNME GDCPSDWKTD STCRMVTSES KNVKLTVSNV LKEIKILNIF GVIKGFVEPD HYVVVGAQRD AWGPGAAKSG VGTALLLKLA QMFSDMVLKD GFQPSRSIIF ASWSAGDFGS VGATEWLEGY LSSLHLKAFT YINLDKAVLG TSNFKVSASP LLYTLIEKTM QNVKHPVTGQ FLYQDSNWAS KVEKLTLDNA AFPFLAYSGI PAVSFCFCED TDYPYLGTTM DTYKELIERI PELNKVARAA AEVAGQFVIK LTHDVELNLD YERYNSQLLS FVRDLNQYRA DIKEMGLSLQ WLYSARGDFF RATSRLTTDF GNAEKTDRFV MKKLNDRVMR EGPQMMLLLT DARPSNHFLS PLLSLHRg

[0107] Enteric neural crest cells express CD121b, in some embodiments, CD121b has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:26, or a fragment thereof. SEQ ID NO:26 MWAQDGALWL LPALQEDSGT YVCTTRNASY CDKMSIELRV FENTDAFLPF ISYPQILTLS TSGVLVCPDL SEFTRDKTDV KIQWYKDSLL LDKDNEKFLS VRGTHLLVH DVALEDAGYY RCVLTFAHEG QQYNITRSIE LRIKKKKEET IPVIISPLKT ISASLGSRLT IPCKVFLGTG TPLTTMLWWT ANDTHIESAY PGGRVTEGPR QEYSENNENY IEVPLIFDPV TREDLHMDFK CVVHNTLSFQ TLRTTVKEAS STFSWGIVLA PLSLAFLVLG GIWMHRRCKH RTGKADGLTV LWPHHQDFQS YPK

[0108] Enteric neural crest cells express CD147. In some embodiments, CD147 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:27, or a fragment thereof. SEQ ID NO:27 MAAALFVLLG FALLGTHGAS GAAGFVQAPL SQQRWVGGSV ELHCEAVGSP VPEIQWWFEG QGPNDTCSQL WDGARLDRVH IHATYHQHAA STISIDTLVE EDTGTYECRA SNDPDRNHLT RAPRVKWVRA QAVVLVLEPG TVFTTVEDLG SKILLTCSLN DSATEVTGHR WLKGGVVLKE DALPGQKTEF KVDSDDQWGE YSCVFLPEPM GTANIQLHGP PRVKAVKSSE HINEGETAML VCKSESVPPV TDWAWYKITD SEDKALMNGS ESRFFVSSSQ GRSELHIENL NMEADPGQYR CNGTSSKGSD QAIITLRVRS HLAALWPFLG IVAEVLVLVT IIFIYEKRRK PEDVLDDDDA GSAPLKSSGQ HQNDKGKNVR QRNSS

[0109] Enteric neural crest cells express CD148. In some embodiments, CD148 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:28, or a fragment thereof. SEQ ID NO:28 MTRGGGSGSS RGSRDRVAAR WGWAPLAPPR EAPARSGTRP PRGSRARLRR VAAAAAAAAM SPGKPGAGGA GTRRTGWRRR RRRRRQEAAT TVPGLGRTAG PDSRVRGTFQ GARGMKPAAR EARLPPRSPG LRWALPLLLL LLRLGQILCA GGTPSPIPDP SVATVATGEN GITQISSTAE SFHKQNGTGT PQVETNTSED GESSGANDSL RTPEQGSNGT DGASQKTPSS TEPIPVSDLR VALTGVRKAA LSWSNGNGTA SCRVLLESIG SHEELTQDSR LQVNISGLKP GVQYNINPYL LQSNKTKGDP LGTEGGLDAS NTERSRAGSP TAPVHDESLV GPVDPSSGQQ SRDTEVLLVG LEPGTRYNAT VYSQAANGTE GQPQAIEFRT NAIQVFDVTA VNISATSLTL IWKVSDNESS SNYTYKIHVA GETDSSNLNV SEPRAVIPGL RSSTFYNITV CPVLGDIEGT PGFLQVHTPP VPVSDFRVTV VSTTEIGLAW SSHDAESFQM HITQEGAGNS RVEITTNQSI IIGGLFPGTK YCFEIVPKGP NGTEGASRTV CNRTVPSAVF DIHVVYVTTT EMWLDWKSPD GASEYVYHLV IESKHGSNHT STYDKAITLQ GLIPGTLYNI TISPEVDHVW GDPNSTAQYT RPSNVSNIDV STNTTAATLS WQNFDDASPT YSYCLLIEKA GNSSNATQVV TDIGITDATV TELIPGSSYT VEIFAQVGDG IKSLEPGRKS FCTDPASMAS FDCEVVPKEP ALVLKWTCPP GANAGFELEV SSGAWNNATH LESCSSENGT EYRTEVTYLN FSTSYNISIT TVSCGKMAAP TRNTCTTGIT DPPPPDGSPN ITSVSHNSVK VKFSGFEASH GPIKAYAVIL TTGEAGHPSA DVLKYTYEDF KKGASDTYVTYLIRTEEKGR SQSLSEVLKY EIDVGNESTT LGYYNGKLEP LGSYRACVAG FTNITFHPQN KGLIDGAESY VSFSRYSDAV SLPQDPGVIC GAVFGCIFGA LVIVTVGGFI FWRKKRKDAK NNEVSFSQIK PKKSKLIRVE NFEAYFKKQQ ADSNCGFAEE YEDLKLVGIS QPKYAAELAE NRGKNRYNNV LPYDISRVKL SVQTHSTDDY INANYMPGYH SKKDFIATQG PLPNTLKDFW RMVWEKNVYA IIMLTKCVEQ GRTKCEEYWP SKQAQDYGDI TVAMTSEIVL PEWTIRDFTV KNIQTSESHP LRQFHFTSWP DHGVPDTTDL LINFRYLVRD YMKQSPPESP ILVHCSAGVG RTGTFIAIDR LIYQIENENT VDVYGIVYDL RMHRPLMVQT EDQYVFLNQC VLDIVRSQKD SKVDLIYQNT TAMTIYENLA PVTTFGKTNG YIA

[0110] In some embodiments, CD193 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to Genbank Accession Nos. XP_011531637.1, XP_006713023.1, NP_001158152.1, NP_847898.1, NP_847899.1, AAI30321.1, AAI10298.1, XP_016861175.1, XP_016861174.1, NP_001828.1, AAI30319.1, or ACN11153.1.

[0111] Enteric neural crest cells express CD193. In some embodiments, CD193 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:29, or a fragment thereof. SEQ ID NO:29 MLLIIVIIVI VNYCDCTCVT DKMCIFFTAA VDWIMPFGIR MLLRAHKPGS SRRSEMTTSL DTVETFGTTS YYDDVGLLCE KADTRALMAQ FVPPLYSLVF TVGLLGNVVV VMILIKYRRL RIMTNIYLLN LAISDLLFLV TLPFWIHYVR GHNWVFGHGM CKLLSGFYHT GLYSEIFFII LLTIDRYLAI VHAVFALRAR TVTFGVITSI VTWGLAVLAA LPEFIFYETE ELFEETLCSA LYPEDTVYSW RHFHTLRMTI FCLVLPLLVM AICYTGIIKT LLRCPSKKKY KAIRLIFVIM AVFFIFWTPY NVAILLSSYQ SILFGNDCER SKHLDLVMLV TEVIAYSHCC MNPVIYAFVG ERFRKYLRHF FHRHLLMHLG RYIPFLPSEK LERTSSVSPS TAEPELSIVF

[0112] In some embodiments, CD193 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to Genbank Accession Nos. XP_011531637.1, XP_006713023.1, NP_001158152.1, NP_847898.1, NP_847899.1, AAI30321.1, AAI10298.1, XP_016861175.1, XP_016861174.1, NP_001828.1, AAI30319.1, or ACN11153.1.

[0113] Enteric neural crest cells express CD243. In some embodiments, CD243 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30, or a fragment thereof. SEQ ID NO:30 MSVNLQGDQR GATEARTFLL EIQPVSQFLE ESAFSQSGPG AVICGLSTKV GVSSSKISRL GGRSKEREVG MDLEGDRNGG AKKKNFFKLN NKSEKDKKEK KPTVSVFSMF RYSNWLDKLY MVVGTLAAII HGAGLPLMNDML VAGNFDML NAGNDLMEMS TGFFMNLEED MTRYAYYYSG IGAGVLVAAY IQVSFWCLAA GRQIHKIRKQ FFHAIMRQEI GWFDVHDVGE LNTRLTDDVS KINEGIGDKI GMFFQSMATF FTGFIVGFTR GWKLTLVILA ISPVLGLSAA VWAKILLASSA DVAKELLAFTA DVAKELLAFTA IRTVIAFGGQ KKELERYNKN LEEAKRIGIK KAITANISIG AAFLLIYASY ALAFWYGTTL VLSGEYSIGQ VLTVFFSVLI GAFSVGQASP SIEAFANARG AAYEIFKIID NKPSIDSYSK SGHKPDNIKG NLEFRNVHFS YPSRKEVKLGKLGVKVGVGNSG CGKSTTVQLM QRLYDPTEGM VSVDGQDIRT INVRFLREII GVVSQEPVLF ATTIAENIRY GRENV™DEI EKAVKEANAY DFIMKLPHKF DTLVGERGAQ LSGGQKQRIA IARALVRNPK ILLLDEATSA LDTESEAVVQ VALDVIKHR TRNADVIAGHR FDDGVIVEKG NHDELMKEKG IYFKLV™QT AGNEVELENA ADESKSEIDA LEMSSNDSRS SLIRKRSTRR SVRGSQAQDR KLSTKEALDE SIPPVSFWRI MKLNLTEWPY FVVGVFCAII NGGLQPAFAI IFSKIIGSNGISKFLIFTFILLQ FLQGFTFGKA GEILTKRLRY MVFRSMLRQD VSWFDDPKNT TGALTTRLAN DAAQVKGAIG SRLAVITQNIANLGTGIIIS FIYGWQLTLL LLAIVPIIAI AGVVEMKMLS GQALKDKKEL EGSGKIATEA IENFRTVVSL TQEQKFEHMY AQSLQVPYRN SLRKAHIFGI TFSFTQAMMY FSYAGCFRFG AYLVAHKLMS FEDVLLVFSA VVFGAMAVGQ VSSFAPDYAK AKISAAHIIM IIEKTPLIDS YSTEGLMPNT LEGNVTFGEV VFNYPTRPDI PVLQGLSLEV KKGQTLALVG SSGCGKSTVV QLLERFYDPL AGKVLLDGKE IKRLNVQWLR AHLGIVSQEP ILFDCSIAEN IAYGDNSRVV SQEEIVRAAK EANIHAFIES LPNKYSTKVG DKGTQLSGGQ KQRIAIARAL VRQPHILLLD EATSALDTES EKVVQEALDK AREGRTCIVI AHRLSTIQNA DLIVVFQNGR VKEHGTHQQL LAQKGIYFSM VSVQAGTKRQ

[0114] In some embodiments, CD243 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to Genbank Accession Nos. NP_001335875.1, NP_001335874.1, NP_001335873.1, NP_000918.2, AAI30425.1, or KIH63939.1.

[0115] Enteric neural crest cells express CD275, hi some embodiments, CD275 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31, or a fragment thereof. SEQ ID NO:31 MVGSDVELSC ACPEGSRFDL NDVYVYWQTS ESKTVVTYHI PQNSSLENVD SRYRNRALMS PAGMLRGDFS LRLFNVTPQD EQKFHCLVLS QSLGFQEVLS VEVTLHVAAN FSVPVVSAPH SPSQDELTFT CTSINGYPRP NVYWINKTDN SLLDQALQND TVFLNMRGLY DVVSVLRIAR TPSVNIGCCI ENVLLQQNLT VGSQTGNDIG ERDKITENPV STGEKNAATW SILAVLCLLV VVAVAIGWVC RDRCLQHSYA GAWAVSPETE LTVSRHGFEQ TTDVLPFILK SSLGASCEPT AFLPPAAPG PCAHLFIWML AECTPCSPVW SSIS

[0116] In some embodiments, CD275 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to Genbank Accession Nos. XP_011527818.1, XP_011527816.1, NP_001382847.1, NP_001269981.1, NP_001269980.1, NP_001269979.1, NP_056074.1, XP_024307828.1, or NP_001352688.1.

[0117] Enteric glial cells express PMP22. In some embodiments, PMP22 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:32, or a fragment thereof. SEQ ID NO:32 MLLLLLSIIV LHVAVLVLLF VSTIVSQWIV GNGHATDLWQ NCSTSSSGNV HHCFSSSPNE WLQSVQATMI LSIIFSILSL FLFFCQLFTL TKGGRFYITG IFQILAGLCV MSAAAIYTVR HPEWHLNSDY SYGFAYILAW VAFPLALLSG VIYVILRKRE

[0118] In some embodiments, PMP22 has at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to Genbank Accession Nos. CAG46751.1, CAG46729.1, NP_001268384.1, NP_001317072.1, NP_001268385.1, NP_696997.1, NP_696996.1, NP_000295.1, XP_024306574.1, or NP_061133.1.

[0119] In some embodiments, the one or more cells are stimulated with a differentiation factor. The differentiation factor may include one or any combination of the following:

[0120] BMP4 (SEQ ID NO:33) MIPGNRMLMV VLLCQVLLGG ASHASLIPET GKKKVAEIQG HAGGRRSGQS HELLRDFEAT LLQMFGLRRR PQPSKSAVIP DYMRDLYRLQ SGEEEEEQIH STGLEYPERP ASRANTVRSF HHEEHLENIP GTSENSAFRF LFNLSSIPEN EVISSAELRL FREQVDQGPD WERGFHRINI YEVMKPPAEV VPGHLITRLL DTRLVHHNVT RWETFDVSPA VLRWTREKQP NYGLAIEVTH LHQTRTHQGQ HVRISRSLPQ GSGNWAQLRP LLVTFGHDGR GHALTRRRRA KRSPKHHSQR ARKKNKNCRR HSLYVDFSDV GWNDWIVAPP GYQAFYCHGD CPFPLADHLN STNHAIVQTL VNSVNSSIPK ACCVPTELSA ISMLYLDEYD KVVLKNYQEM VVEGCGCR

[0121] FGF2 (SEQ ID NO:34) MVGVGGGDVE DVTPRPGGCQ ISGRGARGCN GIPGAAAWEA ALPRRRPRRH PSVNPRSRAA GSPRTRGRRT EERPSGSRLG DRGRGRALPG GRLGGRGRGR APERVGGRGR GRGTAAPRAA PAARGSRPGP AGTMAAGSIT TLPALPEDGG SGAFPPGHFK DPKRLYCKNG GFFLRIHPDG RVDGVREKSD PHIKLQLQAE ERGVVSIKGV CANRYLAMKE DGRLLASKCV TDECFFFERL ESNNYNTYRS RKYTSWYVAL KRTGQYKLGS KTGPGQKAIL FLPMSAKS

[0122] Retinoic Acid TIFF2025502844000017.tif40128 SB431542 TIFF2025502844000018.tif43128 CHIR99021 TIFF2025502844000019.tif37128

[0123] In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 70% sequence identity with the sequence above. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 80% sequence identity with the sequence above. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 85% sequence identity with the sequence above. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 90% sequence identity with the sequence above. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional fragment or variant of the polypeptide disclosed above having at least about 95% sequence identity with the sequence above. In any of the methods or systems disclosed herein, the differentiation factor used may be a functional analog of the small molecule disclosed above. The disclosed methods involve sequentially exposing a culture of cells to two or more different tissue culture media. In some embodiments, the disclosed methods involve sequentially exposing a cell of the present disclosure to Cocktail Me or a tissue culture medium described herein.

[0124] The term "two-dimensional culture" as used herein is defined as a culture of cells disposed on a flat hydrogel comprising Matrigel® and vitronectin disposed in a culture vessel having a height of no more than 1-4 cells. In some embodiments, the two-dimensional culture is no more than 3 cells high. In some embodiments, the two-dimensional culture is no more than 2 cells high. In some embodiments, the two-dimensional culture is no more than 1 cell high.

[0125] As used herein, a "three-dimensional culture" is defined as a culture of cells that assumes a three-dimensional shape in culture. In some embodiments, the three-dimensional culture is more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cells high. In some embodiments, the three-dimensional culture has an organized shape that is self-assembled by simple culture methods. In some embodiments, the three-dimensional culture comprises one or more spheroids or gangliosides.

[0126] As used herein, "spheroid" or "cell spheroid" refers to any group of cells in a three-dimensional shape that generally corresponds to an ellipse or circle rotated about one of its major axes, the major or minor axis, and includes three-dimensional ovoid, oblate and prolate spheroids, spheres, and substantially equivalent shapes.

[0127] The spheroids of the present disclosure may have any suitable width, length, thickness, and / or diameter. In some embodiments, the spheroids may have a width of about 10 μm to about 50,000 μm, or any range therein, such as, but not limited to, about 100 μm to about 200 μm, about 100 μm to about 300 μm, about 100 μm to about 400 μm, about 100 μm to about 500 μm, about 100 μm to about 600 μm, about 100 μm to about 700 μm, about 50 μm to about 200 μm, about 50 μm to about 250 μm, about 100 μm to about 700 μm, about 300 μm to about 600 μm, about 400 μm to about 500 μm, about The nanoparticles may have a width, length, thickness, and / or diameter in the range of 500 μm to about 1,000 μm, about 600 μm to about 1,000 μm, about 700 μm to about 1,000 μm, about 800 μm to about 1,000 μm, about 900 μm to about 1,000 μm, about 750 μm to about 1,500 μm, about 1,000 μm to about 5,000 μm, about 1,000 μm to about 10,000 μm, about 2,000 to about 50,000 μm, about 25,000 μm to about 40,000 μm, or about 3,000 μm to about 15,000 μm. In some embodiments, the spheroids may have a width, length, thickness, and / or diameter of about 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1,000 μm, 5,000 μm, 10,000 μm, 20,000 μm, 30,000 μm, 40,000 μm, or 50,000 μm. In some embodiments, a plurality of spheroids are generated, and each of the plurality of spheroids may have a width, length, thickness, and / or diameter that varies by less than about 20%, such as less than about 15%, 10%, or 5%. In some embodiments, each of the plurality of spheroids may have a different width, length, thickness, and / or diameter within any of the above ranges. In some embodiments, the spheroids of the present disclosure comprise enteric neurons, enteric glial cells, progenitor cells, epithelial cells, mesenchymal cells, smooth muscle cells, and RPE cells. In some embodiments, the spheroids comprise mesenchymal cells, epithelial cells, and enteric neurons, but do not comprise smooth muscle cells, and do not comprise RPE.In some embodiments, the spheroids comprise about 10,000 or more, about 15,000 or more, about 20,000 or more, about 25,000 or more, about 30,000 or more, about 35,000 or more, about 40,000 or more, about 45,000 or more, about 50,000 or more, or about 60,000 or more cells. In some embodiments, the spheroids comprise about 25,000 to about 100,000 cells. In some embodiments, the spheroids comprise about 35,000 to about 100,000 cells. In some embodiments, the spheroids comprise about 45,000 to about 100,000 cells. In some embodiments, the spheroids comprise about 55,000 to about 100,000 cells. In some embodiments, the spheroids comprise about 75,000 to about 100,000 cells.

[0128] The cells within the spheroid may have a specific orientation. In some embodiments, the spheroid may comprise an inner core and an outer surface. In some embodiments, the spheroid may be hollow (i.e., may not contain cells inside). In some embodiments, the inner core cells and the outer surface cells are different cell types. In some embodiments, the spheroid may be composed of one, two, three or more different cell types, including one or more neuronal cell types and / or one or more stem cell types. In some embodiments, the inner core cells may be composed of one, two, three or more different cell types. In some embodiments, the outer surface cells may be composed of one, two, three or more different cell types.

[0129] In some embodiments, the spheroids comprise at least two types of cells. In some embodiments, the spheroids comprise neuronal cells and non-neuronal cells. In some embodiments, the spheroids comprise neuronal cells and astrocytes in a ratio of about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1 neuronal cells to astrocytes. In some embodiments, the spheroids comprise neuronal cells and non-neuronal cells in a ratio of about 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the spheroids comprise neuronal cells and non-neuronal cells in a ratio of about 1:5, 1:4, 1:3, or 1:2. Any combination of cell types disclosed herein may be used in the above specified ratios in the spheroids of the present disclosure.

[0130] Depending on the particular embodiment, the cell groups may be arranged according to any suitable shape, geometry, and / or pattern. For example, the independent cell groups may be deposited as spheroids, and the spheroids may be arranged in a three-dimensional grid, or any other suitable three-dimensional pattern. The independent spheroids may all contain approximately the same number of cells and be approximately the same size, or alternatively, different spheroids may have different numbers of cells and different sizes. In some embodiments, the multiple spheroids may be arranged in shapes such as L or T shapes, radiating from a single point or multiple points, continuous spheroids in single or parallel lines, tubes, cylinders, toroids, hierarchically branched vascular networks, high aspect ratio objects, thin closed shells, organoids, or other complex shapes that may correspond to the geometry of tissues, blood vessels, or other biological structures. In some embodiments, the spheroids are "crest spheres", meaning that they contain one or more neural crest cells as specified herein. In some embodiments, the crestspheres contain greater than about 50% neural crest cells relative to the total number of cells in the spheroid, greater than about 60% neural crest cells relative to the total number of cells in the spheroid, greater than about 70% neural crest cells relative to the total number of cells in the spheroid, greater than about 80% neural crest cells relative to the total number of cells in the spheroid, greater than about 90% neural crest cells relative to the total number of cells in the spheroid, greater than about 95% neural crest cells relative to the total number of cells in the spheroid, greater than about 30% neural crest cells relative to the total number of cells in the spheroid. For example, the spheroid comprises more than about 40% neural crest cells, more than about 10% neural crest cells relative to the total number of cells in the spheroid, more than about 20% neural crest cells relative to the total number of cells in the spheroid, more than about 25% neural crest cells relative to the total number of cells in the spheroid, more than about 30% neural crest cells relative to the total number of cells in the spheroid, more than about 35% neural crest cells relative to the total number of cells in the spheroid, more than about 45% neural crest cells relative to the total number of cells in the spheroid, and more than about 55% neural crest cells relative to the total number of cells in the spheroid.

[0131] The term "subject" as used herein refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, dogs, cats, rodents, and the like. Preferably, the subject is a human subject. The terms "subject," "individual," and "patient" are used interchangeably herein. Thus, the terms "subject," "individual," and "patient" encompass individuals with disorders of gut-brain interaction (e.g., achalasia, Hirschsprung's disease, intestinal pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence).

[0132] As used herein, the term "therapeutic agent" means an agent utilized to treat, combat, mitigate, prevent, or ameliorate an unwanted condition or disease in a patient.

[0133] A "therapeutically effective amount" or "effective amount" of a composition is a predetermined amount calculated to achieve a desired effect, i.e., to treat, address, alleviate, prevent or ameliorate one or more symptoms of intestinal motility. In some embodiments, the activity contemplated by the method includes both medical and / or prophylactic treatment, as appropriate. The specific dose of a compound administered in accordance with the present disclosure to achieve a therapeutic and / or prophylactic effect will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. It will be understood that the effective amount administered will be determined by the physician in light of the relevant circumstances, including the condition being treated, the choice of compound to be administered, and the route of administration selected. Thus, the dosage ranges set forth above are not intended to limit the scope of the present disclosure in any manner. A therapeutically effective amount of a compound of an embodiment of the present disclosure is typically an amount sufficient to achieve an effective systemic or local concentration in a tissue when it is administered in a physiologically acceptable excipient composition. In some embodiments, the effective amount is the amount of material required to impart a biological effect, such as cell differentiation in response to exposure to a PDGFR or PDGFR inhibitor disclosed herein.

[0134] As used herein, the terms "treat", "treated" or "treating" can refer to therapeutic treatment and / or prophylactic or preventative measures, the purpose of which is to prevent or delay (alleviate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. For purposes of the embodiments described herein, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms (reducing the extent of the condition, disorder, or disease), stabilization (i.e., not worsening) of the condition, disorder, or disease state, delay in onset or delay in progression of the condition, disorder, or disease, improvement or remission (whether partial or total) of the condition, disorder, or disease state, whether detectable or undetectable, improvement of at least one measurable physical parameter, not necessarily discernible by the patient, or enhancement or amelioration of the condition, disorder, or disease. Treatment can include eliciting a clinically significant response without excessive levels of side effects. Treatment can also include extending survival compared to expected survival in the absence of treatment.

[0135] As used herein, the terms "preventing" or "prevention" or "prevent" refer to protective or prophylactic measures that prevent or delay the onset of the targeted pathological condition or disorder. Those in need of treatment include those already diagnosed with the disorder, those prone to having the disorder, and those in whom the disorder is to be prevented.

[0136] References in this specification and in the concluding claims to parts by weight of a particular element or component in a composition indicate the weight relationship between the element or component and any other elements or ingredients in the composition or article for which the parts by weight are expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0137] Weight percentages (wt %) of components are based on the total weight of the formulation or composition in which the component is included, unless otherwise specified.

[0138] As used herein, the term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the aforementioned event or circumstance occurs and cases where it does not occur.

[0139] As used herein, the term "diagnosed" means having undergone a physical examination by a skilled artisan, e.g., a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. In some embodiments of the disclosed methods, the subject has been diagnosed as needing treatment for a disorder associated with PDGFR activity, e.g., gut motility disorder, prior to the administering step. As used herein, phrases such as "identified as needing treatment for a disorder" refer to selecting a subject based on need for treatment for a disorder. It is contemplated that the identification, in some embodiments, may be performed by a person different from the person making the diagnosis. In further embodiments, it is also contemplated that the administration may be performed by the person who subsequently performed the administration.

[0140] In some embodiments, the compositions, spheroids or ganglioids are administered in a desired dosage, which in some aspects comprises a desired dose or number of cells and / or a desired ratio of neuronal cell subpopulations. Thus, the dosage of cells is, in some embodiments, based on the total number of cells (or per m2 of body surface area). 2 In some embodiments, the dosage of cells is based on the desired total number of cells (or number per m of body surface area) in each population or each cell type. 2 In some embodiments, dosage is based on a combination of such characteristics, such as the desired total cell number, the desired ratios, and the desired total cell number in an individual population.

[0141] In some embodiments, the compositions, spheroids, or gangliosides are administered at or within an acceptable margin of difference of a desired dose of total cells, e.g., a desired dose of a subtype of neuronal cells (e.g., enteric neurons, glial cells, and mesenchymal cells). In some aspects, the desired dose is determined by a desired number of cells, a desired number of cells per unit of body surface area, or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / m 2 or cells / kg. In some embodiments, the desired dose is equal to or greater than a minimum number per unit of body surface area or body weight. In some embodiments, of the total cells administered at the desired dose, the individual populations or subtypes are at or near the desired output ratios described herein, e.g., within a particular tolerance or error of such ratios.

[0142] In some embodiments, the cells are administered at or within a desired dose tolerance. In some aspects, the desired dose is determined by a desired number of cells, or a desired number of such cells per unit of body surface area or body weight of the subject to whom the cells are administered, e.g., cells / m 2 or cells / kg. In some embodiments, the desired dose is equal to or greater than the minimum number of cells of the population, or the minimum number of cells of the population per unit of body surface area or body weight.

[0143] Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on two or more desired fixed doses, such as each of the individual neuronal subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed dose or a minimum dose of a neuronal subpopulation and a desired ratio thereof.

[0144] In certain embodiments, the composition, spheroid, or ganglioid comprises about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, or a range defined by any two of the foregoing values). cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between those ranges.

[0145] In some embodiments, the dose of total cells and / or the dose of individual neuronal subpopulations of cells is greater than or equal to 10 4 Or about that value ~10 9 or approximately that value of cells / meter 2 (m 2 ) Body surface area, e.g., 10 5 ~10 6 cells / m 2 Body surface area, for example, at or about the following value: 1×10 5 cells / m 2 , 1.5×10 5 cells / m 2 , 2×10 5 cells / m 2 , or 1 × 10 6 cells / m 2 Within the body surface area. For example, in some embodiments, the cells are 4 Or about that value ~10 9or approximately that value of neurons / meter 2 (m 2 ) Body surface area, e.g., 10 5 ~10 6 Neurons or glial cells / m 2 Body surface area, for example, at or about the following value: 1×10 5 Neurons or glial cells / m 2 , 1.5×10 5 Neurons or glial cells / m 2 , 2×10 5 Neurons or glial cells / m 2 , or 1 × 10 6 Neurons or glial cells / m 2 administered by body surface area or within their specified margin of error.

[0146] In some embodiments, the cells are 4 Or about that value ~10 9 or approximately that value of cells / meter 2 (m 2 ) weight, e.g. 10 5 ~10 6 cells / m 2 Body weight, for example, at or about the following value: 1×10 5 cells / m 2 , 1.5×10 5 cells / m 2 , 2×10 5 cells / kg, or 1 x 10 6 cells / m 2 administered by body surface area or within their specified margin of error.

[0147] As used herein, ranges may be expressed as "about" one particular value and / or to "about" another particular value. When such ranges are expressed, the ranges from the one particular value and / or the other particular value are also specifically contemplated and considered to be disclosed, unless the context dictates otherwise. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another specifically contemplated embodiment that is to be considered as disclosed, unless the context dictates otherwise. Moreover, it will be understood that each of the endpoints of a range is significant both in relation to the other endpoint and independently of the other endpoint, unless the context dictates otherwise. The term "about," as used herein when referring to measurable values, such as amounts, time durations, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the specified value, when such variations are appropriate for carrying out the disclosed methods.

[0148] The "percent identity" or "percent homology" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. "Identical" or "identity" as used herein in the context of two or more nucleic acid or amino acid sequences can mean that the sequences have a certain percentage of residues that are the same over a particular region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a particular region, determining the number of positions at which identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the particular region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths or the alignment results in one or more staggered ends and a particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator and not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using computer sequence algorithms such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm stands for Basic Local Alignment Search Tool and is suitable for determining sequence similarity. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). This algorithm involves first obtaining a query sequence that matches or meets a positive threshold score T when aligned with words of the same length in database sequences, which identifies high scoring sequence pairs (HSPs) by identifying words of short length. T is referred to as the neighborhood word score threshold (Altschul et al., supra).These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The extension of the word hits in each direction is stopped when: 1) the cumulative alignment score falls by a quantity X from its maximum achieved value, 2) the accumulation of one or more negative scoring residue alignments causes the cumulative score to fall below 0, or 3) the end of either sequence is reached. The Blast algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, a BLOSUM62 scoring matrix alignment (B) of 50 (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety), an expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered to be similar to another nucleic acid if the minimum total probability in a comparison of the test nucleic acid with the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001. Two single-stranded polynucleotides are "complements" of each other if their sequences can be aligned in an antiparallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide without the introduction of gaps and without unpaired nucleotides at the 5' or 3' ends of either sequence.A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides can hybridize to each other under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.

[0149] The term "functional fragment" refers to any portion of a polypeptide or nucleic acid sequence to which a full-length polypeptide or nucleic acid, respectively, is related, that is of sufficient length and has sufficient structure to confer at least a similar or substantially similar biological effect as the full-length polypeptide or nucleic acid based on the fragment. In some embodiments, a functional fragment is a portion of a full-length or wild-type nucleic acid sequence encoding any one of the nucleic acid sequences disclosed herein, the portion encoding a polypeptide of a certain length and / or structure less than the full length, but still encoding a domain that is biologically functional compared to the full-length or wild-type protein. In some embodiments, a functional fragment may have reduced, approximately equivalent, or enhanced biological activity compared to the wild-type or full-length polypeptide sequence on which the fragment is based. In some embodiments, the functional fragment is derived from a sequence of an organism, e.g., a human. In such embodiments, the functional fragment may retain 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the wild-type human sequence from which the sequence is derived. In some embodiments, a functional fragment may retain 85%, 80%, 75%, 70%, 65%, or 60% sequence identity to the wild-type sequence from which the sequence is derived. In some embodiments, a functional fragment may retain 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% sequence identity to the amino acid sequence encoded by any of the mRNA sequences in Table 2.

[0150] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion preferably contains at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or about 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides or amino acids.

[0151] "Variant" is intended to mean a substantially similar sequence. For nucleic acid molecules, variants include nucleic acid molecules having deletions (i.e., truncations) at the 5' and / or 3' ends, deletions and / or additions of one or more nucleotides at one or more internal sites within the native polynucleotide, and / or substitutions of one or more nucleotides at one or more sites within the native polynucleotide. As used herein, a "natural" nucleic acid molecule or polypeptide includes a nucleotide sequence or amino acid sequence, respectively, that occurs in nature. For nucleic acid molecules, conservative variants include sequences that, due to the degeneracy of the genetic code, code for the amino acid sequence of one of the polypeptides of the present disclosure. Variant nucleic acid molecules also include synthetically derived nucleic acid molecules, such as those generated by using, for example, site-directed mutagenesis, but still code for a protein of the present disclosure. In general, variants of a particular nucleic acid molecule of the present disclosure have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein. Variants of a particular nucleic acid molecule of the present disclosure (i.e., a reference DNA sequence) can also be assessed by comparing the percent sequence identity between the polypeptide encoded by the variant nucleic acid molecule and the polypeptide encoded by the reference nucleic acid molecule. The percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters as described elsewhere herein. When any given pair of nucleic acid molecules of the disclosure is assessed by a comparison of the percent sequence identity shared by the two polypeptides which they encode, the percent sequence identity between the two encoded polypeptides is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.In some embodiments, the term "variant" protein is intended to mean a protein derived from a native protein by deletion (so-called truncation) of one or more amino acids at the N-terminus and / or C-terminus of the native protein, deletion and / or addition of one or more amino acids at one or more internal sites in the native protein, or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, i.e., retain the desired biological activity of the native protein as described herein. Such variants may result, for example, from genetic polymorphism or from human manipulation. Biologically active variants of the proteins of the present disclosure have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence of the native protein as determined by sequence alignment programs and parameters described elsewhere herein. Biologically active variants of the proteins of the disclosure may differ from the protein by 1-15 amino acid residues, 1-10, e.g., 6-10, 5, 4, 3, 2, or even 1 amino acid residue. Proteins or polypeptides of the disclosure may be modified in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of a protein can be prepared by mutation of a nucleic acid sequence recombinantly encoding the amino acid sequence.

[0152] "Optionally" or "optionally" means that the subsequently described event, circumstance, or substance may or may not occur or be present, and the description includes instances where the event, circumstance, or substance occurs or is not present, as well as instances where it does not occur or be present.

[0153] As used herein, the term "salt" refers to an acid or base salt of a compound used in the methods of the present disclosure. Illustrative examples of acceptable salts are salts of mineral acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), salts of organic acids (such as acetic acid, propionic acid, glutamic acid, citric acid, etc.), and salts of quaternary ammonium (such as methyl iodide, ethyl iodide, etc.).

[0154] The terms "subject" and "patient" may be used interchangeably and refer to a mammal in need of treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Typically, the subject is a human in need of treatment. In some cases, the subject is an experimental model, such as a mouse.

[0155] The term "associated with" or "associated with" in the context of a substance or substance activity or function associated with a disease (e.g., a protein-related disease, a condition associated with intestinal motility disorder, a condition associated with NO neuron activity) means that the disease (e.g., intestinal motility disorder) is caused (in whole or in part) or the disease symptoms are caused (in whole or in part) by the substance or substance activity or function. For example, the symptoms of an intestinal motility disease or condition may be symptoms that result (in whole or in part) from regulating NO neuron activity (e.g., induction of colonic motility). As used herein, something that is described as being associated with a disease may be a target for treating the disease if it is a causative agent. For example, intestinal motility disorder may be treated with an agent (e.g., a compound described herein) that is effective in regulating NO neuron activity (e.g., effective in inducing colonic motility).

[0156] "Control" or "control experiment" is used according to its plain and ordinary meaning to refer to an experiment in which an experimental subject or agent is treated as a parallel experiment, except for the omission of an experimental procedure, agent, or variable. In some cases, a control is used as a standard of comparison in evaluating the effect of an experiment.

[0157] "Contacting" is used according to its plain and ordinary meaning and refers to a process that allows at least two different species (e.g., chemical compounds, including biological molecules or cells) to come into sufficient proximity to react, interact, or physically touch. However, it should be understood that the resulting reaction product can be generated directly from the reaction between the added reagents, or from an intermediate from one or more added reagents that can be generated in the reaction mixture. The term "contacting" may include allowing two species to react, interact, or physically touch, and the two species can be a compound described herein and a cell (e.g., a crest sphere containing enteric neurons or enteric glial cells, or one or both). In some embodiments, contacting includes allowing a compound described herein to interact with a protein or enzyme involved in a signaling pathway (e.g., PDGFR).

[0158] As defined herein, the terms "inhibit," "inhibit," "inhibiting," and the like, in reference to the interaction of a protein inhibitor (e.g., an antagonist), refer to adversely affecting (e.g., decreasing) the activity or function of a protein (e.g., PDGFR) relative to the activity or function of the protein in the absence of an inhibitor (e.g., a compound described herein). In some embodiments, inhibition refers to the alleviation of a disease or a symptom of a disease (e.g., intestinal motility disorder). In some embodiments, inhibition refers to the reduction of a signal transduction pathway or the activity of a signaling pathway. Thus, inhibition includes, at least in part, partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating a signal transduction or enzymatic activity or the amount of a protein.

[0159] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. By "co-administered" it is meant that the compositions described herein are administered simultaneously with, immediately before, or immediately after the administration of one or more additional therapies (e.g., a cardiomyopathy therapy, e.g., angiotensin converting enzyme inhibitors (e.g., enalipril, lisinopril), angiotensin receptor blockers (e.g., losartan, valsartan), beta blockers (e.g., lopressor, Toprol-XL), digoxin, or diuretics (e.g., Lasix, or Parkin disease therapies including, e.g., levodopa, dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride), MAO-B inhibitors (e.g., selegiline or rasagiline), amantadine, anticholinergics, anti-inflammatory drugs (e.g., clozapine), cholinergic inhibitors, modafil, or nonsteroidal anti-inflammatory drugs).

[0160] The enteric neurons and / or enteric glial cells of the present disclosure can be administered alone or can be co-administered to a patient. In some embodiments, co-administration is completed with enteric neurons or glial cells in ganglioid or spheroid structures. Co-administration is meant to include administering the compounds individually or in combination (more than one compound or agent), simultaneously or sequentially. Thus, the preparations can also be combined with other active substances (e.g., to reduce metabolic degradation) as needed. The compositions of the present disclosure can be delivered by topical routes, transdermally, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Liquid form preparations include solutions, suspensions, and emulsions, such as water or water / propylene glycol solutions. The compositions of the present disclosure can further include ingredients that provide sustained release and / or comfort. Such ingredients include high molecular weight, anionic mucus-mimetic polymers, gelling polysaccharides, and finely divided drug carrier substrates. These components are discussed in more detail in U.S. Patent Nos. 4,911,920, 5,403,841, 5,212,162, and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present disclosure can also be delivered as microspheres for sustained release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres for sustained release subcutaneously (see Rao, J.Biomater Sci.Polym.Ed.7:623-645,1995), or as biodegradable and injectable gel formulations (e.g., Gao Pharm.Res.12:857-863,1995). In some embodiments, formulations of the disclosed compositions can be delivered by the use of liposomes that fuse with the cell membrane or are endocytosed, i.e., by using a receptor ligand attached to the liposome that binds to a cell's surface membrane protein receptor, resulting in endocytosis.The use of liposomes can focus the delivery of the compositions of the present disclosure to target cells in vivo, especially when the liposome surface carries receptor ligands specific to the target cells or is otherwise preferentially directed to a particular organ. (See, for example, Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46, 1576-1587, 1989). The compositions of the present disclosure can also be delivered as nanoparticles.

[0161] The pharmaceutical compositions provided by the present disclosure include compositions in which the active ingredient (e.g., a compound described herein, including embodiments or examples) is contained in a therapeutically effective amount, i.e., an amount effective to achieve its intended purpose. The present disclosure relates to pharmaceutical compositions comprising any enteric neuronal or glial cell disclosed herein and a pharma- ceutically acceptable carrier. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in a method for treating a disease, such compositions will contain an amount of active ingredient effective to achieve the desired result, e.g., modulate the activity of the subject (e.g., increase the number of nitergeric neurons in the subject) and / or reduce, eliminate, or delay the progression of disease symptoms (e.g., symptoms of intestinal motility disorder). Determination of a therapeutically effective amount of a compound of the present disclosure is well within the capabilities of one of ordinary skill in the art, especially in light of the detailed disclosure provided herein.

[0162] The dosage and frequency (single or multiple doses) administered to the mammal may vary depending on a variety of factors, such as whether the mammal is suffering from another disease and its route of administration, the size, age, sex, health, weight, body mass index, and diet of the recipient, the nature and extent of symptoms of the disease being treated (e.g., symptoms of intestinal motility disorders), types of concurrent treatments, complications from the disease being treated, or other health-related issues. Other treatment regimens or agents may be used in conjunction with the methods and compounds of applicant's disclosure, filed as 63 / 296,15 on January 3, 2022, which is incorporated by reference in its entirety. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the capabilities of one of ordinary skill in the art.

[0163] In some embodiments, the composition is administered to the subject in the form of a pharmaceutical composition, such as a composition comprising a cell or cell population and a pharma- ceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises another pharma- ceutical active agent or drug, such as a chemotherapeutic agent, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the agent is administered in the form of a salt, e.g., a pharma- ceutically acceptable salt. Suitable pharma- ceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and organic acids, such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, such as p-toluenesulfonic acid.

[0164] The choice of carrier in a pharmaceutical composition may be determined in part by the particular method used to administer the cell composition. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0165] In addition, in some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins 21st ed. (May 1, 2005).

[0166] In some embodiments, the pharmaceutical composition comprises a TVM or VM composition in an amount effective to treat or prevent a disease or condition, such as a therapeutically effective amount or a prophylactically effective amount. Thus, in some embodiments, the method of administration comprises administering the composition in an effective amount. In some embodiments, the therapeutic or prophylactic effectiveness is monitored by periodic evaluation of the treated subject. When administered repeatedly over several days or more, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosing regimes may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, multiple boluses of the composition, or continuous infusion administration of the composition.

[0167] In some embodiments, the pharmaceutical composition is administered at a desired dosage, and in some aspects includes a desired dose or number of cells, and / or a desired ratio of intestinal cells and / or glial cell subpopulations. Thus, the dosage of cells is, in some embodiments, based on the total number of cells (or number per m2 of body surface area or per kg of body weight) and the desired amount of each population or subtype. In some embodiments, the dosage of cells is based on the desired total number of cells (or number per m2 of body surface area or per kg of body weight) in each population or each cell type. In some embodiments, the dosage is based on a combination of such features, such as the desired total number of cells in each population and the desired total number of cells.

[0168] In some embodiments, the pharmaceutical composition is administered at or within a range of acceptable differences in the desired dose of total cells, e.g., the desired dose of spheroids, ganglioids, enteric neuron cells and / or glial cells. In some aspects, the desired dose is the desired number of cells, the desired number of cells per unit of body surface area, or the desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / m2 or cells / kg. In some aspects, the desired dose is equal to or greater than a minimum number or is a minimum number per unit of body surface area or body weight. In some aspects, of the total cells administered at the desired dose, the individual populations or subtypes are at or near the desired output ratio described herein, e.g., within a certain tolerance or error of such ratio.

[0169] In some embodiments, the cells are administered at or within a tolerance of a desired dose. In some aspects, the desired dose is a desired number of cells, or a desired number of such cells per unit of body surface area or body weight of the subject to whom the cells are administered, e.g., cells / m2 or cells / kg. In some aspects, the desired dose is equal to or greater than the minimum number of cells of the population, or the minimum number of cells of the population per unit of body surface area or body weight.

[0170] Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and / or based on two or more desired fixed doses, such as each of the enteric neuronal and glial cell subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed dose or a minimum dose of the glial cell subpopulations and a desired ratio thereof.

[0171] composition The present disclosure relates to spheroids comprising multiple cell types, including but not limited to enteric neurons. In some embodiments, the spheroids further comprise enteric glial cells. In some embodiments, the spheroids further comprise progenitor cells. In some embodiments, the spheroids further comprise epithelial cells. In some embodiments, the spheroids further comprise mesenchymal cells. In some embodiments, the spheroids further comprise smooth muscle cells. In some embodiments, the spheroids further comprise retinal pigment epithelial (RPE) cells.

[0172] The present disclosure relates to a spheroid comprising multiple cell types, the cell types comprising at least about 5% enteric neurons. In some embodiments, the spheroid comprises at least about 10% enteric neurons. In some embodiments, the spheroid comprises at least about 15% enteric neurons. In some embodiments, the spheroid comprises at least about 20% enteric neurons. In some embodiments, the spheroid comprises at least about 25% enteric neurons. In some embodiments, the spheroid comprises at least about 30% enteric neurons. In some embodiments, the spheroid comprises at least about 35% enteric neurons. In some embodiments, the spheroid comprises at least about 40% enteric neurons. In some embodiments, the spheroid comprises at least about 45% enteric neurons. In some embodiments, the spheroid comprises at least about 50% enteric neurons. In some embodiments, the spheroid comprises at least about 55% enteric neurons. In some embodiments, the spheroid comprises at least about 60% enteric neurons. In some embodiments, the spheroid comprises at least about 65% enteric neurons. In some embodiments, the spheroids comprise at least about 70% enteric neurons. In some embodiments, the spheroids comprise at least about 75% enteric neurons. In some embodiments, the spheroids comprise at least about 80% enteric neurons. In some embodiments, the spheroids comprise at least about 85% enteric neurons. In some embodiments, the spheroids comprise at least about 90% enteric neurons.

[0173] In some embodiments, the spheroids comprise about 5% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 10% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 15% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 20% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 25% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 30% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 35% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 40% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 45% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 50% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 55% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 60% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 65% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 70% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 75% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 80% to about 90% enteric neurons. In some embodiments, the spheroids comprise about 85% to about 90% enteric neurons.

[0174] The present disclosure relates to a spheroid comprising a plurality of cell types, the cell types comprising at least about 5% enteric glial cells. In some embodiments, the spheroid comprises at least about 10% enteric glial cells. In some embodiments, the spheroid comprises at least about 15% enteric glial cells. In some embodiments, the spheroid comprises at least about 20% enteric glial cells. In some embodiments, the spheroid comprises at least about 25% enteric glial cells. In some embodiments, the spheroid comprises at least about 30% enteric glial cells. In some embodiments, the spheroid comprises at least about 35% enteric glial cells. In some embodiments, the spheroid comprises at least about 40% enteric glial cells. In some embodiments, the spheroid comprises at least about 45% enteric glial cells. In some embodiments, the spheroid comprises at least about 50% enteric glial cells. In some embodiments, the spheroid comprises at least about 55% enteric glial cells. In some embodiments, the spheroids comprise at least about 60% enteric glial cells. In some embodiments, the spheroids comprise at least about 65% enteric glial cells. In some embodiments, the spheroids comprise at least about 70% enteric glial cells. In some embodiments, the spheroids comprise at least about 75% enteric glial cells. In some embodiments, the spheroids comprise at least about 80% enteric glial cells. In some embodiments, the spheroids comprise at least about 85% enteric glial cells. In some embodiments, the spheroids comprise at least about 90% enteric glial cells.

[0175] In some embodiments, the spheroids comprise about 0% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 5% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 10% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 15% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 20% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 25% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 30% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 35% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 40% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 45% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 50% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 55% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 60% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 65% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 70% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 75% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 80% to about 90% enteric glial cells. In some embodiments, the spheroids comprise about 85% to about 90% enteric glial cells.

[0176] The present disclosure relates to a spheroid comprising a plurality of cell types, the cell types comprising at least about 5% progenitor cells. In some embodiments, the spheroid comprises at least about 10% progenitor cells. In some embodiments, the spheroid comprises at least about 15% progenitor cells. In some embodiments, the spheroid comprises at least about 20% progenitor cells. In some embodiments, the spheroid comprises at least about 25% progenitor cells. In some embodiments, the spheroid comprises at least about 30% progenitor cells. In some embodiments, the spheroid comprises at least about 35% progenitor cells. In some embodiments, the spheroid comprises at least about 40% progenitor cells.

[0177] In some embodiments, the spheroids comprise about 0% to about 40% progenitor cells. In some embodiments, the spheroids comprise about 5% to about 40% progenitor cells. In some embodiments, the spheroids comprise about 10% to about 40% progenitor cells. In some embodiments, the spheroids comprise about 15% to about 40% progenitor cells. In some embodiments, the spheroids comprise about 20% to about 40% progenitor cells. In some embodiments, the spheroids comprise about 25% to about 40% progenitor cells. In some embodiments, the spheroids comprise about 30% to about 40% progenitor cells. In some embodiments, the spheroids comprise about 35% to about 40% progenitor cells.

[0178] The present disclosure relates to a spheroid comprising multiple cell types, wherein the cell types comprise at least about 5% epithelial cells. In some embodiments, the spheroid comprises at least about 10% epithelial cells. In some embodiments, the spheroid comprises at least about 15% epithelial cells. In some embodiments, the spheroid comprises at least about 20% epithelial cells.

[0179] In some embodiments, the spheroids comprise about 0% to about 20% epithelial cells. In some embodiments, the spheroids comprise about 5% to about 20% epithelial cells. In some embodiments, the spheroids comprise about 10% to about 20% epithelial cells. In some embodiments, the spheroids comprise about 15% to about 20% epithelial cells.

[0180] The present disclosure relates to a spheroid comprising a plurality of cell types, the cell types comprising at least about 5% mesenchymal cells. In some embodiments, the spheroid comprises at least about 10% mesenchymal cells. In some embodiments, the spheroid comprises at least about 15% mesenchymal cells. In some embodiments, the spheroid comprises at least about 20% mesenchymal cells. In some embodiments, the spheroid comprises at least about 25% mesenchymal cells. In some embodiments, the spheroid comprises at least about 30% mesenchymal cells. In some embodiments, the spheroid comprises at least about 35% mesenchymal cells. In some embodiments, the spheroid comprises at least about 40% mesenchymal cells. In some embodiments, the spheroid comprises at least about 45% mesenchymal cells. In some embodiments, the spheroid comprises at least about 50% mesenchymal cells. In some embodiments, the spheroid comprises at least about 55% mesenchymal cells. In some embodiments, the spheroid comprises at least about 60% mesenchymal cells. In some embodiments, the spheroids comprise at least about 70% mesenchymal cells.

[0181] In some embodiments, the spheroids comprise about 0% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 5% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 10% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 15% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 20% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 25% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 30% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 35% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 40% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 45% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 50% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 55% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 60% to about 70% mesenchymal cells. In some embodiments, the spheroids comprise about 65% to about 70% mesenchymal cells.

[0182] The present disclosure relates to a spheroid comprising multiple cell types, the cell types comprising at least about 5% smooth muscle cells. In some embodiments, the spheroid comprises at least about 10% smooth muscle cells. In some embodiments, the spheroid comprises at least about 15% smooth muscle cells. In some embodiments, the spheroid comprises at least about 20% smooth muscle cells. In some embodiments, the spheroid comprises at least about 25% smooth muscle cells. In some embodiments, the spheroid comprises at least about 30% smooth muscle cells.

[0183] In some embodiments, the spheroids comprise about 0% to about 30% smooth muscle cells. In some embodiments, the spheroids comprise about 5% to about 30% smooth muscle cells. In some embodiments, the spheroids comprise about 10% to about 30% smooth muscle cells. In some embodiments, the spheroids comprise about 15% to about 30% smooth muscle cells. In some embodiments, the spheroids comprise about 20% to about 30% smooth muscle cells. In some embodiments, the spheroids comprise about 25% to about 30% smooth muscle cells.

[0184] The present disclosure relates to a spheroid comprising multiple cell types, wherein the cell types comprise at least about 5% RPE cells.In some embodiments, the spheroid comprises at least about 10% RPE cells.In some embodiments, the spheroid comprises at least about 15% RPE cells.In some embodiments, the spheroid comprises at least about 20% RPE cells.

[0185] In some embodiments, the spheroids comprise about 0% to about 30% RPE cells. In some embodiments, the spheroids comprise about 5% to about 30% RPE cells. In some embodiments, the spheroids comprise about 10% to about 30% RPE cells. In some embodiments, the spheroids comprise about 15% to about 30% RPE cells. In some embodiments, the spheroids comprise about 20% to about 30% RPE cells. In some embodiments, the spheroids comprise about 25% to about 30% RPE cells.

[0186] In some embodiments, the spheroids are substantially free or free of retinal pigment epithelial cells. In some embodiments, the spheroids are substantially free or free of epithelial cells. In some embodiments, the spheroids are substantially free or free of smooth muscle cells. In some embodiments, the spheroids are substantially free or free of mesenchymal cells. In some embodiments, the spheroids are substantially free or free of non-neuronal cells. In some embodiments, the spheroids are substantially free or free of enteric glia. In some embodiments, the spheroids are substantially free or free of progenitor cells. In some embodiments, the spheroids are substantially free or free of RPE.

[0187] In some embodiments, the spheroids comprise about 25% to about 60% enteric neurons and about 25% to about 60% progenitor cells. In some embodiments, the spheroids comprise about 30% to about 60% enteric neurons and about 30% to about 60% progenitor cells. In some embodiments, the spheroids comprise about 35% to about 60% enteric neurons and about 35% to about 60% progenitor cells. In some embodiments, the spheroids comprise about 40% to about 60% enteric neurons and about 40% to about progenitor cells.

[0188] In some embodiments, the spheroids comprise about 10% to about 25% enteric neurons and about 10% to about 35% glial cells. In some embodiments, the spheroids comprise about 15% to about 25% enteric neurons and about 10% to about 35% glial cells.

[0189] In some embodiments, the spheroids comprise a percentage of the cell types found in Table 1.

[0190] [Table 1] TIFF2025502844000021.tif77167

[0191] The present disclosure relates to a composition comprising a spheroid comprising enteric neurons, the enteric neurons comprising SOX10 and CD24. The composition, in some embodiments, comprises cells expressing a biomarker disclosed in FIG. 3E.

[0192] The present disclosure also relates to a system comprising: (i) a cell culture vessel, optionally comprising a hydrogel; (ii) one or more stem cells or neural crest cells, either in suspension or as a component of a spheroid; and (iii) one or more differentiation factors.

[0193] In some embodiments, the system further comprises one or a combination of culture media disclosed herein. The present disclosure also relates to a method of culturing enteric neurons in a system, the system comprising (i) a cell culture vessel, optionally comprising a hydrogel; (ii) one or more stem cells or neural crest cells, either in suspension or as a component of a spheroid; and (iii) one or more differentiation factors. In some embodiments, the system further comprises one or a combination of culture media disclosed herein. In some embodiments, the method relates to (i) exposing the one or more stem cells to a first cell culture medium for a period sufficient to differentiate the one or more stem cells into neural crest cells, and successively exchanging the medium, and (ii) exposing the one or more neural crest cells to a second cell culture medium for a period sufficient to differentiate the one or more neural crest cells into enteric neurons, with at least one medium change during a culture period of about 12 to about 21 days.

[0194] method In some embodiments, the compounds and compositions described herein are useful for treating intestinal motility disorders.Therefore, provided herein is a method for treating intestinal motility disorders, comprising administering to a subject in need thereof a therapeutically effective amount of enteric neurons, enteric glial cells, or spheroids comprising them as described herein, or a composition comprising enteric neurons, enteric glial cells, or spheroids comprising them.Disorders treatable by the compounds and compositions of the present invention include, for example, achalasia, Hirschsprung's disease, intestinal pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence.

[0195] The present disclosure relates to a method of implanting one or more compositions herein in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of enteric neurons, enteric glial cells, or spheroids comprising them as described herein, or a pharma- ceutically acceptable salt thereof, or a composition comprising enteric neurons, enteric glial cells, or spheroids comprising them. Disorders treatable by the compounds and compositions of the present invention include, for example, achalasia, Hirschsprung's disease, pseudo-obstruction, gastroesophageal reflux disease (GERD), functional dysphagia, functional dyspepsia, irritable bowel syndrome (IBS), gastroparesis, functional constipation, functional diarrhea, and fecal incontinence. In some embodiments, the subject is a rodent, for example, a mouse. Thus, the present disclosure relates to a mammalian subject comprising a composition comprising enteric neurons, enteric glial cells, or spheroids, including those disclosed herein.

[0196] The present disclosure relates to methods of enriching a cell culture with a plurality of enteric neurons by exposing the cell culture to one or more PDGFR inhibitors. In some embodiments, the PDGFR inhibitor comprises an effective amount of a platelet-derived growth factor receptor (PDGFR) inhibitor or a pharma- ceutically acceptable salt thereof.Examples of PDGFR inhibitors include, but are not limited to, (Z)-orantinib, AC710, AC710 mesylate, AG1295, amuvatinib, amuvatinib hydrochloride, avapritinib, axitinib, AZD2932, cediranib, cediranib maleate, tiauranib, CHIR-124, CP-673451, crenolanib, dovitinib, dovitinib lactate, dovitinib lactate hydrate, dovitinib-D8, ENMD-2076, ENMD-2076 tartrate, flumatinib ... Lumatinib mesylate, GZD856, GZD856 formate, HG-7-85-01, hypothemycrin, ilorasertib, ilorasertib hydrochloride, imatinib, imatinib D4, imatinib D8, imatinib mesylate, JI-101, JNJ-10198409, KG5, Ki20227, lenvatinib, lenvatinib mesylate, linifanib, masitinib, masitinib mesylate, methylnisoline, multikinase inhibitor 1, N-(p-coumaroyl) )Serotonin, nintedanib, nintedanib esylate, NVP-ACC789, orantinib, pazopanib, pazopanib hydrochloride, PD-089828, PD-161570, PDGFRα kinase inhibitor 1, ponatinib, ponatinib D8, PP121, PP58, regorafenib, regorafenib D3, regorafenib hydrochloride, regorafenib monohydrate, ripretinib, sennoside B, seraltinib, SU5402, SU14813, SU14813 maleate, SU16f, SU 4312, SU4984, sunitinib, sunitinib D10, sunitinib malate, sunitinib-d4, TAK-593, tandutinib, tandutinib hydrochloride, telatinib, telatinib mesylate, TG100572, TG100572 hydrochloride, TG100801, TG100801 hydrochloride, toceranib, toceranib salt, toceranib-d8, trapidil, tyrosine kinase-IN-1, tyrphostin AG1296, tyrphostin AG1433, and borolanib.

[0197] In a further embodiment, the PDGFR inhibitor is TIFF2025502844000022.tif61128TIFF2025502844000023.tif196130TIFF202550284 4000024.tif210133TIFF2025502844000025.tif206135TIFF2025502844000026.tif20 2144TIFF2025502844000027.tif166128TIFF2025502844000028.tif205140TIFF2025502844000029.tif220125TIFF2025502844000030.tif26128, or a pharma- ceutically acceptable salt thereof.

[0198] In a further embodiment, the PDGFR inhibitor is a hydrate. In yet a further embodiment, the PDGFR inhibitor is Selected from TIFF2025502844000031.tif70128.

[0199] In a further embodiment, the PDGFR inhibitor is isotopic. In yet a further embodiment, the PDGFR inhibitor is deuterated. In yet a further embodiment, the PDGFR inhibitor is TIFF2025502844000032.tif250136 or a pharma- ceutically acceptable salt thereof.

[0200] In a further embodiment, the PDGFR inhibitor is administered as a pharma- ceutically acceptable salt, including, but not limited to, mesylate, hydrochloride, maleate, lactate, tartrate, formate, esylate, phosphate, or malate. In yet a further embodiment, the pharma- ceutically acceptable salt is TIFF2025502844000033.tif205143TIFF2025502844000034.tif206134TIFF2025502844000035.tif202143TIFF2025502844000036.tif114128.

[0201] In some embodiments, the present disclosure relates to a method of generating or enriching NO enteric neurons in culture by first exposing a cell culture comprising pluripotent stem cells to a series of tissue culture media, in some embodiments, the tissue culture media is one of the following:

[0202] E8-C, hPSC medium for maintenance Essential 8-Flex Supplement (20μl ml -1 ) and Essential 8™ Flex Medium. Store at 4°C (use within 2 weeks).

[0203] Cocktail A, first ENC differentiation medium BMP4 (1ng ml -1 ), SB431542 (10 μM), CHIR99021 (600 nM) and Essential 6™ medium. Store at 4° C. (use within 2 weeks).

[0204] Cocktail B, second ENC differentiation medium Combine SB431542 (10 μM), CHIR99021 (1.5 μM) and Essential 6™ medium. Store at 4° C. (Use within 2 weeks).

[0205] Cocktail C, third ENC differentiation medium Combine SB431542 (10 μM), CHIR99021 (1.5 μM), retinoic acid (1 μM) with Essential 6™ medium. Store at 4° C. (use within 2 weeks).

[0206] NC-C and ENC media for maintaining spheroids FGF2 (10 ng ml -1 ), CHIR99021 (3 μM), N2 supplement (10 μl ml -1 ), B27 supplement (20 μl ml -1 ), Glutagro (10 μl ml -1 ), MEM non-essential amino acids (10 μl ml -1) with Neurobasal® medium. Store at 4°C (use within 2 weeks).

[0207] EN-C and EN medium for differentiation and maintenance GDNF (10 ng ml -1 ), ascorbic acid (100 μM), N2 supplement (10 μl ml -1 ), B27 supplement (20 μl ml -1 ), Glutagro (10 μl ml -1 ), MEM non-essential amino acids (approximately 10 μl ml -1 ) with Neurobasal® medium. Store at 4°C (use within 2 weeks).

[0208] The present disclosure relates to a method of culturing any of the compositions disclosed herein with one or more of the cell culture media disclosed herein and one or more PDFR inhibitors.The present disclosure relates to a method of differentiating neural crest cells into two-dimensional or three-dimensional ganglioids or spheroids, comprising exposing one or more human pluripotent stem cells to GDNF, ascorbic acid, Neurobasal™ (ThermoFisher), n2 and B27 complement, and one or more PDGFR inhibitors.The present disclosure relates to a method of differentiating neural crest cells into two-dimensional or three-dimensional ganglioids or spheroids, comprising exposing one or more human pluripotent stem cells to the differentiation factor of FIG. 1E and one or more PDGFR inhibitors.In some embodiments, the PDGFR inhibitor does not include PP121 or a salt thereof.

[0209] The present disclosure relates to a method of purifying enteric neurons comprising exposing neurons in culture from human pluripotent stem cells to one or more antibodies specific for biomarkers expressed by the cells. In some embodiments, the biomarkers on the enteric neurons are at least 70% sequence identity to at least one or a combination of CD24, CD45RA, CD57, CD63, CD71, CD121b, CD147, CD164, CD184, CD193, CD243, and CD275.

[0210] The present disclosure also relates to a method of screening for agents that induce NO induction in cells, or NO induction in cells, comprising exposing an agent, e.g., a pharmaceutical compound, that is a candidate for the treatment of intestinal motility disorders, to one or more compositions disclosed herein.The present disclosure also relates to a method of screening for toxicity of therapeutic efficacy of an agent, comprising exposing an agent, e.g., a pharmaceutical compound, that is a candidate for the treatment of intestinal motility disorders, to one or more compositions disclosed herein.

[0211] Other embodiments are described in the following non-limiting examples. Various publications, including patents, published applications, technical papers, and academic papers, are cited throughout this specification. Each of these cited publications is incorporated herein by reference in its entirety. Publications include a co-pending provisional application, U.S. Application No. 63 / 296,151, entitled "Methods of Treating Gut Motility Disorders," filed on January 3, 2021, which is incorporated herein by reference in its entirety. Publications also include a co-pending PCT application, PCT Application No. PCT / US19 / 68447, which is incorporated herein by reference in its entirety. EXAMPLES

[0212] Example 1 hPSC-derived intestinal ganglioid model Human ENS development and function Here, we describe an experimental system for inducing ENS tissue from human pluripotent stem cells (hPSCs) that recapitulates the remarkable cellular diversity of the human ENS. These three-dimensional (3D) cultures (termed enteric ganglioids), together with two-dimensional (2D) ENS cultures, provide a scalable source of human enteric neurons and glia that are compatible with a wide range of high-throughput applications. Using single-cell transcriptomics, we map cell type-specific molecular features of human enteric neurons and glia, affording new strategies for enriching, isolating, or functionally targeting human enteric neurons and glia. We exploit hPSC-derived enteric ganglioids as a model system to investigate the development of NO neurons, characterize their molecular and physiological properties, and identify clinically relevant strategies to modulate their function in vitro and ex vivo in the mouse colon. Furthermore, we demonstrate the widespread engraftment and regenerative potential of NO neuron ganglioids in the colon of adult mice, providing a new xenograft model for studying the human ENS in vivo.

[0213] Induction of gut ganglioids from hPSCs for model development, functional and molecular diversity of the human ENS The ENS originates from the vagus nerve and the sacral neural crest (NC). Vagal NC cells migrate extensively and colonize the entire digestive tract, whereas sacral NC cells colonize only the most distal end of the colon (Serbedzija et al., 1991; Burns and Douarin, 1998; Heanue and Pachnis, 2007; Nagy and Goldstein, 2017). We have previously established a hPSC differentiation method to induce enteric neural crest cells (ENCs) under highly defined conditions (Figure 1A) (Barber et al., 2019; Fattahi et al., 2016). This protocol involves two steps following embryonic NC development. In step 1, enteric neural crest is induced by activating bone morphogenetic protein (BMP) and Wnt signaling in combination with retinoic acid (RA) treatment. RA caudates the differentiating NC and specifies the identity of vagal NC. Step 2 involves the generation of intestinal crestspheres in the presence of Wnt and fibroblast growth factor (FGF) signaling ( Barber et al., 2019 , Fattahi et al., 2016 ).

[0214] To characterize these developmental processes in the human ENS lineage at the molecular level, we performed single-cell RNA-seq (scRNA-seq) on the enteric neural crest and enteric crestspheres. During the enteric neural crest stage, enteric neural crest (ENC) (SOX10 + , FOXD3 + ), neuroepithelial precursors (NEPs) (WNT2B + , PAX6 + ), cranial placode (CP) (SIX1 + , EYA2 + ) and non-neural ectoderm (NNE) (EPCAM + , CDH1 + In the next step, suspension cultures of enteric neural crest cells are differentiated into four transcriptionally distinct cell types: NEP, two CP clusters (CP1 and CP2), and mesenchymal (Mes) (TWIST1). + , MSX1 +) serves as a purification strategy resulting in enteroclastospheres consisting primarily of ENCs with small populations of clusters (Figure 1B bottom, Figure 8A right). Modular scoring of the transcriptional signatures of cell types in step 1 and step 2 validates the shared transcriptional identity of the ENC clusters (Figure 8B).

[0215] Further subclustering of the ENC population identified four distinct subtypes (ENC1-4 for the enteric neural crest stage and ENC1'-4' for the enteric crestsphere) that differentially express classical ENC markers such as SOX10, EDNRB, TFAP2B and FOXD3 and transition over time from PHOX2B to PHOX2A expression (Figure 1C and D). To study how ENCs progress during the differentiation steps, we modularly scored the ENC1-4 transcriptional signature in the enteric crestsphere ENC1'-4' (Figure 8C). ENC1' and 2' showed high transcriptional similarity to ENC4, the three most transcriptionally distinct ENC subtypes, and ENC2 and 3, respectively (Figure 8C, Figure 8D and E).

[0216] hPSC-derived ENCs have previously been shown to recapitulate key migratory features of ENS precursors in health and disease states and can give rise to enteric neurons upon further differentiation (Barber et al., 2019; Fattahi et al., 2016), but their ability to generate the diverse array of neuronal and glial subtypes that constitute the human ENS remains to be characterized. To determine the potential of enteroclastospheres to differentiate into ENS cell types, we established two-dimensional and three-dimensional culture conditions that facilitate the transition of ENCs to mature ENS cell types (Figure 1E). Although two-dimensional cultures confer inherent technical advantages for applications such as high-content imaging assays, we choose to focus primarily on three-dimensional cultures, termed enteric ganglioids, given their scalability and potential to capture higher order cell-cell interactions that occur in developing and adult ENS tissues. In addition, three-dimensional culture platforms are technically advantageous for applications such as cell therapy.

[0217] To define the cellular composition of intestinal ganglioids, we performed single-nucleus RNA-seq (snRNA-seq) on stage 1 (differentiation days 35–50) and stage 2 (differentiation days 70–90) intestinal ganglioids (Figure 1E). Unbiased clustering of stage 1 intestinal ganglioids revealed a large population of enteric neurons, two progenitor populations, contaminating epithelial cells, a small population of mesenchymal cells, and one cluster of unknown identity (Figure 1F, Figure ​Figure8F). 8F). Phylogenetic analysis based on module scoring revealed that the progenitor 1 population shared high transcriptional similarity with ENC2' and 4'. Furthermore, the mesenchymal population was highly similar to ENC3' (Figure ​Figure8G). 8G. Importantly, stage 2 enteric ganglioids contained enteric glia in addition to enteric neurons, indicating that gliogenesis follows neurogenesis during in vitro differentiation, consistent with the in vivo developmental timeline (Figure 1G) (Rothman et al., 1986; Young et al., 2003). Immunostaining for GFAP confirmed the presence of glia in our stage 2 enteric ganglioids (Figure 1H). Stage 2 ganglioids contained a greater proportion of contaminating epithelial cells, mesenchymal cells, and two unknown clusters (Figure 1G, Figure 8H). At stage 2, the epithelial and mesenchymal populations showed higher transcriptional diversity compared to stage 1 clusters and could be further subclustered into two unique epithelial populations and five unique mesenchymal populations (Figures 8I and J). In addition, at this latter stage, contaminating retinal pigment epithelium (RPE) and smooth muscle cell populations appeared (Figure 1G, Figure 8H). To assess the functional maturation state of enteric neurons over the differentiation process, we assessed the expression of the neuronal activity marker cFOS. Neuronal depolarization leads to the expression of cFOS, a proto-oncogene that has been used as a marker of neuronal activity (Hunt et al., 1987; Bullitt, 1990; Santos et al., 2018). cFOS expression increased as enteric gangliosides progressed during differentiation (Figure 1I and J).To demonstrate synaptic maturation and electrical excitability of enteric neurons within ganglioids, we used optogenetics by differentiating a reporter hESC line expressing enhanced yellow fluorescent protein (EYFP)-tagged channelrhodopsin-2 under the control of the human synapsin promoter (Steinbeck et al., 2016). EYFP was readily detectable as early as day 43 (Figure 1K). Light stimulation of stage 1 ganglioids increased electrical firing rates as detected by microelectrode arrays (MEAs) (Figure 1L, Figures 9A and B) and led to increased cFOS expression compared to unstimulated enteric ganglioids (Figure 9C). Thus, stage 1 and stage 2 enteric neurons are functional and continue to mature over time.

[0218] We next investigated the transcriptional differences, lineage relationships, and functional properties of stage 1 and stage 2 enteric ganglioids. Many genes, including transcription factors, neurotransmitter receptors, neuropeptide receptors, cytokines and their receptors, secreted signaling ligands and their receptors, and surface markers, were exclusively expressed by each population of stage 1 and stage 2 enteric ganglioids (detected in more than 25% of cells in a single cluster) (Figure 10A-P). Other genes in these categories were not exclusively expressed but showed differential expression between cell types (Figure 11A-H, Figure S5A-H). Data in Figure S5 are not shown but describe the expression profiles of selected gene categories in stage 2 enteric ganglioid cell types. Data in S5 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. To determine the shared lineage between stage 1 and stage 2 enteric ganglioids, we performed a lineage analysis based on module scoring. Transcriptional signatures were highly conserved between stage 1 and stage 2 enteric neurons and mesenchymal cells (Figure 1M). Interestingly, the glial population was most transcriptionally similar to the two progenitor populations from stage 1 (Figure 1M). By generating a similarity-weighted non-negative embedding (Wu et al., 2018) by projecting stage 2 ganglioid cells onto the stage 1 SWNE (SWNE), we confirmed these transcriptional similarities between stage 1 and stage 2 enteric ganglioid cell types (Figure 1N). Stage 2 cell types mapped to similar SWNE spatial locations to matched stage 1 cell types, suggesting similar expression patterns and lineage continuity (Figure 1N).

[0219] To compare cellular diversity between our 2D ENS cultures and intestinal ganglioids, we performed scRNA-seq on 2D cultures at stage 2. Similar to 3D ganglioids, clustering and annotation by expression of key marker genes revealed enteric neurons, glia, epithelial cells, and mesenchymal cells, as well as two unknown populations (Figures S6A and B). Although the data in Figure S6 is not shown, we will describe a comparison between stage 2 intestinal ganglioids and 2D ENS cultures. The data in S6 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. Projection of 2D cells into the 3D ganglioid SWNE space showed conserved expression patterns between 2D and 3D enteric neurons, glia, mesenchymal cells, and unknown cluster 1 (Figure S6C). These observations were confirmed by calculating the Spearman correlation of expression of 3000 shared variably expressed genes (Seurat anchor features) between stage 2 ganglioside and 2D ENS cultured cell types (Figure S6D). Importantly, 2D and 3D enteric neurons and glia showed high correlations of 0.83 and 0.76, respectively (Figure S6D). In addition, the mesenchymal and unknown 1 clusters showed high correlations (0.82 and 0.80, respectively), while the epithelial cluster showed a modest correlation of 0.29 (Figure S6D). Interestingly, the 2D specific unknown cluster 2 showed a moderate correlation to the glial cluster (Figure S6D). Taken together, these data indicate that enteric neurons and glia generated by the new 3D differentiation format are highly metabolically similar to their 2D counterparts, while off-target / contaminating cell types may change in composition and transcriptional identity between the two formats.

[0220] hPSC-derived gut ganglioids recapitulate neuronal diversity in the human ENS Recent characterization of human and mouse primary enteric neurons at single-cell resolution has revealed many transcriptionally distinct clusters of enteric neurons (Drokhlyansky et al., 2020, Morarach et al., 2021). To characterize the diversity of our hPSC-derived enteric neurons, we subclustered neuronal populations in stage 1 and 2 gangliosides. We identified eight transcriptionally distinct neuronal subtypes (EN1-8) in stage 1 enteric neurons (Figure 2A, Figures S7A and B). The data in Figure S7 is not shown, but describes the profiling of enteric neuron subtypes present in stage 1 and 2 enteric gangliosides. The data in S7 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. Module scoring revealed that EN1 and EN6 showed higher similarity with enteric crestsphere ENCs potentially representing earlier stage neuronal populations (Fig. S7C). For stage 2 enteric neurons, subclustering analysis similarly revealed eight distinct subtypes of enteric neurons (EN1'-8') (Fig. 2B, Fig. S7D and E). Comprehensive analysis of functionally and technically relevant gene categories revealed transcription factors, neuropeptides and their receptors, neurotransmitter receptors, cytokines and their receptors, secreted signaling ligands and their receptors, and surface markers exclusively expressed by enteric neuron subtypes in stages 1 and 2 (Fig. S8A-Q). Data in Fig. S8 are not shown, but describe the identification of cluster-specific markers by gene categories in enteric ganglioid neuron subtypes in stages 1 and 2.The data for S8 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. Many genes in these categories were not exclusively expressed, but showed differential expression between enteric neuron subtypes (Figures S9A-J, Figures S10A-J). The data for Figures S9 and S10 are not shown, but S9 describes the expression profile of selected gene categories in stage 1 enteric ganglioid neuron subtypes, and S10 describes the expression profile of selected gene categories in stage 2 enteric ganglioid neuron subtypes. The data for S9 and S10 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. This implies a remarkable functional diversity of cell types in differentiated ganglioids.

[0221] Next, we determined the lineage similarity between stage 1 and stage 2 enteric neuron subtypes. Module scoring revealed the highest transcriptional similarity between EN1 and EN2' (Figure 12A). Other stage 2 enteric neuron subtypes shared minor transcriptional similarity with multiple stage 1 enteric neuron subtypes (Figure 12A). To confirm this observation, we projected stage 2 enteric neurons into stage 1 enteric neuron SWNE space. Consistent with this, many stage 1 and 2 enteric neurons showed similar expression patterns in SWNE space based on the regional overlap of EN1 and EN2', EN2 and 6 with EN3', EN4 and 8 with EN1' and 7', and EN7 with EN6' (Figure 2C). Interestingly, stage 2 enteric neurons had very little overlap with stage 1 EN3 and 5, suggesting that they may be transient neuron subtypes (Figure 2C).

[0222] To compare neuronal diversity between 2D cultures and ganglioids, a parallel subclustering analysis of 2D stage 2 enteric neurons was performed (Figure 12B). 2D enteric neurons clustered into five distinct enteric neuron populations (Figure 12B and C). Module scoring revealed that all ganglioid neuron signatures were present in 2D enteric neurons, but EN3'-5' and EN7' and 8' clustered together within the 2D enteric neuron dataset (Figure 12D and E). These results were further supported by Spearman correlation analysis of 3000 anchor features shared between 2D and ganglioid enteric neurons, which revealed a positive correlation between 2D EN3'-5' and ganglioid EN3' and EN5', as well as 2D EN7' / 8' and ganglioid EN7' (Figure 12F).

[0223] To verify that hPSC-derived gut ganglioids recapitulate in vivo ENS biology, we compared our stage 1 and stage 2 ganglioids to a previously published snRNA-seq dataset of primary human colon by Aviv Regev and Colleagues (Drokhlyansky et al., 2020) (Figure 12G). Notably, modular scoring of the ganglioid cell type signature in relevant primary cell types showed that in vitro and in vivo gut neurons were highly similar (Figure 2D). Similarly, correlating the expression of 3000 anchor features shared between the three datasets showed Spearman correlation values ​​of 0.89 and 0.87 between primary neurons and stage 1 and 2 gut neurons, respectively (Figure 12H, top). Furthermore, module scoring and Spearman correlation analysis revealed that stage 1 and 2 enteric neuron subtypes represent transcriptional signatures of primary neuron subtypes from all neuronal classes (Figure 2E, bottom of Figure S12H). These data indicate that our ganglioids capture the diversity of neuronal transcriptional identity in the human ENS.

[0224] Another important component of the identity of a enteric neuron is its location within the myenteric or submucosal plexus. To generate myenteric and submucosal gene signatures, we utilized metadata associated with human samples sequenced by Drokhliansky et al., which indicates the tissue layer from which each sample was collected. Modular scoring of primary enteric neuron subtypes using these plexus gene modules found that the tissue layer signatures were mutually exclusive, with each neuron subtype having a positive score for either one module or the other (Figure 12I, left). Interestingly, this analysis suggests that the PEMN, PIMN, PSN, and PSVN categories include both myenteric and submucosal subtypes. However, both PIN subtypes scored positive for the submucosal signature (Figure 12I, left). Modular scoring of our stage 1 and 2 enteric neurons suggests that our gangliosides generate both myenteric and submucosal neurons, largely recapitulating the mutual exclusivity of these signatures (FIG. 12I, middle and right).

[0225] The identity of enteric neurons is often described based on their neurochemical properties, including nitrergic, cholinergic, glutamatergic, catecholaminergic, GABAergic, or serotonergic. First, we confirmed the presence of neurons with various neurochemical characteristics in our stage 1 and stage 2 ganglioids by immunostaining (Figure 2F). These neurochemical markers were consistently expressed in both two-dimensional and ganglioid culture formats (Figure 2G). Interestingly, our transcriptional analysis of both stages showed that multiple enteric neuron subtypes express the same neurotransmitter markers (Figure S9B, Figure S10B). Data in Figure S9 are not shown, but describe the expression profiles of selected gene categories in stage 1 enteric ganglioid neuron subtypes. The data for S9 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. For example, neurons in EN4, 5, and 8 neurons expressed NOS1, an enzyme that produces NO and a marker for nitrergic neurons. In addition, individual enteric neuron subtypes expressed markers for multiple neurotransmitters. For example, EN5 expressed the GABAergic markers NOS1 and GAD1, and EN8 expressed the cholinergic marker SLC5A7 and glutamatergic marker SLC17A6 (Figure S9B). Next, flow cytometry was performed to verify the co-expression of neurotransmitter markers within neuronal subclusters. As proof of principle, we used two surface markers, CCR6 and GYPB, that were specifically expressed in EN8 at stage 1 (fig. S9J), to label this subcluster and express serotonin. + , CHAT + , GABA + , and NOS1 + The proportion of enteric neurons was quantified. All four neurotransmitter markers were expressed in the CCR6 + and GYPB +We confirmed that transcriptionally distinct enteric neuron subclusters detected in the population were not defined by a single neurotransmitter identity (Figure 2H), suggesting that a single neurotransmitter cannot serve as a specific marker for annotation of transcriptionally distinct enteric neuron subtypes and reaffirming the hypothesis that individual neurons can adopt multiple neurochemical identities.

[0226] To further explore this hypothesis at single-cell resolution, we designed a rigorous two-step approach to define the neurochemical identity of enteric neurons. In the first step, we identified neurons that express characteristic rate limiting neurotransmitter synthesis enzymes (Figure 13A and B). In the second step, our module scored these neurons based on their expression of a curated list of neurotransmitter metabolic enzymes and transport proteins (Table S1, Figure 13C and D). Neurons that passed both steps were binned into specific classes of neurotransmitter identity (Figure 13E and F). For example, neurons were annotated as nitrergic if they expressed NOS1 and scored high for the NO metabolism and transport genes NOS1AP, ARG1 / 2, ASL, and ASS1. In both two and three dimensions, we found that all EN subtypes contained neurons from all neurotransmitter identity classes (Figure 13G-I). Moreover, at the single-cell level, many neurons were equipped to synthesize multiple neurotransmitters (FIGS. 13A-F).

[0227] [Table S1]

[0228] A crucial aspect of the function of enteric neurons is their ability to sense and respond to specific neurotransmitters released by other neurons. As a first step to map the neuronal communication network in the enteric ganglioside, we profiled the expression of neurotransmitter receptor gene families in EN subtypes. Interestingly, neurons in stages 1 and 2 showed the same phenomenon, classified into one of three major neurotransmitter responsive groups: NO / serotonin / GABA / glutamate responsive, acetylcholine responsive, or dopamine responsive (Figure 13J and K). For example, in stage 1, neurons in EN3-5, 7, and 8 are predicted to be responsive to NO, serotonin, GABA, and glutamate, neurons in EN2 and 6 are predicted to be responsive to acetylcholine, and a subset of EN1 neurons is predicted to be responsive to dopamine (Figure 2A, Figure 13J). It is important to note that many individual neurotransmitter receptor genes within the same family were differentially expressed among our enteric neuron subtypes (Figure S9C, Figure S10C). For example, within the family of acetylcholine receptors expressed by stage 1 enteric neuron subtypes, CHRM1 and CHRNA10 are exclusively expressed by EN6, whereas CHRNB3 is exclusively expressed by EN5 (Figure S9C). These observations, together with the multiple neurotransmitter synthesis properties present in individual enteric neurons, highlight the complexity of the ENS circuitry in that a single neuron can synthesize and respond to multiple neurotransmitters. Furthermore, neurons can exhibit subtype-specific responses to neurotransmitters depending on the expression of receptor family members.

[0229] To validate that these complex neurochemical and transcriptional identities are physiologically relevant, we applied the same characterization criteria to both primary mouse and human ENS datasets (Drokhlyansky et al., 2020; Morarach et al., 2021) (Figure 14A and B). Interestingly, previously annotated subtypes were predicted to contain enteric neurons that synthesize multiple neurotransmitters, confirming our in vitro observations (Figure 14A and B). We then compared the overall abundance of neurons within each neurochemical class across each dataset, regardless of whether the neurons were predicted to synthesize multiple neurotransmitters (Figure 2I). We found that our ganglioids recapitulated the temporal characteristics of ENS development and maturation, such as the increase in NO neurons and the loss of catecholaminergic neurons over time (Baetge and Gershon, 1989; Baetge et al., 1990; Bergner et al., 2014; Lake and Heuckeroth, 2013; Obermayr et al., 2013) (Figure 2I). In addition, by connecting the individually predicted neurochemical identities, we found that primary enteric neurons also contain complex neurochemical identities, with neurons predicted to synthesize one, two, three, or more neurotransmitters (Figures 2J and K). We confirmed this by immunostaining primary human colon and identifying neurons that were positive for both GABA and NOS1, or both GABA and CHAT (Figure 2L). Interestingly, hPCS-derived enteric ganglioid neurons displayed relatively similar proportions of neurotransmitter complexity categories as primary neurons, and neurochemical complexity appears to change during mouse development (Figure 2K). Decomposition of neurons belonging to each single and dual neurochemical class confirms the presence of similar types of neurons across all datasets (Figure 2M).

[0230] hPSC-derived gut ganglioids recapitulate glial diversity in the human ENS Enteric glia play a key role in ENS physiology and disease, yet their molecular and functional characteristics remain elusive. We have shown that stage 2 enteric ganglioid and 2D ENS cultures contain glia (Figure 1G and H, Figure S6A). To characterize these hPSC-derived enteric glia and determine whether they recapitulate the transcriptional properties of primary enteric glia (Drokhlyansky et al., 2020; Morarach et al., 2021), we subclustered glial populations in enteric ganglioid and 2D ENS datasets (Figure 3A) and performed independent subclustering analyses of primary glia sequenced by Drokhlyansky et al. and Morarach et al. In two independent replicates, we identified four glial subtypes (glia 1-4) in enteric ganglioid (Figure 3B). Similarly, clustering of primary glia in Drokhlyansky et.al.'s human dataset showed four distinct subtypes (pGlia14) that differ from the six subtypes originally annotated by the authors (three shared and three patient-specific subtypes) (Figure 3C). Interestingly, visualizing the proportion of glial subtypes isolated from each patient sample in the primary human dataset suggests that the representation of glial subtypes varies from patient to patient, likely due to differences in sample collection (Figure 3D). Furthermore, we subclustered transcriptionally distinct glial lineages in our 2D ENS cultures and in the previously published mouse dataset (Figures 15A-D). All glial subtypes in 2D ENS cultures, gut ganglioids and primary datasets expressed classical glial markers (Figure 3E, Figure 15E). We confirmed this using immunofluorescence staining of S100 and GFAP in gut ganglioids and primary human colon tissue (Figure 3F). Interestingly, GFAP transcripts were undetectable in all glial populations, restricted to glia 1 in gut ganglioids, glia 1 and glia 4 in 2D ENS cultures, and low in primary human glial populations (Figure 3E, Figure S15E).Immunofluorescence staining for S100 and GFAP confirmed that these markers were not co-expressed in all glial cells (Figure 3G). To determine the similarity between glial subtypes between 3D and 2D subclusters, we next compared their transcriptional signatures. Module scoring and Spearman correlation revealed that a single 2D subtype shared the glia 2 and 3 signatures in 3D gut ganglioids (Figures 15F and G), confirming that all glial subtypes in gut ganglioids are present in 2D ENS cultures (Figure 15F). Module scoring showed that pGlia 1 was most transcriptionally similar to glia 1 and 4 subtypes, while pGlia 4 was most similar to glia 2 and 3 subtypes (Figure 3H). These data indicate that our 2D ENS cultures and gut ganglioids capture the glial diversity of the human ENS.

[0231] Surprisingly, we detected high levels of expression of the myelination markers PMP22, MPZ, and MBP in our cultures (Figure 3E). Similarly, MBP transcripts were present in all four human primary enteric glial cell subtypes, with pGlia 3 displaying MPZ expression (Figure 3E). Immunofluorescence staining confirmed expression of stage 2 ganglioside and myelin markers in human primary colonic tissue (Figure 3I). This is intriguing given the long-held assumption that myelination does not occur in the ENS.

[0232] Although S100B and PLP1 were expressed by all p-glial subtypes, they were only detected in glia 1 and glia 4 (Fig. 3E). On the other hand, MPZ and MBP were mainly expressed by the other two subtypes, glia 2 and 3 (Fig. 3E). The mutually exclusive expression patterns of some of the classical glial markers in our intestinal ganglioids prompted us to explore their developmental origins. We aimed to infer the lineage relationships between our stage 1 progenitor populations and glial subtypes. We found that glia 2 and 3 shared a similar signature with progenitor 1, while glia 1 and 4 were most similar to the progenitor 2 population (Fig. 3J). This suggests that unique intestinal progenitor populations are distinct MPZ subtypes. + / MBP + and GFAP + / AQP4 + / S100B + / PLP1 + This may suggest that they give rise to enteric glia (Figure 3E).

[0233] Given that enteric glial diversity has not been comprehensively transcriptionally profiled, we undertook a deeper characterization of our stage 2 enteric ganglioid glial population. Many transcription factors, neurotransmitter receptors, neuropeptide receptors, cytokines and their receptors, secreted signaling ligands and their receptors, and surface markers were exclusively expressed by each glial subtype (Figures S16A-H), whereas other genes in these categories were differentially expressed between subtypes (Figures S17A-H).

[0234] Furthermore, we developed an approach to compare the functional characteristics of glial subtypes in our gut ganglioids with primary glial subtypes. Gene set enrichment analysis (GSEA) was performed using biological functional Gene Ontology (GO) gene sets for the significantly upregulated gene list of each mature glial subtype. We then performed hierarchical clustering of glial subtypes across all datasets based on the normalized enrichment scores of all enriched GO terms present in at least one glial subtype. This analysis revealed three overarching classes of gut glia that were conserved between mouse and human (Figure 18A). Examination of the GO terms enriched in all glia of a particular class revealed diverse predicted functions for each class (Figure 3K). Class 1 glia are enriched for terms related to synaptic regulation and ion transport, while class 2 glia exhibit terms related to adhesion and immune functions. Both class 1 and 2 glia also contain terms related to epithelial and endothelial regulation. Interestingly, class 3 glia also exhibit terms related to synaptic regulation, but uniquely contain terms specific to sensory processes. We then generated myenteric and submucosal glial signatures based on differentially expressed genes of all primary glia isolated from each plexus. These signatures were used to predict the plexus identity of each glial subtype in the human dataset. Similar to neurons, scoring of primary human glial subtypes showed mutual exclusion of tissue layer signatures, with p-glia 1 and 2 scoring positive for submucosal and p-glia 3 and 4 scoring positive for myenteric (Figure 18B). Interestingly, p-glia 4 is located primarily in the myenteric signature (Figure 18C). Similar to neurons, scoring glia 1-4 with the plexus signature suggests that our cultures generate glia from both myenteric and submucosal layers (Figure 18D). Taken together, these data demonstrate that our hPSC-derived enteric glia recapitulate the transcriptional, functional, and topographical characteristics of primary human enteric glia.

[0235] Gut ganglioids enable comprehensive characterization of human NO neurons Gastrointestinal motility is directly controlled by excitatory and inhibitory motor neurons in the intestine. The majority of inhibitory neurons use NOS1 to synthesize the neurotransmitter NO, which induces relaxation in smooth muscle tissue (Bredt et al., 1990; Bult et al., 1990; Ward et al., 1992; Young et al., 1992). NO is also a key regulator of mucosal integrity and barrier function. Enteric NO neurons are of particular importance due to their involvement in a wide range of motility disorders. Selective loss and dysfunction of NO neurons is associated with muscle hypercontractility, which underlies many motility disorder conditions such as achalasia, gastroparesis, pseudo-obstruction, and colonic inertia (Bodi et al., 2019; Rivera et al., 2011).

[0236] Our hPSC-derived 2D ENS cultures and intestinal ganglioids contain diverse neuronal populations, including NO neurotransmitter identity. Access to this subtype of neurons prompted us to perform deeper characterization of their molecular and functional identity and develop assays to understand and modulate their activity. To facilitate a strategy to study NO neurons in vitro, we generated hESC NOS1::GFP lines by inserting a GFP cassette under the control of the endogenous NOS1 promoter using CRISPR / Cas9 knock-in technology (Figure 19A). Following our ENS induction protocol, we generated mature cultures with NO neurons co-expressing GFP and NOS1 from NOS1::GFP hESCs (Figures 19B and C). CD24 was used as a marker for neurons to identify FACS-purified NOS1::GFP neurons. + / CD24 + and NOS1::GFP - / CD24 +We performed bulk RNA sequencing (bulk RNA-seq) on NO neurons to identify genes differentially expressed in NO neurons (Figure 19D). In parallel, our snRNA-seq profiling of stage 1 gut ganglioids identified NO neurons by expression of the key marker gene NOS1, and selected metabolic and NO transport genes (Figures 13A-F, Figures 4A and B, Table S1). To determine molecular diversity within NO neurons, we performed further subclustering and identified five subtypes (NO1-5, Figures 4B and C, Figure 19E and F). In addition to NOS1, these clusters showed enrichment of other NO biosynthetic pathway genes, confirming their shared NO identity (Figure 4D). Our module did not score NO neuron enriched genes identified by bulk RNA-seq in our snRNA-seq clusters, revealing positive enrichment for all NO1-5, especially NO3, compared to other neurons further validating the reliability of the reporter line (Figure 19G). By combining bulk and snRNA-seq data, we assessed the transcriptional profile of several gene categories in NO neurons and revealed that many transcription factors, neuropeptides and their receptors, neurotransmitter receptors, cytokines and their receptors, secreted ligands and their receptors, and surface markers were differentially expressed in NO neurons compared to other neurons (Figure 20A-J). Bulk RNAseq data with greater sequencing depth confirmed many of the expression patterns observed with snRNA-seq. For example, neurotransmitter receptor GABRA3 and neuropeptide SCG2 were enriched, while secreted ligand SEMA3A and ligand receptor DDR1 were depleted in NO neurons (Figure 20C, H and I).

[0237] Similar to gut ganglioids, subclustering of NO neurons in the adult primary ENS dataset generated by (Drokhlyansky et al., 2020) identified five primary NO neuron subtypes (pNO1-5 Figure 4E and F). Module scoring revealed similarity between NO1-5 and pNO1-5 (Figure 4G). Similarly, Spearman correlation analysis based on the expression of 3000 anchor features showed correlations between NO2-5 and pNO3, as well as NO2, 4, 5 and pNO4 (Figure 4H). Deeper characterization of these subclusters revealed differential expression patterns of several NO neuron-specific features, such as serotonin receptors (HTR2C, HTR5A, and HTR3E), GABA receptors (GABRA2 and GABRG1), glutamate receptors (GRIN1, GRIN2A, GRM1, and GRM8), and the opioid receptor OPRM1 (Figure 4I and J, Table S2). For example, we checked the expression of surface markers, transcription factors, neuropeptides, and their receptors and identified genes that were specific to NO neurons but showed subcluster-specific expression patterns, such as POU5F1, CARTPT, HTR3E, NPFFR2, and BTLA (Figure 4I and J, Table S2). These novel markers of NO neuron subtypes can be utilized for the identification and further functional characterization of unique NO neurons, both in in vitro cultures and primary tissue samples. We further detected cholinergic, glutamatergic, catecholaminergic, GABAergic and serotonergic identities within NO neuron subtypes in both datasets (Figure 4K-M). These data indicate that enteric NO neurons are transcriptionally diverse and may have distinct functional characteristics.

[0238] [Table S2] TIFF2025502844000039.tif77168TIFF2025502844000040.tif198167TIFF2025502844000041.tif107167

[0239] To determine whether these transcriptional differences might reflect differences in tissue localization, we used previously generated neuron-specific myenteric and submucosal gene modules to identify their plexus identity. In the primary dataset, this analysis revealed one highly specific myenteric cluster (pNO2) and one highly specific submucosal cluster (pNO4) (Figure 4N, left). Our ganglioid neurons similarly showed alignment with either myenteric or submucosal identity (Figure 4N, right).

[0240] An hPSC-derived ENS model identifies regulators of NO neurons that promote colonic motility Given the important role of enteric NO neurons in gastrointestinal motility and their selective vulnerability in a wide range of congenital and acquired enteric neuropathy (Bodi et al., 2019; Rivera et al., 2011), there is great interest in establishing strategies to modulate their function. Factors that modulate NO neuron activity and increase NO release would facilitate the identification of potential drug targets for the treatment of enteric neuropathy. Therefore, we took advantage of our scalable ENS culture platform to screen for compounds that induce NO neuron activity.

[0241] We developed a screening strategy for NO neuron activity based on induction of cFOS expression as a readout. To evaluate cFOS as an accurate readout of neurochemically induced activity, cFOS flow cytometry analysis and MEA neuron firing measurements were performed in parallel in cultures treated with epinephrine, which is known to stimulate enteric neurons. Epinephrine induced neuronal cFOS expression and led to increased electrical firing of ganglioid neurons. This provides a scalable readout of activity suitable for high-throughput screens. (Figures S20A-C). The data in Figure S20 is not shown, but we describe the identification of enteric NO neuron modulators by functional high-throughput screening. The data in S20 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. First, we performed a cFos transduction screen, exposing NOS1::GFP gut ganglioside cells to a library of 582 neuromodulators (the Selleck neuronal signaling library™) and quantifying NO neuronal activity by measuring co-expression of cFOS and GFP ( Fig. 5A , fig. S20D). + We identified 20 compounds that increase the proportion of NO neurons in cells. To identify the mechanisms involved in cFOS expression in NO neurons, we compiled and categorized a list of target proteins and discovered multiple shared protein classes. Notably, these proteins converged on serotonin receptors, sodium channels, acetylcholine receptors, glutamate receptors, adrenergic receptors, histamine and opioid receptors, and dopamine receptors (Figure 5B).

[0242] In an independent functional screen, we established a high-throughput readout to assess NO neuronal activity. We utilized a commercially available kit that allows for NO detection in culture medium. Upon release into the medium, NO is spontaneously oxidized to nitrate. The kit uses nitrate reductase to convert nitrate to nitrite, which is then detected as a colored azo dye. 2D ENS cultures were incubated with a neuromodulator library and NO release was measured using calorimetry (Figure 5C). We identified 17 compounds that significantly enhanced NO concentrations in the supernatant with a z-score greater than 2.0 (Figure S20E). The neuromodulators that induced NO release in our ENS cultures were diverse but predicted common target protein classes, including serotonin receptors, sodium channels, acetylcholine receptors, glutamate receptors, adrenergic receptors, and opioid receptors (Figure 5D).

[0243] Interestingly, there was a high degree of similarity between predicted targets from cFOS induction and the NO release screen (Fig. 5B and D, Fig. S20F). These targets included receptors for neurotransmitters such as serotonin and dopamine. Modular scoring of neurotransmitter receptor gene families in our snRNA-seq data of hPSC-derived stage 1 enteric NO neurons confirmed that NO neurons express receptors for NO, serotonin, GABA, glutamate, acetylcholine, or dopamine extensively (Fig. 5E). Interestingly, compared to other neuronal subtypes, the stage 1 NO neuron cluster was enriched for all predicted hit targets (Fig. S20G). Notably, the NO3 cluster scored highly for expression of the majority of NO neuron modulator target classes (Fig. S20H). Profiling the expression of individual genes in each target protein category in ganglioids and primary human ENS revealed striking subtype-specific expression patterns among NO neurons. For example, GABA receptor genes were expressed primarily by the NO2, NO3, and pNO4 subtypes, whereas acetylcholine receptor gene expression was not specific to any particular subtype (Figure 5F).

[0244] We then selected a subset of hits representing different target classes, prioritizing FDA-approved compounds for follow-up analysis (Figure 5K, Figure S20F). For selected compounds, we performed a more comprehensive and integrated target analysis by combining reported experimental data (Combined DB) and computational methods (SEA, Carlsbad, Dinies, Swisstarget, Superdrug, Pubchem Bioassays, Figure 5G). We then tested the effect of these compounds on colonic motility in organ bath assays (Figure 5H). In these assays, we maintained excised segments of mouse colon in physiological buffer to study motility patterns using video recordings. In a first experiment, we tested the effect of all selected drug candidates on mouse colonic motility, ex vivo (Figure 5I). In each experiment, untreated control and drug-treated colon samples were tested in parallel during five consecutive 10-min acquisitions. Instead of analyzing the generally variable fecal output for each acquisition, we performed a more sophisticated contractile analysis by generating spatiotemporal maps from the video data based on the change in colon diameter over time, which were used to calculate the velocities of colonic migratory motor complexes (CMMCs) and slow waves (SWs). CMMCs are regular propulsive contractions initiated by the ENS, whereas SWs are mediated through the pacemaking activity of interstitial cells of Cajal (Barajas-Lopez and Huizinga, 1989; Burns et al., 1996; Fida et al., 1997; Lyster et al., 1995; Smith et al., 1987). To track the dynamics of CMMC and SW events during each acquisition, we generated cumulative percentage graphs (data not shown) and calculated the intervals at the 75th percentile (Figure 5J, data not shown). Compounds that showed promising effects on lowering the CMMC interval compared to the untreated condition were selected for follow-up evaluation (i.e., aripiprazole, dexmedetomidine, matrine, MPEP) (Figure 5J and data not shown).To assess whether the compounds mediated their effects on CMMC by modulating NO release, sequential drug treatments were performed in the presence and absence of the NOS1 inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Each experiment consisted of four 6-min acquisitions for five independent pairs of samples in control and drug-treated groups (Figure 5K). Of the drugs tested, the adrenergic receptor agonist dexmedetomidine reduced CMMC intervals in four of five colonic samples, but had no effect when colons were treated simultaneously with dexmedetomidine + L-NAME (Figure 5L, data not shown). SW was not affected by drug treatment (data not shown). In addition to CMMC, the effects of the compounds on colonic motility were quantified by annotating and measuring anterograde contractile events detected in the spatiotemporal maps. These events were termed "longitudinal muscle contractile events" (LCEs) and are highlighted by representative arrows in Figure 5K. In dexmedetomidine-treated colons, we observed a decrease in the total number of LCEs (Figure 5M) and a trend toward an increase in their average duration (Figure 5N) in all five replicates. These effects were reversed after drug removal and blocked by L-NAME cotreatment (Figures 5M and N). These results provide a blueprint for leveraging in vitro human ENS models to uncover mechanisms regulating gut motility that can identify therapies targeting specific ENS populations.

[0245] High-throughput small molecule screen reveals PDGFR inhibition as a driver of NO neuronal induction To evaluate the potential of hPSC-derived cultures to model human ENS development, we set out to define the mechanisms of NO neuron specification in vitro. Searching for pathways that regulate NO neuron differentiation, we performed a high-throughput small molecule screen. Identifying distinct pathways and chemical modulators that promote NO neuron induction can provide insights into NO neuron development and provide strategies for the induction of NO neuron-enriched ENS cultures. To identify compounds that induce NO neuron differentiation, we treated enteroclastospheres with 1694 compounds from the Selleck inhibitor library™ and induced NOS1 + We identified 12 hit compounds that increased the proportion of neurons by at least 8-fold (Figure 6A, Figure S25A and B). Although the data in Figure S25 is not shown, we describe a small molecule high-throughput screen to identify compounds that enrich NO neurons in hESC-derived ENS cultures. The data in S25 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. To clarify the mechanism by which these hit compounds enhanced NO neuron induction, we performed a target prediction analysis by combining reported experimental data (Joint DB) and computational methods (SEA, Carlsbad, Dinies, Swisstarget, Superdrug, Pubchem Bioassays) (Figure 6B). After clustering the predicted protein targets, common patterns emerged for a subset of compounds. For example, PP121, ibrutinib, afatinib, and AMG-458 were all predicted to interact with EGFR, ERBB, MAP, and TEC family kinases, among others (Figure 6B). For follow-up analysis, this subset of compounds showed the highest %NOS1 + We chose to focus on PP121, a hit with a fold increase. PP121 showed a dose-dependent effect on NO neuron induction efficiency as measured by flow cytometry (Figure S25C). To find the most effective treatment window for PP121-induced NO neuron induction, we treated differentiated cultures for 5 days at various time points. Measurement of GFP signal in stage 1 NOS1::GFP intestinal gangliosides showed the highest induction efficiency for cells treated between days 15 and 20 (Figure 6C and D, Figure S25D).

[0246] For the enrichment protocol to be reliable, it was important to ensure that PP121 treatment did not alter the identity of our cell types. To compare PP121-treated stage 1 intestinal ganglioids with untreated ones at single-cell resolution, snRNA-seq was performed and both datasets were combined. This analysis revealed that all cell types were represented in both conditions (Figures 6E and E, Figure S26A). The data in Figure S26 is not shown, but describes that PP121 treatment enriches NO neurons without affecting their overall cellular diversity. The data in S26 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. Importantly, comparison of the average expression of all genes for matched PP121-treated and untreated cell types showed highly similar transcriptomes (R2 correlation > 0.91), indicating that PP121 treatment did not alter the transcriptional identity of the cell types (Figure S26B). Interestingly, subclustering of the combined control and PP121-treated neurons revealed nine neuronal subtypes, EN A-I (Figure 6G). The PP121-treated dataset subtypes showed high transcriptional similarity with EN1-8 in the control-only dataset (Figure 6H). EN cluster I, consisting mostly of PP121-treated cells and a few control cells, showed moderate transcriptional similarity with control-only EN cluster 4, suggesting that this neuronal subtype, although present, is rare in control cultures clustering these neurons with the most similar subtype, EN4 (Figure 6H, Figure S26C). Along with EN I, which was approximately 25% nitrergic, PP121 treatment also enriched the cultures for neuronal subtypes EN D and H (nearly 50% and 25% nitrergic, respectively), whereas EN A and G were less represented ( Fig. 6I ; fig. S26C).Again, despite changes in subtype abundance, control and PP121-treated neurons of the same subtype displayed similar transcriptomes (R2 correlation > 0.88) (fig. S26D). Further subclustering of combined control and PP121-treated nitrergic neurons revealed enrichment for nitrergic B (most similar to control-only nitrergic 2) and the rarer population nitrergic C (most similar to control-only nitrergic 3) (Fig. 6J and K, fig. S26E). Transcriptome comparison again showed highly similar gene expression of control and PP121-treated nitrergic neurons of the same subtype (R2 correlation > 0.7), with the highest variance between nitrergic C neurons, likely due to the low number of neurons in this cluster (fig. S26F). Taken together, the data suggest that early ganglioid treatment with PP121 induces changes in the abundance of neuronal subtypes typically seen in untreated cultures, without affecting the gene expression patterns of the resulting subtypes.

[0247] The ability to purify enteric NO neurons is of great interest, especially for applications such as cell therapy. Access to the NOS1::GFP reporter line, and the ability to direct differentiation into NO neurons using PP121, allowed us to search for FACS-compatible surface markers for these cells. We screened a panel of 242 antibodies for human cell surface molecules (BD Lyoplate) and measured GFP and surface antigen expression signals by flow cytometry (Figure S27A). The data in Figure S27 is not shown, but we do describe that the huma surface marker antibody screen identifies NO neuron-specific surface markers. The data in S27 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. We identified 27 antibodies that stained at least 50% of NOS1 neurons (GFP + %CD in the population + GFP + , top of Figure S27B). The data in Figure S27 are not shown, but we describe a human surface marker antibody screen to identify NO neuron-specific surface markers. The data in S27 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. To identify the most specific candidates among these hits, we performed a 3-fold increase in CD4+ expression of 5-HT4+ β-lactamase inhibitors (5-HT4+ β-lactamase inhibitors) with ... + GFP + CD +We looked for antibodies with staining ratios to CD47 (Figure S27B, bottom). CD47, CD49e, CD59, CD90, and CD181 fulfilled both criteria (Figure S27C). We further confirmed the expression and enrichment of CD47, CD49e, CD59, and CD90 in stage 1 ganglioid and NO neuron clusters in human primary snRNA-seq datasets (Figures S27D and E). As an example, we further confirmed the localization of CD47 in NO neurons of human primary colonic myenteric ganglia using immunohistochemistry (Figure S27F). In addition to identifying antibodies that specifically enrich for NO neurons, we found 12 antibodies that stained more than 70% of ganglioid cells and could serve as pan-enteric neuron surface markers (CD24, CD45RA, CD57, CD63, CD71, CD121b, CD147, CD164, CD184, CD193, CD243, and CD275) (fig. S27G), confirming enriched expression of CD24 in our enteric neurons (snRNA-seq data) and primary human colonic myenteric ganglions (figs. S27H and I).

[0248] To determine the mechanism by which PP121 induced NO neuronal enrichment in gangliosides, a combination of pharmacological and genetic approaches was used. PP121 is a multitargeted receptor tyrosine kinase (RTK) inhibitor with known inhibitory activity against PDGFR, VEGFR, and EGFR (Apsel et al., 2008). Our CrestSphere snRNA-seq analysis confirmed expression of PDGFRA, PDGFRB, ERBB2, and ERBB3, whereas no mRNA for VEGFR could be detected (Figure 6L). The efficiency of induction of NO neurons in response to PDGF (PDGFR agonist), sunitinib (PDGFR and VEGFR antagonist), NRG1 (ERBB agonist), and sapitinib (ERBB antagonist) was evaluated (Figure 6M). NRG1 and sapitinib had no significant effect on NO neuron induction, whereas treatment with PDGF and sunitinib resulted in lower and higher NO neuron ratios, respectively (Figure 6N). To genetically confirm the role of PDGFR signaling in NO neuron induction, we used CRISPR-Cas9 to knock out PDGFRA and PDGFRB in our intestinal crestspheres and analyzed the percentage of NO neurons in stage 1 gangliosides. In both PDGFRA and PDGFRB knockout cultures, NO neurons were enriched, further confirming the mechanism of action of PP121 (Figures 6O and P).

[0249] hESC-derived NOS1 neurons express Nos1 - / - Engraftment in mouse colon Developing an experimental system to study the human ENS in vivo opens a wide range of basic science and clinical opportunities. For example, human ENS xenografts allow to study human neuronal circuits in vivo and to investigate ENS-CNS and ENS-immune system-microbiota communication. They also provide a platform for disease modeling and drug development. In addition, the limited regenerative capacity of the ENS highlights the importance of developing cell therapy approaches to replace lost populations of neurons. There are currently no clinical interventions to replace damaged or lost neurons caused by inherited and acquired ENS pathologies such as Hirschsprung's disease and diabetes. We have previously shown that hPSC-derived ENC precursors can be successfully engrafted in vivo (Fattahi et al., 2016). McCann et al. also showed that transplantation of ex vivo cultured mouse intestinal neurospheres can rescue gastrointestinal motility defects in Nos1- / - mice (McCann et al., 2017). However, these neurospheres are heterogeneous populations that contain only a small percentage of NO neurons. In addition, obtaining sufficient numbers of neurospheres from human primary tissues is an important limitation for ultimate regenerative applications. Compared to ENC precursors, transplantation of mature neurons provides a postmitotic source of cells with a low clinical risk of tumor formation. Obtaining highly enriched NO neuron cultures prompted us to evaluate the transplantation potential of intestinal gangliosides. PP121-treated intestinal gangliosides were injected into the distal colonic wall of immunodeficient Nos1- / - (B6.129S4-Nos1tm1Plh / J) mice. Eight weeks after surgery, animals were euthanized and colonic longitudinal myenteric plexus (LMMP) preparations were evaluated by fluorescence microscopy (Figure 7A). Transplanted cells were distinguished by expression of the human cytoplasmic marker SC121. Notably, we observed a notable number of SC121+ cells incorporated along the length of the colon (Figure 7B). Transplanted cells were detected inside and outside the intermyelin ganglia, and many expressed NOS1, confirming the fate of NO ( Fig. 7C ; fig. S28).Data in Figure S28 are not shown, but describe the engraftment of h-ESC-derived enteric ganglioids in adult mouse colon. The data in S28 are disclosed in Majd et al., “hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function”, bioRxiv, posted on January 3, 2022, and are incorporated by reference in their entirety. In addition to clinically significant cell therapy applications, the developed human enteric ganglioid xenografts provide a previously unattainable opportunity to understand the development, physiology, and pathophysiology of the human ENS in vivo.

[0250] Consideration The ENS is a complex network of enteric neurons and glia that controls all aspects of gastrointestinal physiology (Long-Smith et al., 2020; Schneider et al., 2019; Yoo and Mazmanian, 2017) and plays a central role in the initiation and progression of enteric neuropathies and diseases of the gut-brain axis (Camilleri, 2021; Niesler et al., 2021; Pesce et al., 2018). Nevertheless, understanding of the ENS has been disproportionately affected by long-standing technical challenges. Gaining access to the human ENS requires invasive biopsies or surgery because these cells constitute only 1% of the intestinal tissue (Drokhlyansky et al., 2020) and reside deep within the muscle and mucosal layers. Moreover, large-scale isolation and purification of ENS cells is extremely challenging. The majority of neuronal cell bodies are located within the ganglia, with fragile processes extending to other parts of the intestinal tissue. In addition, there are no well-established surface markers for FACS-based purification of specific subtypes of enteric neurons or glia. Moreover, animal models do not fully recapitulate human ENS (patho)physiology. For example, rodents tolerate well mutations that cause life-threatening enteric neuropathy in humans (Bondurand and Southard-Smith, 2016). Here, we propose hPSC-derived ENS cultures as an alternative model that overcomes many of these challenges and allows for significant advances in the field of enteric neurobiology.

[0251] We thoroughly compared the composition of our hPSC-derived ENS platform with recently published primary ENS datasets by snRNA-seq (Drokhlyansky et al., 2020; Morarach et al., 2021), revealing diverse neuronal and glial subtypes that resemble the cellular diversity seen in vivo. For example, hPSC-derived enteric neurons express key markers and receptors for numerous hormones, neuropeptides, and neurotransmitters known to be present in primary human ENS, supporting the reliability and utility of our hPSC-based platform for modeling the human ENS. Clustering and further subclustering of our dataset identified novel markers for each subtype that offer opportunities for immunochemistry-based detection, purification, genetic manipulation, and reporter line development. Furthermore, studies of primary and hPSC-derived neurons shed light on an array of neurons with multiple neurochemical identities. These neurochemically diverse neurons significantly exceed the traditional and widely accepted belief that one neuron expresses one neurotransmitter. Although there are immunohistochemistry-based reports of enteric neurons with multiple neurochemical identities (Qu et al., 2008), a comprehensive characterization has not been performed to date. The level of complexity revealed here could not be easily recognized and characterized by common lower-throughput staining-based detection methods. This will be of great scientific and medical value, advancing our understanding of human ENS circuitry and autonomy, and aiding in the development of more targeted therapies with fewer side effects.

[0252] The autonomy of the ENS and its ability to independently perform diverse tasks depends on the diversification of its neuronal and glial components through an elaborate fate specification process. The precise developmental patterns that drive the differentiation of vagal neural crest into ENS precursors, which then mature into a myriad of neuronal and glial subtypes, remain elusive, especially in humans. Studying these complex developmental patterns is extremely challenging due to the transient nature of many developmental states, technical limitations in isolating tissue, and differences between species. Leveraging a stepwise ENS induction system, we generated high-resolution time maps that reveal the complex developmental programs that give rise to enteric neurons and glia. We initiated in vitro differentiation by inducing vagal and enteric neural crests, which develop into enteric crestspheres and further differentiate into enteric neurons and glia. Interestingly, in long-term cultures, we observed the emergence of enteric glial subclasses that resemble adult primary glia. This resembles the developmental timeline in the CNS, where gliogenesis is followed by neurogenesis. Given the complexity of processes affecting ENS development, it is not surprising that defects at any developmental stage lead to enteric neuropathies such as Hirschsprung's disease (Lake and Heuckeroth, 2013; Rao and Gershon, 2018). Investigating broad and cell type-specific developmental programs in the ENS offers opportunities to understand developmental neuropathies and facilitate the directed induction of disease-relevant cell types.

[0253] An unparalleled advantage of hPSC-derived cultures is their scalability. This is especially important when the desired cell type is rare and has very limited regenerative and proliferation capabilities, such as neural tissue. Our ENS culture platform has repeatedly proven reliable in providing a scalable source of ENS cell types that are compatible with applications that are otherwise very difficult to implement, such as high-throughput screening. In particular, we used our 2D ENS cultures to screen thousands of inhibitors to identify compounds that direct differentiation toward clinically valuable NO neurons. Examination of the mechanism of action of our top hits revealed pathways that are important in NO neuron fate specification. Using a combination of pharmacological and genetic approaches, we discovered that one such pathway, PDGFR signaling, contributes to the induction of NO neurons, highlighting the remarkable potential of hPSC-based platforms to uncover developmental mechanisms.

[0254] Our two- and three-dimensional ENS cultures are electrically active. Access to functional enteric neurons is highly advantageous as it facilitates a fundamental understanding of neuronal circuits and cellular electrophysiology. Furthermore, identifying cell type-specific neurochemical and functional characteristics is invaluable in drug development as it allows for the identification of targeted neuromodulators and the prediction of potential side effects due to direct and indirect neurochemical mechanisms. As a proof of concept, we developed a functional screening platform to uncover candidate drugs that specifically modulate the activity of NO neurons. Interestingly, our hit compounds generally target adrenergic, cholinergic and serotonergic receptors, and sodium channels. Notably, these targets are over-represented in NO neurons, highlighting the specificity of these compounds and their potential for further therapeutic development for gastrointestinal indications. By testing a subset of these neuromodulators, we further demonstrated that these candidate drugs can affect colonic motility patterns in an in vivo organ bath assay. This is the first example of identifying a candidate drug for regulating gut motility by targeting a specific enteric neuron subtype. These findings demonstrate the reliability, robustness, and scalability of the hPSC-derived ENS model.

[0255] Derivation of intestinal ganglioids from hPSCs provides a scalable source of human ENS tissue for regenerative applications. In addition, the development of human ENS xenografts paves the way for a wide range of basic science and clinical research. In the past two decades, developing cell-based therapies for enteric neuropathy has been a major area of ​​research (Alhawaj, 2021; Burns et al., 2016). However, achieving a scalable source of human ENS cells suitable for transplantation is difficult. Here, we report the development of Nos1 xenografts, which are a type of xenograft that is capable of producing enteric gangliosides. - / -We provide proof-of-concept results on widespread engraftment of NO neurons in mice by transplanting ganglioids enriched for this neuronal subtype. Beyond cell therapy, these human ENS xenograft models provide new experimental systems for various purposes. First, they allow the study of the human ENS in vivo, facilitating the identification and development of therapeutic candidates with high specificity, efficacy and potency. Second, they allow the models to be used to study pathologies of the human ENS in vivo using strategies such as transplantation of ganglioids with specific mutations, ganglioids exposed to specific stressors, or ganglioids derived from patient iPSCs. Third, transplanting ganglioids at different stages of differentiation allows comprehensive studies on cell fate specification and maturation in the human ENS. Finally, human ENS xenografts provide a promising model to study crosstalk between the human ENS and local intestinal tissue, the CNS, and the microbiota.

[0256] Enteric neuropathy can affect any part of the gastrointestinal tract at any stage of life and represents some of the most challenging clinical disorders for which there is no effective therapy. Enteric neuropathy can result from congenital defects affecting ENS development, can occur in response to changes in the tissue environment (toxins, microbes, immune system), or can appear secondary to systemic diseases such as diabetes and obesity (Camilleri et al., 2011; Niesler et al., 2021; Yarandi and Srinivasan, 2014). The lack of efficient therapies stems from an inadequate understanding of ENS development, cellular architecture, and function. Our hPSC-derived 2D ENS cultures and enteric gangliosides provide a human-based platform to model enteric neuropathy. Genetic manipulation of neurons, glia, and their specific subtypes at different stages of development is now possible. The effects of genetic background (healthy and patient-derived iPSCs) (Lai et al., 2017) and specific mutations, as well as environmental stressors, infectious agents, and metabolic toxins, can now be studied through targeted and unbiased approaches. Furthermore, functional and fully characterized ENS cultures pave the way to investigate further layers of complexity represented in gut physiology, such as crosstalk with surrounding and distant tissues. For example, motor function can be studied by developing cocultures with smooth muscle cells, ENS-immune system communication can be investigated by setting up cocultures with immune cells, and ENS-gut microbiota interactions can be examined by exposing ENS cells to by-products of the gut microbiota.

[0257] Our hPSC differentiation strategy provides robust 2D and 3D ENS culture systems that enable developmental, molecular and functional mapping of the human ENS. We provide important insights into the physiological properties of NO neurons and identify the development programs required to identify this clinically relevant enteric neuron subtype. These models open new avenues for drug discovery and regenerative medicine and provide a new framework for basic research into enteric neurobiology.

[0258] method Cultivation and maintenance of undifferentiated human stem cells Human embryonic stem cell (hESC) line H9 (WAe009-A and reporter-expressing derivatives hSYN::ChrR2-EYFP, NOS1::GFP) and induced pluripotent stem cell (hiPSC) line WTC-11 (UCSFi001-A) were plated on geltrex™ coated plates and maintained in chemically defined medium (E8) as previously described (Barber et al., 2019). Maintenance cultures were tested for mycoplasma every 30 days.

[0259] Enteric neural crest (ENC) induction When the monolayer culture of hPSCs reached approximately 70% confluency, the maintenance medium (E8) was aspirated and replaced with fresh neural crest induction medium A [essential 6 medium containing BMP4 (1 ng ml -1 We initiated a previously established 12-day neural crest (ENC) induction protocol (Barber et al., 2019, Fattahi et al., 2016) by replacing the medium A with medium B [SB431542 (10 μM) and CHIR99021 (1.5 μM)] in Essential 6 medium (D0). We then fed the cultures with neural crest induction medium B [medium B with retinoic acid (1 μM)] on D2 and D4, the days of ENC induction, and medium C [medium B with retinoic acid (1 μM)] on D6, D8, and D10. We then formed ENC crest spheres between D12 and D15 to facilitate the selection of ENC lineages and prevent contamination of our cultures. At that time, ENC induction at D12 was performed by removing the neural crest medium C and incubating with Accutase (30 min, 37 °C, 5% CO 2 ) was used to separate the ENC monolayer. After centrifugation of the samples at 290 × g for 1 min, the ENC cells were cultured in NC-C medium [neurobasal medium supplemented with FGF2 (10 ng ml -1 ), CHIR99021 (3 μM), N2 supplement (10 μl ml -1 ), B27 supplement (20 μl ml -1 ), glutagro (10 μl ml -1), and MEM NEAA (10 μl ml -1 )] and transferred to ultra-low attachment plates to form free-floating three-dimensional enterocrestospheres. On D14, when free-floating enterocrestospheres could be observed, they were gently collected in the center of each well using a vortex motion. The old medium was then carefully aspirated from the periphery of each well without removing the crestospheres. After adding fresh NC-C medium, the cultures were incubated for 24 h (37 °C and 5% CO) before the enteric neuron induction stage. 2 ) incubated.

[0260] Enteric neuron induction from enteric neural crest On D15, using a vortex motion, the intestinal crestspheres were collected in the center of the well and the NC-C medium was removed using a P1000 micropipette with a slow swirling motion, avoiding the free-floating crestspheres. At this step, the protocol varied depending on the final desired culture layout (2D ENS cultures vs. 3D intestinal gangliosides). For 2D ENS cultures, after washing the intestinal crestspheres with PBS, Accutase (Stemcell Technologies, 07920) was added and the plate was incubated at 37 °C for 30 min to dissociate the crestspheres. The remaining spheroids were then cultured in ENC medium [Neurobasal medium, GDNF (10 ng ml -1 ), ascorbic acid (100 μM), N2 supplement (10 μl ml -1 ), B27 supplement (20 μl ml -1 ), glutagro (10 μl ml -1 ), and MEM NEAA (10 μl ml -1 The cells were disrupted by pipetting 500 ml of PBS (pH 7.5). The cells were spun down (2 min, 290 × g, 20–25 °C) and the supernatant was removed. The pellet was resuspended in ENC medium and the cells were plated on poly-L-ornithine (PO) / laminin / fibronectin (FN) plates at 1 cm 2For three-dimensional intestinal gangliosides, Accutase treatment was avoided and intestinal crestspheres were plated at 100,000 viable cells per well. For three-dimensional intestinal gangliosides, Accutase treatment was avoided and intestinal crestspheres were plated at 100,000 viable cells per well in the same volume of ENC medium [neurobasal medium containing GDNF (10 ng ml -1 ), ascorbic acid (100 μM), N2 supplement (10 μl ml -1 ), B27 supplement (20 μl ml -1 ), glutagro (10 μl ml -1 ), and MEM NEAA (10 μl ml -1 Continue feeding with ENC medium every other day until D30–D40, after which the frequency of feeding may be reduced to 1–2 times per week, but the volume of medium fed will be larger.

[0261] Immunofluorescence For immunofluorescence (IF) staining, cells were first fixed with 4% PFA in PBS (30 min at room temperature (RT) and then blocked with permeabilization buffer (PB) (Foxp3 / Transcription Factor Staining Buffer Set, 00-5523) and permeabilized for another 30 min at RT. After the fixation and permeabilization steps, cells were incubated in primary antibody solution overnight at 4°C, then washed three times with PB, followed by incubation with fluorophore-conjugated secondary antibodies at RT. Before imaging, stained cells were incubated with DAPI fluorescent nuclear stain and washed three more times. A list of antibodies and working dilutions is shown in Table S3.

[0262] [Table S3]

[0263] Preparation of intestinal ganglioid frozen sections hPSC-derived gangliosides were collected at stage 1 (days 37-50) and stage 2 (days 70-90), rinsed twice in PBS, fixed in 4% PFA (SCBT sc-281692) for 3 h, and then stored at 4°C for up to 6 months with 90% of the supernatant replaced with PBS. Gangliosides were treated with 5% sucrose (RPI Research Products 524060) in PBS for 10 min at room temperature, followed by 10% sucrose in PBS for 2 h at room temperature and 20% sucrose overnight at 4°C. Sucrose-treated ganglioids were placed in cryomolds (Tissue-Tek® Cryomold® medium, VWR 25608-924), all 20% sucrose was removed, and incubated in 2:1 20% sucrose:OCT (Tissue Plus OCT Compound Fisher HealthCare 5484) for 2 hours at room temperature before being snap frozen in ethanol / dry ice. 1220 μm sections were taken on a cryostat (Leica 3050S) attached to Superfrost® Plus Micro Slides, Premium (VWR 48311-703) and dried in a slide dryer at 42°C for up to 2 hours before being stored at -80°C for up to 1 year.

[0264] Preparation of paraffin-embedded human colon sections Human sigmoid colon tissue was received from the International Institute for the Advancement of Medicine (IIAM), which provides non-transplantable organs from Organ Procurement Organizations for biomedical research purposes. Colon tissue was obtained under sterile conditions, flushed with isotonic solution, immersed in organ transplantation solution, and transported to the laboratory on ice within 24 hours after death. Full-thickness tissue pieces (approximately 2 cm2) were fixed overnight (<24 hours) in 10% neutral buffered formalin (Cancer Diagnostics, FX1003). Samples were transferred to 70% ethanol prior to paraffin embedding (Leica ASP6025, tissue processor). Transverse tissue sections approximately 5 μM thick were cut on coated glass slides (Superfrost® Plus Micro Slide, VWR, 48311-703) and air-dried overnight. All of the following slide preparation steps were performed at room temperature. Slides with paraffin sections were washed three times in clean xylene substitute (Sigma A5597), then once each in 100% ethanol, 95% ethanol, and 70% ethanol. Slides were then washed under in-house deionized water for 5 minutes before being placed in 1x PBS for storage at 4°C for up to 4 weeks. Prior to staining, paraffin sections underwent antigen retrieval in either citrate buffer (Vector Laboratories Antigen Unmasking Solution H-3300) or TE buffer (Thermo 17890, pH 9.0 with 1M NaOH). Slides were incubated in the buffer at 95°C for 10 minutes using a Pelco BioWave Pro+ set at 400 watts.

[0265] Intestinal ganglioids: staining of frozen sections and paraffin-embedded human colon sections. All steps were performed at room temperature unless otherwise specified. Ganglioid cryosections and paraffin-embedded human normal colon sections were prepared as described above, then washed three times with PBS and blocked for 1–2 h in serum (10% donkey or 10% goat) containing 0.5% (v / v) Triton X-100 (VWR 0694). Slides were then incubated for 12–20 h at 4°C with primary antibodies diluted in serum (10% donkey or 10% goat) containing 0.1% Triton X-100. Slides were washed six times for 20 min each in PBS containing 0.1% Tween-20 (Sigma P1379) and incubated with Alexa Fluor-conjugated secondary antibodies for 1 h. The diluted secondary antibody solution was removed and replaced with 1.0 μg / mL DAPI in water for 10 min. Slides were washed six times for 20 min each in PBS containing 0.1% Tween-20 and coverslips were mounted with Fluoromount-G (Southern Biotech 0100-01). A list of antibodies and working dilutions is given in Table S3. Images were acquired on a Leica SP8 inverted confocal or on an Echo Revolve. For stitched images, Leica's LAS X tiling function or FIJI's Grid / Pairwise Stitching plugin (PMID 19346324) were used.

[0266] Two-photon fluorescence imaging Imaging experiments were performed on a custom-built upright two-photon microscope operated with μManager software (San Francisco, CA). The excitation source was a two-photon Coherent Chameleon Vision II laser operating at 760 nm (Coherent, Santa Clara, CA). Images were collected using an Olympus LWD 1.05 NA water immersion objective (Olympus, Tokyo Japan). DAPI was imaged using an emission filter (Chroma, Bellow Falls VT) that collected light from 380 nm to 420 nm, while the fluorescence emission of Alexa 568 was collected using a 565 nm to 635 nm filter (Chroma, Bellow Falls VT).

[0267] Spectral fluorescence imaging Images were taken with a Nikon AZ100M "Macro" laser scanning confocal configured with a long-range low-magnification lens. The microscope is equipped with standard 405 nm, 488 nm, 561 nm, and 640 nm laser lines and has a PMT detector with a detection range of 400-700 nm. An optical zoom factor of 2.1x was used to reduce signal falloff at the image edges, and a digital zoom factor of 1.873x was used to increase lateral resolution.

[0268] Flow cytometry For preparation of samples for flow cytometry analysis, cells were first treated with Accutase (Stemcell Technologies, 07920, for 30–60 min at 37 °C and 5% CO. 2) into a single cell suspension and then fixed and permeabilized using Fixation / Permeabilization Buffer (Foxp3 / Transcription Factor Staining Buffer Set, 00-5523). Cells were stained with primary and secondary antibodies as described above for immunofluorescence. Flow cytometry was performed using a BD LSRFortessa cell analyzer and data were analyzed using FlowjoTM (FlowJoTM Software Version 8.7). A list of antibodies and working dilutions is provided in Table S3.

[0269] Blue light activation of human synapsin::channelrhodopsin2-EYFP gut ganglioids Intestinal ganglioids were either exposed to blue light (100% laser intensity, 3 x 1 minute exposures with 30 second intervals, EVOS FL) or left in ambient light. Intestinal ganglioids were then incubated at 37°C for 45 minutes before dissociation, fixation and permeabilization for flow cytometry (see above). Cells were stained using antibodies against cFos (abcam, ab190289) and TUBB3 (Biolegend, 801202).

[0270] Bulk RNA-seq data analysis Total RNA was extracted using the PureLinkTM RNA Mini Kit. First strand cDNA was then synthesized with the Quantseq Forward Library Preparation Kit from Lexogen. Illumina-compatible RNA sequencing libraries were prepared on Quantseq, pooled, and sequenced on an Illumina Hiseq 4000 platform at the UCSF Center for Advanced Technology. UMIs were extracted from fastq files using umi_tools, and short, low-quality reads were removed using cutadapt. Reads were aligned against the human GENCODE v.34 reference genome using STAR aligner, and duplicate reads were collapsed using umi_tools. Gene-level counts were measured using HTSeq and compared using DESeq2.

[0271] Single-cell and single-nucleus RNA sequencing sample preparation and data collection All tubes and pipette tips used for cell harvest were pretreated with 1% BSA in 1x PBS. Cells were dissociated in Accutase (Stem Cell) at 37°C with end-to-end rotation for 10 min increments until a single cell suspension was obtained. Cells were washed with cell staining buffer (Biolegend) and stained with TotalSeq HTO antibody for 30 min on ice. Cells were washed twice in cell staining buffer and filtered through a 40 μm pipette tip strainer (BelArt). Cells were counted using trypan blue dye and a hemocytometer and pooled for sequencing. scRNA-seq libraries were prepared using Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics) with custom amplification of TotalSeq HTO sequences (Biolegend). Libraries were sequenced on an Illumina NovaSeq sequencer at the Center for Advanced Technologies (UCSF). A cell feature matrix was extracted using kallisto / bustools and demultiplexed using seurat.

[0272] Quality Control and Cell Filtration Datasets were analyzed using Seurat v4 in R v4.0.3 (Hao et al., 2021). The number of reads mapping to mitochondrial and ribosomal gene transcripts per cell was calculated using the "PercentageFeatureSet" function. Cells were identified as low quality and then removed independently for each dataset based on the number of unique features captured per cell, the number of UMIs captured per cell, and the percentage of reads mapping to mitochondrial transcripts per cell. Dataset-specific quality control metric cutoffs can be found in Table S4.

[0273] [Table S4]

[0274] Dimensionality reduction, clustering, and annotation Where applicable, biological replicate samples were first merged using the base R “merge” function. Count matrices were log-normalized with a scaling factor of 10,000 and 2,000 variable features were identified using the “vst” method. For the datasets specified in Table S5, count matrices of biological replicate samples were merged using the Seurat merge function with default parameters. Cell cycle duration was predicted using the “CellCycleScoring” function with Seurat’s S and G2M features provided in “cc.genes”. The variable feature set was scaled and centered and regressed on the following variables: nFeatures, nCounts, mitochondrial gene percentage, ribosomal gene percentage, S score and G2M score. Principal component analysis (PCA) was performed using default settings and uniform manifold approximation projection (UMAP) dimensionality reduction was performed using PCA reduction. Shared nearest neighbor (SNN) graphs were calculated using default settings and cell clustering was performed using the default Louvain algorithm. Quality control metrics were visualized for each cluster to identify and remove clusters of low-quality cells (less than average nFeatures or nCounts, and greater than average mitochondrial and ribosomal gene percentages) (Table S5). The above pipeline was run again on the datasets for sub-clustering analysis of enteric neural crest, enteric neurons, nitrergic neurons, and enteric glia after removing any low-quality cell clusters. The number of principal components used for UMAP reduction and SNN calculations was determined by the standard deviation of the principal components and varied for each dataset. The number of principal components used for SNN and UMAP calculations, as well as the resolution used for clustering for each dataset, can be found in Table S5. Cluster markers were found using Wilcoxon Rank Sum test, and clusters were annotated based on the expression of known cell type marker genes (Table S6). Following cell type annotation, gene dropout values ​​were imputed using adaptive thresholded low-rank approximation (ALRA) (Linderman et al., 2018).The rank-k approximation was automatically selected for each dataset, and all other parameters were set as default values. Imputed gene expression is shown in all plots and used in all downstream analyses unless otherwise specified.

[0275] [Table S5] TIFF2025502844000045.tif210167TIFF2025502844000046.tif208167TIFF2025502844000047.tif209167

[0276] [Table S6]

[0277] Analysis of published datasets Quality control. Low-quality cells were identified and removed using criteria used by the original authors of each dataset. Dataset-specific quality control metric cutoffs can be found in Table S4.

[0278] Dimensionality Reduction and Clustering. The dataset was analyzed with Seurat using the methods and parameters described by the original authors.

[0279] Morarach et al.: For all datasets, count matrices were normalized and regressed on the percentage of mitochondrial genes, returning 3000 variable features using the "SCTransform" function. Prior to running PCA, highly expressed specific and immediate early genes (Xist, Gm13305, Tsix, Eif253y, Ddx3y, Uty, Fos, Jun, Junb, Egr1) were removed from the variable feature list. Dataset-specific parameters used for the "RunUMAP", "FindNeighbors" and "FindClusters" functions can be found in Table S5. Cell annotations determined by the authors were used for cell types and neuronal subtypes.

[0280] Drokhlyansky et al.: For all datasets, count matrices were log-normalized with a scaling factor of 10,000 and 2,000 variable features were identified using the "vst" method. Batch correction by "Unique_ID" was performed using mutual nearest neighbor correction (MNN) with the "RunFastMNN" Seurat Wrappers function. Dataset-specific parameters used for the "RunUMAP", "FindNeighbors" and "FindClusters" functions can be found in Table S5. Cell annotations determined by the authors were used for cell types and neuronal subtypes. For consistency of comparisons, gene dropout values ​​were imputed using ALRA for all published datasets using automatically determined rank-k approximation and all other default values. Imputed gene expression is shown in all plots and used in all downstream analyses unless otherwise specified.

[0281] Glial subclustering analysis. Glia were subclustered using methods similar to the original analysis pipeline described by each of the authors above.

[0282] Morarach et al.: Because the E18 dataset contained a single transcriptionally homogenous glial cluster, glial and progenitor populations were subclustered together to provide the comparative cell populations required for downstream analyses. The subset dataset was then normalized and regressed on the percentage of mitochondrial genes, returning 3000 variable features using the "SCTransform" function. Prior to running PCA, highly expressed specific and immediate early genes (Xist, Gm13305, Tsix, Eif253y, Ddx3y, Uty, Fos, Jun, Junb, Egr1) were removed from the variable feature list. Dataset-specific parameters used for the "RunUMAP", "FindNeighbors" and "FindClusters" functions can be found in Table S5.

[0283] Drokhlyansky et al.: The glial subset dataset was log-normalized with a scaling factor of 10,000 and 2,000 variable features were identified using the "vst" method. Batch correction by "Unique_ID" was performed using mutual nearest neighbor correction (MNN) with the "RunFastMNN" Seurat Wrappers function. Dataset-specific parameters used for the "RunUMAP", "FindNeighbors" and "FindClusters" functions can be found in Table S5.

[0284] Characterization of gene group expression Gene lists were compiled for genes belonging to 10 different functional groups (transcription factors, neurotransmitter synthesis, neuropeptides, neurotransmitter receptors, neuropeptide receptors, cytokines, cytokine receptors, secreted signaling ligands, ligand receptors, and surface markers) (Table S7). For each dataset, gene lists were filtered to remove low abundance genes (detected in less than 25% of cells in each cluster). Genes from these lists were determined to be exclusively expressed by a cluster if more than 25% of cells in only a single cluster expressed the gene.

[0285] [Table S7] TIFF2025502844000050.tif148159TIFF2025502844000051.tif148159TIFF2025502844000052.tif148159TIFF20255028440 00053.tif148159TIFF2025502844000054.tif144160TIFF2025502844000055.tif144160TIFF2025502844000056.tif129160

[0286] Cell-type transcriptional signature scoring To find transcriptionally similar cell populations between the two datasets, first, differentially expressed (DE) genes of the reference dataset were calculated from the unimputed gene counts using the "FindAllMarkers" function using a Wilcoxon Rank Sum test to return only genes with positive fold changes. The DE gene list is first filtered to remove genes not present in the query dataset. Then, for each cell cluster in the reference dataset, a transcriptional signature gene list is created from the top 100 DE genes sorted by increasing adjusted p-values. The query dataset is then scored for the transcriptional signature gene list of each reference dataset cell cluster based on the imputed gene counts of the query dataset using the "AddModuleScore" function.

[0287] Spearman correlation Transcriptional correlations of cell clusters in the two datasets were calculated from the unsubstituted gene counts, utilizing Seurat's integration function to first find 3,000 anchor features based on the first 30 dimensions of the classical correlation analysis, and then merge the two datasets using the same number of dimensions. The representations of these 3000 anchor features were then scaled and centered on the merged data object, and the scaled average expression of each anchor feature was calculated for cell clusters in each dataset of interest using the "AverageExpression" function. A Spearman correlation matrix comparing all cell clusters to all cell clusters was generated based on the scaled average expression of the 3000 anchor features.

[0288] SWNE projection The reference and query dataset count matrices are first filtered to include only genes detected in both datasets. Similar weighted nonnegative embeddings (SWNE) are then generated for the reference dataset using the SWNE v0.6 package. First, nonnegative matrix factorization (NFM) generates component factors from 3000 variable features calculated from the unsubstituted gene counts of the reference dataset. Two-dimensional component factor embeddings are calculated using Sammon mapping, where cells and designated key genes are embedded in two dimensions for the component factors. Finally, an SNN network is calculated from the reference dataset and used to smooth the cell locations. Next, the query dataset is mapped to the two-dimensional component factor space of the reference dataset by first projecting the query dataset onto the NFM factors of the reference dataset. The resulting query dataset cell embeddings are then smoothed by projection onto the SNN network of the reference dataset.

[0289] Intermuscular and Submucosal Scoring Neurons or glia were grouped separately by tissue layer origin using patient metadata published by the authors. Pan-neuronal and pan-glial myenteric and submucosal gene signatures were created by performing Wilcoxon rank-sum tests to identify DE genes between myenteric and submucosal cell groups. Neuronal and glial datasets were scored with the cell type-specific tissue layer signatures by first ordering the gene list by increasing the adjusted p-value and removing genes not detected in the dataset being scored. Cells were then scored for the 100 most significantly enriched genes in each tissue layer using the "AddModuleScore" function.

[0290] Neurochemical identification of neurons Neurochemical identification of neurons was performed independently for each neurotransmitter to accommodate multiple neurochemical identities. For each neurotransmitter, a core set of genes consisting of the rate-limiting synthesis enzyme(s), metabolic enzymes, and transport proteins was selected (Table S1). First, cells were scored for each neurotransmission-related gene set using the “AddModuleScore” function. Then, cells were annotated as “x-ergic” if the cell’s expression of the rate-limiting enzyme was greater than 0 and the cell’s module score for the corresponding gene set was greater than 0. If both criteria were not met, the cell was annotated as “other.” Multiple neurochemical identities were determined by concatenating each cell’s individually determined single neurochemical identities. The overall prevalence of each neurochemical identity per dataset was calculated by summing the total number of cells annotated for each single identity and calculating the percentage of each “x-ergic” identity from that sum.

[0291] Neurotransmitter response scoring Separate gene lists were created containing all receptors activated by each neurotransmitter. Cells were scored for expression of each neurotransmitter receptor family gene set using the "AddModuleScore" function.

[0292] Glial GSEA hierarchical clustering For each subclustered glial dataset, the DE genes for each glial subtype were calculated using the "FindAllMarkers" function. Gene set enrichment analysis (GSEA) on the MSigDB gene ontology sets was performed on the upregulated DE genes (positive log2 fold change only) for each glial subtype sorted by decreasing log2 fold change using fgsea v1.16. Normalized enrichment scores (NES) were calculated for gene sets containing a minimum of 15 genes in the DE gene list with scoreType set to "positive". The GSEA results for each glial subtype were filtered to include only biological process gene sets, but not filtered based on significance to avoid limiting the results to pathways enriched with the highest fold change genes. The NES of the filtered gsea results for all glial subtypes were then merged and pathways not detected in any glial subtype were assigned an NES of 0. Hierarchical clustering was then performed based on NES to cluster both gene ontology pathways and glial subtypes. After the glial classes were determined by clustering, pathways enriched in each class were identified by filtering pathways with an NES greater than 1.1 in all subtypes of a given class.

[0293] Expression correlation of PP121 to control genes To compare gene expression for control and PP121-treated cell types, neuronal subtypes, and NO neuronal subtypes, subset data sets for each cell type and subtype annotation were first created. Then, for each subset, the unimputed average expression of all genes was calculated for control and PP121-treated cells using the "AverageExpression" function and natural log transformed for plotting. R was used to compare control and PP121 natural log expression values. 2 Values ​​were calculated from linear modeling using the "y~x" formula.

[0294] cFOS expression screening Stage 2 intestinal gangliosides were dissociated using Accutase and single cell suspensions (in ENC medium) were distributed into wells of a V-bottom 96-well plate. Compounds from the Neuronal Signaling Compound Library (Selleckchem, USA) were added at 1 pM using a pin tool and cells were incubated for 75 min at 37 °C. Cells were then washed with PBS and immediately fixed for flow cytometry.

[0295] NO release assay For high-throughput measurements of nitric oxide (NO) release, stage 1 two-dimensional ENS cultures (96-well plates) were used. Cells were washed with Tyrode's solution [NaCl (129 mM), KCl (5 mM), CaCl2 (2 mM), MgCl2 (1 mM), glucose (30 mM) and HEPES (25 mM) at pH 7.4], after which 70 pl / well of Tyrode's solution was added to each well. Neuronal signaling compounds (Selleckchem, USA) were added at 1 pM using a pin tool. After 45 min of incubation at 37 °C, the supernatants were used to determine NO release using a NO assay kit (Invitrogen, EMSNO). Briefly, the kit uses nitrate reductase to convert nitrate to nitrite, which is then detected as a colored azo dye that absorbs light at 540 nm. NO release for each compound was measured as a function of A relative to vehicle (DMSO). 540nmwas presented as:

[0296] A high-throughput screen to identify compounds that enrich for NO neurons Day 15 H9 hESC-derived intestinal crestspheres were dissociated into single cells (Accutase, Stemcell Technologies, 07920, 30 min, 37°C), resuspended in ENC medium and transferred to 384-well plates. Plates were incubated for 2 h to allow cells to attach. Using the pin tool, drugs from the 1694 inhibitor library (SelleckChem, USA) were added to the wells at a final concentration of 1 pM and plates were incubated with drugs until D20, at which time the medium was changed to drug-free ENC. At day 40, cells were fixed, stained for NOS1 and imaged using an InCellAnalyzer 2000 (GE Healthcare, USA). Hits showed significantly higher NOS1 expression compared to vehicle (DMSO)-treated wells. + Selection was based on fold increase in percentage of cells.

[0297] Surface marker screening For human surface marker screening, PP121-treated NOS1::GFP intestinal gangliosides from four independent differentiations were pooled and dissociated into single cells (Accutase, Stemcell Technologies, 07920, for 30–60 min at 37 °C and 5% CO). 2) and fixed (Foxp3 / Transcription Factor Staining Buffer Set, 00-5523, 30 min, 4°C). Cells were permeabilized and blocked (same staining kit) before incubation with anti-GFP antibody (abcam, ab13970, 4°C). After washing three times, cells were stained with Alexa Fluor 488-conjugated secondary antibody (40 min, room temperature). Secondary antibody solution was removed (washed three times) and cells were incubated with blocking buffer containing PBS and 2% FBS (30 min, on ice). Cells were split in a ratio of 240:16 corresponding to the number of library antibodies raised in mouse and rat and received anti-mouse and anti-rat Alexa Fluor 647-conjugated secondary antibodies, respectively. They were then distributed into V-bottom 96-well plates and treated with library antibodies for 30 min on ice (BD Biosciences, 560747). After two washes, surface marker and GFP signals were quantified by high-throughput flow cytometry (BD LSRFortessa). + GFP + cells) and highest specificity (lowest percentage of CD + GFP - Based on these results, we identified NO neuron-specific surface markers.

[0298] Drug-target interaction prediction We generated a list of their known and predicted targets by taking the classical SMILES of our hits from PubChem (De Giorgio et al., 2016; Niesler et al., 2021) and combining data from the following databases: BindingDB (https: / / www.bindingdb.org / ), Carlsbad (http: / / carlsbad.health.unm.edu / ), DINIES (https: / / www.genome.jp / tools / dinies / ), PubChem BioAssay (https: / / pubchem.ncbi.nlm.nih.gov / , filtered for valid interactions), SEA (http: / / sea.bkslab.org / , filtered for MaxTC>0.4), SuperDRUG2 (http: / / cheminfo.charite.de / superdrug2 / ) and SwissTargetPrediction (http: / / www.swisstargetprediction.ch / ).

[0299] In vivo cell transplantation Specific pathogen-free (SPF) homozygous neuronal nitric oxide synthase knockout mice (B6.129S4-Nos1 tm1Plh / J;nNos1 - / - ) were bred and maintained for use as recipients in individually ventilated cages (IVC). Animals used in these studies were kept and experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and approved by the University College London Biological Services Ethical Review Process. Animal husbandry at UCL Biological Services was in accordance with the UK Home Office Certificate of Designation. Nos1 - / -Because the mice were immunocompetent, they were orally administered cyclosporine A (250 jig / ml in drinking water) 2 days before transplantation to reduce possible rejection of the donor human cells. - / - Mice were randomly selected within litter groups and stage 1 intestinal ganglioids were transplanted into P23-P27 mice by laparotomy under isoflurane anesthesia. Briefly, the distal colon was exposed and then intestinal ganglioids containing 0.5-1M cells were transplanted onto the serosal surface of the distal colon using a pulled glass micropipette by mouth pipette. Each transplanted tissue typically received three types of ganglioids and was manipulated on the surface of the distal colon by the beveled tip of a 30G needle to ensure proper positioning. The transplanted Nos1 - / - Mice were maintained with continuous free access to drinking water treated with cyclosporine A (250 jig / ml) for up to 8 weeks after transplantation to ensure extended immunosuppression before euthanasia and removal of the colon for analysis. Because cyclosporine A can affect several signaling pathways and induce gene expression changes, it will be important to validate the immunofluorescence results using appropriate controls, such as tissues from cyclosporine A-treated untransplanted animals, in follow-up studies. In addition, other immunocompromised backgrounds (e.g., NSG) will be important to further validate these engraftment results.

[0300] Tissue preparation and fixation After resection, the entire colon was pinned onto a Petri dish lined with Sylgard (Dow, MI, USA) and opened along the mesenteric border, after which the mucosa was removed by sharp dissection and the tissue was fixed in 4% PFA in PBS (45 min to 1 h, 22 °C) for further processing.

[0301] tissue staining Colonic longitudinal myenteric plexus (LMMP) tissue was fixed with 4% PFA (1 h on ice, Thermo scientific, J19943-K2), blocked and permeabilized with a buffer containing 1% BSA and 1% triton X-100 (in PBS, 45 min, RT). Tissue was then incubated with primary antibody solution (in the same buffer, overnight, 4°C) and washed three times before treatment with fluorophore-conjugated secondary antibodies (1 h, RT). Samples were stained with DAPI and washed using vectashield (Vector Laboratories, H-1400) before mounting. Antibodies are listed in Table S3.

[0302] Multielectrode array (MEA) analysis Data Acquisition: Neuronal activity was recorded with an Axion Maestro Edge on a Cytoview MEA 24-well plate with 1-hour recording sessions for each condition. Neuromodulators or vehicle were added by removing the plate from the Maestro Edge, replacing half of the medium with 2x concentrated neuromodulators or vehicle in pre-warmed medium, and immediately placing the plate back on the Axion to resume recording. Optogenetic stimulation was performed with an Axion Lumos attachment by stimulating all wells of the plate with 488 nm light at 50% intensity, 1 second on, 4 seconds off, 30 times.

[0303] Data processing: Raw data were first sorted by a modified version of SpikeInterface (https: / / github.com / SpikeInterface) using MountainSort to identify high quality units by manually scoring based on amplitude, waveform shape, firing rate, and interspike interval contamination. For pharmacology experiments, neurons were matched between vehicle and neuromodulator recordings by examining all units detected on a particular electrode after spike scoring and identifying units with identical waveforms. The firing rates of these "paired" units from all treated wells were compared across control and neuromodulator conditions. Positive responders were units with firing rate changes greater than +0.1 Hz, negative responders had firing rate changes less than -0.1 Hz, and neutral responders had firing rate changes between -0.1 Hz and +0.1 Hz. For optogenetic experiments, individual units were again extracted in SpikeInterface and manually scored. Recordings were divided into "on" times, when the LED was active, and "off" times, when the LED was inactive. All units were compiled, and the firing rate of each unit was compared between the on and off windows.

[0304] Ex vivo colonic motility assay Preparation of solutions: Krebs buffer [NaCl (117 mM), KCl (4.7 mM), NaH 2 PO 4 (1.2 mM), MgCl 2 (1.5 mM), CaCl 2 .2H 2 O (2.5 mM), NaHCO 3 (25 mM), glucose (11 mM, pH 7.4)] in a 37 °C water bath and incubate at 95% O for at least 30 min before the start of the experiment. 2 and 5% CO 2The cells were aerated with a carbogen gas mixture. The "drug" treatment solutions were freshly prepared by adding the drug compound to the Krebs buffer before data acquisition began. A solution containing an NOS1 inhibitor was prepared by adding N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) to the drug solution making "drug + L-NAME."

[0305] Tissue ablation: For each experimental replicate, a pair of 8-week-old wild-type C57BL6 mice (male) was placed in a sealed chamber and incubated with CO. 2 Mice were euthanized by asphyxiation followed by cervical dislocation. The lower gastrointestinal tract (cecum and colon) was removed and immediately transferred to 37°C carbonate-containing Krebs buffer with feces still inside. After removing adipose tissue and mesentery, the colon was placed in the organ bath reservoir of a gastrointestinal motility monitor (GIMM) device. The GIMM has two reservoirs, allowing for simultaneous acquisition of control and drug-treated colons.

[0306] Experimental setup and procedure: The GIMM was designed based on a previously reported model (Swaminathan et al., 2016). The organ reservoir of the GIMM has two chambers for simultaneous recording of two specimens. It is connected to the working solution kept at 37 °C via a four-channel peristaltic pump (WPI, PERIPRO-4LS). The lower GI tract was harvested and transferred to an organ bath with flowing Krebs buffer. The cecum was pinned at the proximal tip and the distal end of the colon was pinned through the serosa / septum membrane. Five 10-min (for the first experiment) or consecutive 6-min (for consecutive drug treatments in the presence and absence of L-NAME) video recordings were recorded using IC Capture software (Imaging Source) with a high-resolution monochromatic Firewire industrial camera (Imaging Source®, DMK41AF02) connected to a 2 / 3 inch 16 mm f / 1.4 C-Mount Fixed Focal Lens (Fujinon HF16SA1). Tissue in the control chamber was exposed only to Krebs solution, while the order of solutions in the experimental chambers was as follows: Krebs, drug compound, Krebs (6 min each), L-NAME (2 min), L-NAME in the presence of drug compound (6 min) and Krebs (6 min). Chambers were cleaned after each collection.

[0307] Data and statistical analysis: Spatiotemporal maps (STM) of each acquisition were generated using VolumetryG9a (Spear et al., 2018). Slow wave (SW) and colonic migratory motor complex (CMMC) data were generated from the STM. Statistical analysis was performed using PRISM.

[0308] Generate schematic diagrams of diagrams Adobe Illustrator (version 25.4.1) was used to generate schematic diagrams for the figures.

[0309] Table 2 is a list of those biomarkers that are specific to one or more cells disclosed in the present application. Most of these biomarkers are expressed as proteins on the surface of the cell. In some embodiments, the biomarkers are expressed as mRNAs within the cell. The biomarkers in Figures 8-20 (including 8) are disclosed in Figures 8E and 8F and correspond to the cell types disclosed in those panels. When a cell type is matched to a gene name, it is understood that the cell type contains a protein or expresses the disclosed gene. In some embodiments, the cell types disclosed in Figures 8-20 express an mRNA that contains at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an mRNA associated with an accession number in Table 2 or an mRNA identified by an accession number in Table 2. In some embodiments, the cell types disclosed in Figures 8-20 express a protein associated with an accession number in Table 2 or a protein that comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a protein identified by an accession number in Table 2. The sequences associated with an accession number in Table 2 are incorporated by reference in their entirety. The sequences associated with an accession number in Table S7 are incorporated by reference in their entirety.

[0310] [Table 2] TIFF2025502844000058.tif217167TIFF2025502844000059.tif219167TIFF2025502844000060.tif219167TIFF2025502844000061.tif222167TIFF2025502844000062.tif225167TIFF2025502844000063.tif219167TIFF2025502844000064.tif226167TIFF2025502844000065.tif222167TIFF2025502844000066.tif225167TIFF2025502844000067.tif218167TIFF2025502844000068.tif227167TIFF2025502844000069.tif223167TIFF2025502844000070.tif225167TIFF2025502844000071.tif221167TIFF2025502844000072.tif223167TIFF2025502844000073.tif217167TIFF2025502844000074.tif223167TIFF2025502844000075.tif223167TIFF2025502844000076.tif223167TIFF2025502844000077.tif223167TIFF2025502844000078.tif223167TIFF2025502844000079.tif223167TIFF2025502844000080.tif226167TIFF2025502844000081.tif226167TIFF2025502844000082.tif227167TIFF2025502844000083.tif223167TIFF2025502844000084.tif219167TIFF2025502844000085.tif226167TIFF2025502844000086.tif225167TIFF2025502844000087.tif219167TIFF2025502844000088.tif224167TIFF2025502844000089.tif225167TIFF2025502844000090.tif226167TIFF2025502844000091.tif224167TIFF2025502844000092.tif220167TIFF2025502844000093.tif226167TIFF2025502844000094.tif226167TIFF2025502844000095.tif227167TIFF2025502844000096.tif224167TIFF2025502844000097.tif224167TIFF2025502844000098.tif225167TIFF2025502844000099.tif225167TIFF2025502844000100.tif226167TIFF2025502844000101.tif227167TIFF2025502844000102.tif224167TIFF2025502844000103.tif222167TIFF2025502844000104.tif224167TIFF2025502844000105.tif224167TIFF2025502844000106.tif224167TIFF2025502844000107.tif224167TIFF2025502844000108.tif224167TIFF2025502844000109.tif224167TIFF2025502844000110.tif226167TIFF2025502844000111.tif218167TIFF2025502844000112.tif217167TIFF2025502844000113.tif225167TIFF2025502844000114.tif224167TIFF2025502844000115.tif226167TIFF2025502844000116.tif227167TIFF2025502844000117.tif222167TIFF2025502844000118.tif227167TIFF2025502844000119.tif223167TIFF2025502844000120.tif223167TIFF2025502844000121.tif223167TIFF2025502844000122.tif228167TIFF2025502844000123.tif226167TIFF2025502844000124.tif226167TIFF2025502844000125.tif223167TIFF2025502844000126.tif222167TIFF2025502844000127.tif222167TIFF2025502844000128.tif223167TIFF2025502844000129.tif217167TIFF2025502844000130.tif221167TIFF2025502844000131.tif221167TIFF2025502844000132.tif224167TIFF2025502844000133.tif226167TIFF2025502844000134.tif220167TIFF2025502844000135.tif224167TIFF2025502844000136.tif225167TIFF2025502844000137.tif224167TIFF2025502844000138.tif218167TIFF2025502844000139.tif222167TIFF2025502844000140.tif222167TIFF2025502844000141.tif224167TIFF2025502844000142.tif223167TIFF2025502844000143.tif219167TIFF2025502844000144.tif223167TIFF2025502844000145.tif224167TIFF2025502844000146.tif225167TIFF2025502844000147.tif224167TIFF2025502844000148.tif226167TIFF2025502844000149.tif219167TIFF2025502844000150.tif226167TIFF2025502844...

Claims

1. (i) enteric glial cells containing PMP22 or a cell line containing said enteric glial cells; or (ii) an enteric neuron comprising SOX10 and at least one or a combination of CD24, CD45RA, CD57, CD63, CD71, CD121b, CD147, CD164, CD184, CD193, CD243, or CD275, or a cell line comprising said enteric neuron; or (iii) enteric neurons comprising SOX10 and at least one or a combination of CD24, CD45RA, CD57, CD63, CD71, CD121b, CD147, CD164, CD184, CD193, CD243, or CD275, or a cell line comprising said enteric neurons, and enteric glial cells comprising PMP22 or a cell line comprising said enteric glial cells. The composition comprising:

2. 1. A method for differentiating neuronal cells into enteric neuronal cells, comprising exposing the neuronal cells to an effective amount of a platelet-derived growth factor receptor (PDGFR) inhibitor or a pharmaceutically acceptable salt thereof for a period of time sufficient to differentiate the neuronal cells into enteric neuronal cells.

3. The PDGFR inhibitor is (Z)-orantinib, AC710, AC710 mesylate, AG1295, amuvatinib, amuvatinib hydrochloride, avapritinib, axitinib, AZD2932, cediranib, cediranib maleate, tiauranib, CHIR-124, CP-673451, crenolanib, dovitinib, dovitinib lactate, dovitinib lactate hydrate, dovitinib-D8, ENMD-2076, ENMD-2076 tartrate, flumatinib, flumatinib mesylate , GZD856, GZD856 formate, HG-7-85-01, hypotemicline, ilorasertib, ilorasertib hydrochloride, imatinib, imatinib D4, imatinib D8, imatinib mesylate, JI-101, JNJ-10198409, KG5, Ki20227, lenvatinib, lenvatinib mesylate, linifanib, masitinib, masitinib mesylate, methylnisoline, multikinase inhibitor 1, N-(p-coumaroyl)serotonin, nintedanib, nintedanib esil acid salt, NVP-ACC789, orantinib, pazopanib, pazopanib hydrochloride, PD-089828, PD-161570, PDGFRα kinase inhibitor 1, ponatinib, ponatinib D8, PP121, PP58, regorafenib, regorafenib D3, regorafenib hydrochloride, regorafenib monohydrate, ripretinib, sennoside B, seraltinib, SU5402, SU14813, SU14813 maleate, SU16f, SU4312, SU4984, sunitinib, sunitinib 3. The method of claim 2, wherein the agonist is selected from sunitinib D10, sunitinib malate, sunitinib-d4, TAK-593, tandutinib, tandutinib hydrochloride, telatinib, telatinib mesylate, TG100572, TG100572 hydrochloride, TG100801, TG100801 hydrochloride, toceranib, toceranib salt, toceranib-d8, trapidil, tyrosine kinase-IN-1, tyrphostin AG1296, tyrphostin AG1433, borolanib, or a hydrate thereof.

4. The PDGFR inhibitor is or a salt thereof.

5. The PDGFR inhibitor is The method of claim 2, wherein the compound is selected from the group consisting of:

6. 3. The method of claim 2, wherein the PDGFR inhibitor is exposed to the stem or neuronal cells is isotopic, deuterated, or as a pharmaceutically acceptable salt.

7. A method for enriching NO enteric neurons in a cell culture, comprising exposing a composition of neural crest cells or crestspheres to an effective amount of a PDGFR inhibitor or a pharmaceutically acceptable salt thereof for a period of time sufficient to cause the cells to differentiate into enteric neurons.

8. The method of claim 7 , wherein the cell culture comprises neural crest cells in two or three dimensions.

9. The method of claim 7, wherein the method modulates NO neuronal activity nitric oxide responsiveness.

10. a PDGFR inhibitor, or a pharmaceutically acceptable salt thereof; a. instructions for treating intestinal motility disorders, and b. Instructions for administering the compound in connection with treating intestinal motility disorders and one or more selected from Includes a kit.

11. (i) A cell line comprising neural crest cells or crestspheres; (ii) stem cells or differentiated human stem cells; and (iii) one or more enteric neurons 11. The kit of claim 10, further comprising one or a combination of:

12. 2. The composition of claim 1, wherein the enteric glial cells further comprise one or a combination of SB100, PLP1, AQP4, GFAP, MBP, and / or MPZ.

13. the one or more enteric glial cells are derived from human induced pluripotent stem cells; the one or more enteric glial cells are present in a ganglioid or spheroid or substantially spherical cell composition; and / or the one or more enteric glial cells have been in cell culture for about 5, 10, 12, or 15 days or more; The composition of claim 1.

14. one or more enteric neuron cells; (i) one or more mesenchymal cells; (ii) one or more epithelial cells, the epithelial cells being arranged within a spheroid or ganglioid; The composition of claim 1 further comprising:

15. 15. The composition of claim 14, wherein the mesenchymal cells express one or a combination of PRRX1, RNX2, TWIST1, COL11A1, COL1A2, COL1A1, COL3A1, COL5A2, FN1, LAMA4, EDNRA, PDGFRA, and PDGFRB.

16. 15. The composition of claim 14, further comprising one or more progenitor or stem-like cells that express one or a combination of the biomarkers of FIG. 8F.

17. the glial cells express one or a combination of GFAP, ERBB4, NTRK2, NTRK3, PAX3, EDNRB, FZD3, and SOX2; the epithelial cells express one or a combination of CDH1, EPCAM, KRT119, and the enteric neurons express one or a combination of NRXN3, NRXN1, DCX, MAPT, ELAVL2, NRCAM, RBFOX3, NCAM1, NRG1, SYN1, and SYP; the mesenchymal cells express one or a combination of PRRX1, RNX2, TWIST1, COL11A1, COL1A2, COL1A1, COL3A1, COL5A2, FN1, LAMA4, EDNRA, PDGFRA, and PDGFRB; 15. The composition of claim 14.

18. The mesenchymal cells KRT119 and and one or a combination of PRRX1, RNX2, TWIST1, COL11A1, COL1A2, COL1A1, COL3A1, COL5A2, FN1, LAMA4, EDNRA, PDGFRA, and PDGFRB. The composition of claim 14, wherein the composition expresses

19. 10. The composition of claim 1, wherein the composition is free of or substantially free of retinal pigment epithelial (RPE) cells.

20. A composition described in any one of claims 1 and 12 to 19 for treating intestinal motility disorders.

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