Isolation of enteric neurons and progenitors from the enteric ganglia for cell therapy

Abstract

Disclosed are compositions including a population of human enteric neural stem cells (ENSCs), wherein the population comprises at least one of a cell expressing nerve growth factor receptor (NGFR), a cell expressing integrin alpha 6 (ITGA6), a cell expressing L1 cell adhesion molecule (L1CAM), a cell that expresses proteolipid protein 1 (PLP1), and / or a cell that expresses paired-like homeobox 2B (PHOX2B), methods of producing the compositions, and their uses.

Description

[0001]PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 ISOLATION OF ENTERIC NEURONS AND PROGENITORS FROM THE ENTERIC GANGLIA FOR CELL THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of U.S. Provisional Application No.63 / 659,137, filed on June 12, 2024, the contents of which are incorporated herein by reference in their entirety. BACKGROUND OF THE INVENTION This invention relates to enteric neural stem cells and their use in treating enteric neuropathies. The enteric nervous system (ENS) is composed of an extensive interconnected network of enteric neurons and enteric glial cells (EGCs) that reside within the gut wall and regulate the numerous complex functions of the gastrointestinal (GI) tract, including absorption, secretion, barrier function, motility, and immunity. Acquired and congenital enteric neuropathies, including Hirschsprung disease, gastroparesis, esophageal achalasia, and others result in severe morbidity due to severe GI dysmotility. Current treatment options for these conditions do not address the absence or loss of enteric neurons and EGCs underlying their pathophysiology, but rather focus on symptom management or surgical removal of affected GI segments. Regenerative cell therapy is a promising treatment option that directly targets the fundamental problem of enteric neuropathies by replacing the missing neurons and glia with the potential to re-establish normal gut innervation and restore GI homeostasis. Accordingly, there is a need in the art to develop such therapies. SUMMARY OF THE INVENTION In one aspect, the disclosure features a composition including a population of human enteric neural stem cells (ENSCs), where the population includes at least one of a cell expressing nerve growth factor receptor (NGFR), a cell expressing integrin alpha 6 (ITGA6), and a cell expressing L1 cell adhesion molecule (L1CAM). In one embodiment, the population of human ENSCs further includes a cell expressing proteolipid protein 1 (PLP1) and / or a cell expressing paired-like homeobox 2B (PHOX2B). In one embodiment, the composition includes a cell expressing NGFR, a cell expressing ITGA6, and a cell expressing L1CAM. In another embodiment, the composition includes a cell expressing NGFR, a cell expressing ITGA6, a cell expressing L1CAM, a cell expressing PLP1, and a cell expressing PHOX2B. In one embodiment NGFR, ITGA6, and / or L1CAM are expressed at the protein level, and expression of NGFR, ITGA6, or L1CAM protein is detected using an immunohistochemistry assay, flow cytometry, or Western blotting. In an embodiment, PLP1 and / or PHOX2B are expressed at the mRNA level, and expression of PLP1 or PHOX2B mRNA is detected using a polymerase chain reaction (PCR) assay or a quantitative PCR assay, or by RNA sequencing. In another embodiment, expression of PLP1 and / or PHOX2B mRNA in the population of human ENSCs is at a higher level than in an equivalent population of human fibroblast or myofibroblast cells. In a further embodiment, the ENSCs, or a portion of the ENSCs, are cryopreserved. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 In another aspect, the disclosure features a container including a composition including a population of human ENSCs, where the population includes at least one of a cell expressing NGFR, a cell expressing ITGA6, a cell expressing L1CAM, a cell that expresses PLP1 and / or a cell that expresses PHOX2B. In one embodiment, the composition included in the container is cryopreserved. In another aspect, the disclosure features a method of producing a human ENSC from intestinal tissue. This method includes the steps of: a) dissociating the intestinal tissue using an enzyme b) passing the dissociated tissue through a series of cell strainers having pore sizes between 1000 μm and 40 μm and collecting material that passes through the 1000 μm pore size strainer, but not the 40 μm pore size strainer to collect material enriched for myenteric ganglia; and c) culturing myenteric ganglia; thereby producing a human ENSC from intestinal tissue. In one embodiment, the intestinal tissue comprises muscularis propria tissue. In another embodiment, the enzyme is a collagenase and / or a dispase. In additional embodiments, the intestinal tissue is from a pediatric patient or is from an adult patient. In an embodiment where the intestinal tissue is from an adult patient, the method includes collecting material that passes through the 1000 μm pore size strainer, but not a 100 μm pore size strainer. In a further embodiment of this aspect, the intestinal tissue is cryopreserved and thawed prior to step (a). In another embodiment, culturing myenteric ganglia includes culturing in a medium containing fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF) and / or retinoic acid. In a further embodiment, the method further includes harvesting the human ENSC. In another embodiment, the human ENSC expresses NGFR, ITGA6, L1CAM, PLP1, and / or PHOX2B, where NGFR, ITGA6, and / or L1CAM are expressed at the protein level, and where PLP1 and / or PHOX2B are expressed at the mRNA level. In another embodiment, the method further includes cryopreserving the human ENSC. In another aspect, the disclosure features a method of cryopreserving a population of human ENSCs produced from intestinal tissue. The method includes cryopreserving the population of human ENSCs produced from intestinal tissue in cryopreservation medium, where the population of human ENSCs comprises at least one of a cell expressing NGFR, a cell expressing ITGA6, and a cell expressing L1CAM, where NGFR, ITGA6, and / or L1CAM are expressed at the protein level. In another embodiment, the population of human ENSCs further includes a cell that expresses PLP1 and / or a cell that expresses PHOX2B, where PLP1 and / or PHOX2B are expressed at the mRNA level. In a further embodiment, the cryopreservation medium is Bambanker serum-free cryopreservation media. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 In a further aspect, the disclosure features a tissue transplant comprising a population of human ENSCs, where the population includes at least one of a cell expressing NGFR, a cell expressing ITGA6, and a cell expressing L1CAM, where NGFR, ITGA6, and / or L1CAM are expressed at the protein level. In another embodiment, the population of human ENSCs further comprises a cell that expresses PLP1 and / or a cell that expresses PHOX2B, where PLP1 and / or PHOX2B are expressed at the mRNA level. In an additional embodiment, the tissue transplant is a neurosphere. In another aspect, the disclosure features a method of enhancing a function of an enteric nervous system in a patient in need thereof. This method includes administering a composition or a tissue transplant including a population of human ENSCs, where the population includes at least one of a cell expressing NGFR, a cell expressing ITGA6, a cell expressing L1CAM, a cell that expresses PLP1 and / or a cell that expresses PHOX2B to a patient. In one embodiment, the composition or tissue transplant includes an ENSC that is autologous to the patient. In another aspect, the disclosure features a method of treating gastroparesis in a patient in need thereof. This method includes administering a composition or a tissue transplant including a population of human ENSCs, where the population includes at least one of a cell expressing NGFR, a cell expressing ITGA6, a cell expressing L1CAM, a cell that expresses PLP1 and / or a cell that expresses PHOX2B to a patient. In another aspect, the disclosure features a method of treating Hirschsprung disease (HSCR) in a patient in need thereof. This method includes administering a composition or a tissue transplant including a population of human ENSCs, where the population includes at least one of a cell expressing NGFR, a cell expressing ITGA6, a cell expressing L1CAM, a cell that expresses PLP1 and / or a cell that expresses PHOX2B to a patient. In one embodiment, the composition or tissue transplant includes an ENSC that is autologous to the patient. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Definitions “Enhancing a function of an enteric nervous system” refers to providing one or more neuronal activities of the enteric nervous system. Neuronal activities of the enteric nervous system include regulation of a digestive process, such as motility, secretion, and blood flow. Neuronal activity can be determined, for example, by monitoring calcium influx following electric field stimulation (EFS). A function PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 of the enteric nervous system may be enhanced in a patient with a congenital disorder that affects the enteric nervous system (e.g., Hirschsprung disease or gastroparesis). As used herein, the term “enteric nervous system” or “ENS” refers to the nerves that are present in the lining of the gastrointestinal system starting from the esophagus to the anus. As used herein, a “pediatric patient” is a patient under the age of 18. The pediatric patient may be an infant or a child under 1 year old. As used herein, a “tissue transplant” is a cluster of cells, e.g., a neurosphere. A “neurosphere” as used herein is a cluster of cells including neural stem cells. In one embodiment, a neurosphere includes a cell expressing NGFR, a cell expressing ITGA6, a cell expressing L1CAM, a cell expressing PLP1, and / or a cell expressing PHOX2B. Expression of the marker genes may be at the protein level or at the mRNA level. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee. FIG.1A – FIG.1H show BAF53b-Cre selectively targets post-mitotic ENs in the intestinal wall. FIG.1A- FIG.1Aʹ show BAF53b::tdT expression in the myenteric plexus of the colon. Dotted insert indicates magnified field of view. Scale bar = 1mm. FIG.1B shows binarization of BAF53b::tdT fluorescence along the entire colon. Inserts show high magnification images. FIG.1C shows visualization of the neuronal soma marker HuC / D in the myenteric plexus of BAF53b::tdT mice. Scale bars = 50 µm. FIG.1D shows visualization of the neuronal soma and nerve fiber marker TUBB3 in the myenteric plexus of BAF53b::tdT; Plp1-EGFP mice. Scale bars = 50µm. FIG.1E shows normalized gene expression (z-score) and percentage of expressing cells in scRNA-seq data of the colon for select neuronal and EGC markers. FIG.1F shows UMAP visualization of single-cell RNA-seq data generated from postnatally derived neurospheres. From top left panel to right: data shows unsupervised clustering of cell populations, cell cycle phases and gene expression levels of Plp1, Col6a2, Elavl4, Ascl1, Actl6b (BAF53b) and Mki67 (Ki-67). EGC, enteric glial cell; EMC, enteric mesenchymal cell. FIG.1G shows an immunocytochemistry analysis for Ki-67 in cultures from BAF53b::tdT; Plp1- EGFP mice. Scale bars = 200 µm. FIG.1H shows a quantification of the percentage of cells in culture expressing Ki-67, BAF53b::tdT and Plp1-EGFP. Data presented as mean ± SEM. n = 12 independent cultures with 473.5±79.3 cells per culture. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test *P<0.05, **P<0.01, ****P<0.0001. FIG.2A- FIG.2K show that EGC contact directs neurites and governs higher order branching of post-mitotic ENs. FIG.2A shows cell cultures derived from the muscularis propria of BAF53b::tdT mice. Scale bars = 500µm. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.2B shows a strategy for the isolation of EGCs (Plp1-EGFP), ENs (BAF53b::tdT) and the remaining EMCs. FIG.2C shows purified EGCs in co-culture with purified ENs in vitro. Scale bars = C, 200µm; Ci, 50µm. FIG.2D shows a quantification of the number of ENs and EGCs in co-cultures in vitro. n = 4 independent cultures. FIG.2E shows the number of neurites >3rdorder and ≤ 3rdorder in post-mitotic ENs in co-culture with EGCs or EMCs in low density and low FBS conditions. n = number of neurons from 4 cultures of Neurons alone (n = 118), Neurons + EGCs (n = 105) and Neurons + EMCs (n = 69). FIG.2F shows quantification as of above in post-mitotic ENs in co-culture with EGCs with data segregated by the presence of direct EGC contact with neurites and those having no contact. n = number of neurons from 4 cultures of Neurons alone (n = 118), EGC contact (n = 72) and EGC no contact (n = 33). FIG.2G - FIG.2H show EN with neurites making direct contact (FIG.2G) with EGCs or having no contact (FIG.2H) with EGCs in purified co-cultures. Scale bars = 200µm. FIG.2I shows the percentage of neurons making neurites, the average time to first neurite production and the percentage of neurons that undergo cell death after projecting neurites in cultures of neurons alone and neurons co-cultured with EGCs directly (Neurons + EGC) or in transwell inserts (Neurons + transwell EGC), in high density and high FBS conditions. n = number of independent cultures of Neurons alone (n = 19) Neurons + EGC (n = 16-17) and Neurons + transwell EGC (n = 12-17). FIG.2J shows EN in paracrine culture with EGCs. Scale bars = 50µm. FIG.2K shows filamentous neurites of an EN in direct EGC culture with elongated neurite projections and their branch points (arrows) coinciding with the processes of EGCs. Scale bars = 50µm. Quantification data presented as mean ± SEM. One-way ANOVA was performed with Holm-Sidak post hoc test for multiple comparisons *P<0.05, **P<0.01, ****P<0.0001. See also FIG.8 -FIG.1. FIG.3A – FIG.3F show nerve fiber projections from ENs closely associate with the architecture of EGCs in the myenteric plexus and intramuscular space. FIG.3A- FIG.3B show transplanted ENs / nerve fibers (BAF53b::tdT), transplanted EGCs (Plp1- EGFP) and S100B immunohistochemistry in the wildtype colon (FIG.3A) and aganglionic colon of Ednrb KO mice (FIG.3B). Dotted inserts show magnified regions. White arrows show neurites along transplanted EGCs and yellow arrows show neurites along the endogenous EGCs of the recipient. Scale bars = 100µm. FIG.3C shows myenteric plexus grafts from BAF53b::tdT; Plp1-EGFP mice to the colonic muscularis of a Plp1-EGFP recipient mouse. Scale bars = 200µm. FIG.3D shows time-series imaging of the above showing nerve fiber projections of the graft traversing through the myenteric plexus of the recipient colon. Scale bars = 200µm. FIG.3E – FIG.3F show localized administration of diphtheria toxin to Plp1::CreERT2-iDTR mice to selectively ablate EGCs showing the unaffected proximal region (FIG.3E) and the injection site (FIG. 3F). Scale bars = 200µm.. FIG.4A - FIG.4I shows the BAF53b::iDTR model of aneural intestine preserves EGCs with limited off-target effects. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.4A shows an experimental protocol to specifically ablate ENs in vivo. FIG.4B shows expression of Plp1-EGFP and TUBB3 in the myenteric plexus after focal ablation of neurons in the colon. Scale bar = 1mm. FIG.4C shows Plp1-EGFP, TUBB3 and Hu expression at the injection site of DT in littermate control mice lacking iDTR expression (top), the injection site of DT in mice with the BAF53b::iDTR genotype (middle), and 1-2 cm proximal to the DT injections site (bottom). Scale bar = 200µm. FIG.4D shows ENs (Hu) after DT administration to: control mice after 2 weeks and BAF53b::iDTR mice after 3 days, 1 week and 2 weeks. Scale bars = 200µm. FIG.4E shows Hu expressing ENs in the myenteric plexus per mm2. Bar charts are reported as mean ± SEM. n = number of mice per group: DT to control (n = 4), DT to BAF53b::iDTR after 3 days, 1 week and 2 weeks (n = 3). Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test *P<0.05, **P<0.01. FIG.4F shows Experimental protocol of bulk RNA-seq of the colons of mice injected with DT with the BAF53b::iDTR genotype and littermates lacking iDTR expression (top) and heatmap representations of significant changes in gene expression (bottom). FIG.4G shows an UMAP representation of the Drokhlyansky et al.202022mouse colonic atlas showing the original authors annotations of colonic cell populations (left). Gene set signature scores for the genes significantly downregulated in the colons of BAF53b::iDTR (right). FIG.4H shows a heatmap representations of pan-neuronal markers, markers of EN subpopulations, and neurotransmitter receptors. FIG.4I shows over-representation analysis of genes downregulated in BAF53b::iDTR mice against the Gene ontology database. Data presented as fold enrichment (y axis), gene counts (size) and - Log10 p value (color). See also FIG.12. FIG.5A – FIG.5I show limited neurogenesis in the aneural intestine 3 months after ablation. FIG.5A shows ENs (Hu) 2 weeks, 4 weeks, 8 weeks and 3 months after DT administration to the colons of BAF53b::iDTR mice. Scale bars = 200µm. FIG.5B shows Hu expressing ENs in the myenteric plexus per mm2. Bar charts are reported as mean ± SEM. n = number of mice per group: DT to BAF53b::iDTR after 2 weeks (n = 3), 4 weeks (n = 5), 8 weeks (n = 3) and 3 months (n = 6). FIG.5C shows TUBB3 (pan-nerve fiber and soma), Hu (pan-neuronal soma) and Calretinin (neuronal subtype intrinsic to the gut) expression in the myenteric plexus of BAF53b::iDTR mice at the injection site (top panels), 1-2cm proximal to the DT injection site (bottom left) and littermate controls (bottom right). Yellow arrows indicate ganglia-like structures lacking ENs. Scale bar = 200µm. FIG.5D shows Hu expressing ENs in the myenteric plexus per mm2in the DT injected region and 10-15 mm proximally. n = number of mice per group: DT control and Adjacent BAF53b::iDTR (n = 4), and DT BAF53b::iDTR (n = 7). FIG.5E shows Change in force (g) of colonic smooth muscle contractions in response to neuron- specific electric field stimulation after 2 weeks and 3 months after neuronal ablation. n = number of mice per group: 2 weeks control (n = 8), 2 weeks BAF53b::iDTR (n = 10) and 3 months BAF53b::iDTR (n = 6). FIG.5F shows Site-specific labeling of ENs by local administration of AAV1-FLEX-mCherry to BAF53b::iDTR mice. Scale bars = 5mm. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.5G shows Experimental protocol to ablate ENs distally and fluorescently label incoming nerve fiber projections in vivo. FIG.5H shows mCherry fluorescence in BAF53b::iDTR mice (top to bottom) without DT at the site of local AAV1-FLEX-mCherry injection after 8 weeks, the corresponding distal segment, and the distal segment post DT administration after 2 and 8 weeks. Scale bars = 100µm. FIG.5I shows an Area covered by mCherry labeled nerve fiber projections in the ablated segment. n = 3 mice per group. Bar charts are reported as mean ± SEM. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test (FIG.5B, FIG.5D), One-way ANOVA was performed with Holm-Sidak post hoc test (FIG.5E) or Unpaired t test (FIG.5I). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. See also FIG.13. FIG.6A – FIG.6N show transplanted post-mitotic ENs reinnervate the postnatal ganglionated and aganglionic colon in vivo. FIG.6A shows neuronal ablation and ganglia transplantation experimental overview. FIG.6B shows ganglia isolated from BAF53b::tdT mice. Dotted insert indicates magnified image. Scale bar = 200µm. FIG.6C shows transplanted enteric ganglia from BAF53b::tdT mice engrafted into the muscularis 2 weeks after injection into the colon of a wildtype recipient. Scale bar = 500µm. FIG.6D – FIG.6D’ show intramuscular projections of nerve fibers from transplanted ENs in between smooth muscle fibers visualized by bright field microscopy. Scale bar = 200µm. FIG.6E shows transplantation of enteric ganglia from BAF53b::tdT showing nerve fiber extensions along the endogenous EGCs of the myenteric plexus in a Plp1-EGFP recipient mouse. Scale bar = 200µm. FIG.6F shows enteric ganglia isolated from the BAF53b::tdT; Plp1-EGFP dual reporter mouse. Scale bar = 200µm. FIG.6G shows expression of Plp1-EGFP and BAF53b::tdT derived from myenteric ganglia transplants in the muscularis. Scale bar = 500µm. FIG.6H – FIG.6H’ show transplanted neurons (BAF53b::tdT) in mice after EN ablation (BAF53b::iDTR). Scale bar = 200µm. FIG.6I - FIG.6J shows transplanted ENs in the aneural BAF53b::iDTR mouse. Scale bar = 50µm. FIG.6J shows a 3D rendering of transplanted ENs forming interconnected ganglia. FIG.6K shows constipation scores in mice lacking iDTR expression with DT injection (R26-iDTR), mice injected with DT with the BAF53b::iDTR genotype to ablate ENs and those transplanted with myenteric ganglia from large bowel (LB) or small bowel (SB). Constipation score = +1 for every 20min until 120min. n = number of mice per group: R26-iDTR (n =15), Baf53b::iDTR (n = 20), Baf53b::iDTR + LB / SB (n = 6), Baf53b::iDTR + LB (n = 3) and Baf53b::iDTR + SB (n = 3). FIG.6L shows fecal water content from the mice described above. n = number of mice per group: R26-iDTR (n =10), Baf53b::iDTR (n = 16), Baf53b::iDTR + LB (n = 3) and Baf53b::iDTR + SB (n = 3). FIG.6M shows force of contraction produced by colonic smooth muscle in response to electric field stimulation (EFS) of ENs in organ bath experiments. LB, large bowel; SB, small bowel. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.6N shows force of smooth muscle contraction in response to EFS relative to baseline. LB, large bowel; SB, small bowel. n = number of mice per group: R26-iDTR (n = 7), Baf53b::iDTR (n = 6), Baf53b::iDTR + LB (n = 4) and Baf53b::iDTR + SB (n = 3). Bar charts are reported as mean ± SEM. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test (FIG.6K, FIG.6L, FIG.6N). *P<0.05, **P<0.01. FIG.7A – FIG.7G show post-mitotic ENs acquire functional connections with smooth muscle in a time-dependent manner in the postnatal environment. FIG.7A shows a schematic of experiments (top) and images of myenteric ganglia transplantation from BAF53b::tdT-ChR2 mice to the colons of wildtype mice at the time of injection (D0, left panel) and after engraftment (D14, right panel). White arrow indicates transplantation site (left panel). Scale bar = 500µm. FIG.7B shows an in vivo electromyography (EMG) in the smooth muscle and intra luminal pressure of the colon in response to blue light stimulation of transplanted ENs 3, 7, and 14 days post- engraftment. FIG.7C shows an increase in electrical activity by EMG in response to blue light stimulation. n = number of mice per group: Day 3 (n = 3), Day7 (n = 4) and Day14 (n = 5). FIG.7D shows intraluminal pressure in response to blue light stimulation. n = number of mice per group; Day 3 (n = 3), Day7 and 14 (n =4). FIG.7E shows force of contraction produced by colonic smooth muscle in response to blue light stimulation of transplanted ENs in organ bath experiments. FIG.7F shows a negative deflection from baseline smooth muscle tone in response to blue light stimulation. n = number of mice per group; Day 3 (n = 3), Day7 and 14 (n = 4). FIG.7G shows smooth muscle contraction in response to blue light stimulation relative to baseline. n = number of mice per group; Day 3 (n = 3), Day7 and 14 (n =4). Bar charts are reported as mean ± SEM. Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test (FIG.7C, FIG.7D, FIG.7F, FIG.7G). *P<0.05, **P<0.01. FIG.8A -FIG.8C show characterization of enteric mesenchymal cells (EMCs) in vitro. See also FIG.2. FIG.8A shows UMAP visualization of single-cell RNA-seq data generated from postnatally derived neurospheres showing the gene expression levels of fibroblast markers Pdgfra, Col6a3 and Col6a2. FIG.8B shows representative images of immunocytochemistry for Collagen VI (purple) and PDGFRɑ (green) in enteric mesenchymal cells (EMCs) purified by FACS sorting of the double negative fraction from BAF53b::tdT; Plp1-EGFP mice and cultured in monolayer conditions. Scale bar = 200µm. FIG.8C shows representative images of BAF53b::tdT+ ENs co-cultured with EMCs. Bottom panels show high magnification visualization of BAF53b::tdT+ neurites. Scale bar = 200µm. FIG.9A – FIG.9I show properties of in vitro neurite projections after EN co-culture with EGCs and EMCs. See also FIG.2. FIG.9A shows a quantification of the number of ENs per field of view when cultured alone or in the presence of EGCs or EMCs after 10 days. Data presented as mean ± SEM. n = 4 independent cultures per group. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.9B shows a quantification of the number of Ens with neurites when cultured alone or in the presence of EGCs or enteric EMCs after 10 days. Data presented as mean ± SEM. n = 4 independent cultures per group. One-way ANOVA was performed with Holm-Sidak post hoc test for multiple comparisons ***P<0.001. FIG.9C- FIG.9E show a quantification of neurite metrics in cultures of purified post-mitotic ENs in co-culture with EGCs or EMCs. Quantification includes the sum of the lengths of neurites per neuron (FIG.9C), the average highest order neurite in each neuron (FIG.9D), and the number of neurites per neuron (FIG.9E). Bar charts are reported as mean ± SEM. n = number of neurons from cultures of Neurons alone (n = 118), Neurons + EGCs (n = 105) and Neurons + EMCs (n = 69). One-way ANOVA was performed with Holm-Sidak post hoc test for multiple comparisons *P<0.05, ****P<0.0001. FIG.9F shows representative images of ENs with neurites in direct contact with EGCs (top panels) and ENs without making EGC contact (bottom panels). Dotted inserts show high magnification of the high order BAF53b::tdT neurites (right panels). Scale bar = 250μm. FIG.9G – FIG 9I show quantification of neurite metrics in cultures of purified post-mitotic ENs in co-culture with EGCs with data segregated by the presence of direct EGC contact with neurites. Quantification includes the sum of the lengths of neurites per neuron (FIG.9G), the average highest order neurite in each neuron (FIG.9H) and the number of neurites per neuron (FIG.9I). Bar charts are reported as mean ± SEM. n = number of neurons from cultures of Neurons alone (n = 118), EGC contact (n = 72) and EGC no contact (n = 33). One-way ANOVA was performed with Holm-Sidak post hoc test for multiple comparisons *P<0.05, **P<0.01, ****P<0.0001. FIG.10A – FIG.10F show characterization of in vitro neurite projections after co-culturing ENs with EGCs directly or in paracrine transwell inserts. See also FIG.2. FIG.10A - FIG.10B show representative images of co-cultures between BAF53b::tdT+ ENs and Plp1-EGFP+ EGCs in high density and high FBS conditions with EGCs seeded either in direct co-culture with neurons (FIG.10A), or in transwell inserts (FIG.10B). Images from top to bottom represent merged image of BAF53b::tdT and Plp1-EGFP fluorescence, inverted BAF53b::tdT fluorescence for greater visualization of neurites, and neurite traces colored by path orders. Scale bars = 200µm. FIG.10C - FIG.10F show quantification of the sum of the lengths of neurites per neuron (FIG. 10C), the average highest order neurite in each neuron (FIG.10D), the sum of the lengths of neurites per neuron greater than the third order (FIG.10E), and those including the third order and below (FIG.10F). Data presented as mean ± SEM. n = number of neurons from direct EGCs co-culture (C, n = 13; D, n = 13; E, n = 12; F, n = 13) or transwell culture (C, n = 13; D, n = 14; E, n = 3; F, n = 15). Unpaired t test *P<0.05, ***P<0.001, ****P<0.0001. FIG.11A – FIG.11C show directional EN and EGC paracrine co-cultures. See also FIG.2. FIG.11A shows representative images of dual chamber culture channels loaded with BAF53b::tdT+ ENs and Plp1-EGFP+ EGCs. Chambers were seeded with either ENs and EGCs in one chamber (contact), or in opposing chambers of the channel with shared media (paracrine). FIG.11B shows representative images of the morphology of neurites in the above culture conditions. Scale bars = 200µm. FIG.11C shows quantification of the direction of the primary neurite projections from ENs in relation to the EGC seeded well (0°) in paracrine experiments shown as the frequency. n = 19 individual PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 neurons. FIG.12A – FIG.12D show characterization of the region of enteric neuronal ablation. Related to FIG.4. FIG.12A shows representative image of a colonic ring utilized in organ bath force contraction experiments from a BAF53b::iDTR mouse two weeks post DT administration after processing for immunohistochemical labeling with Hu. Panels include high magnification images of regions with Hu immunoreactive cells and quantification of Hu immunoreactive cells counted from the entire preparation. FIG.12B shows quantification of the number of Hu expressing ENs in the myenteric plexus per mm2 10-15 mm proximally to the DT injected region at two weeks post administration. Bar charts are reported as mean ± SEM. n = 4 mice per group. Unpaired t test **P<0.01. FIG.12C shows representative image showing the DT injection site and the region of tissue dissected for RNA-seq studies two weeks later. Scale bar = 10mm. FIG.12D shows a heatmap representations of key markers of inflammation, intestinal smooth muscle cells (Mus), ECGs and ENs in the colons of BAF53b::iDTR mice and littermate controls. Data are presented as Z-scores. FIG.13 shows the morphological association of reinnervating nerve fiber projections with the EGC architecture. Related to FIG.5. Representative images of AAV1-FLEX-mCherry nerve fiber projections in the ablated distal colonic segment of BAF53b::iDTR mice after 8 weeks. Images show the close association between EGCs and infiltrating mCherry projections in the myenteric plexus and intramuscular space. Scale bars = 200µm. FIG.14A – FIG.14K show the isolation of myenteric ganglia from the mouse intestine. FIG.14A shows representative images of the myenteric ganglia (MyGa) of BAF53b::tdT; Plp1- EGFP mice with Plp1-EGFP serving as a EGC marker and BAF53b::tdT as a neuronal marker. Scale bar = 50µm. FIG.14B shows Depth coded projection of the Z-axis of Plp1-EGFP fluorescence showing EGCs within the MyGa (white arrows) and EGCs in the intramuscular space above (yellow arrows). Scale bar = 50µm. FIG.14C - FIG.14G shows representative images of the myenteric plexus from intestinal regions of BAF53b::tdT; Plp1-EGFP mice. Scale bars = 500µm. FIG.14Cʹ shows the minimum and maximum Feret’s diameter of a MyGa. Scale bar = 100µm. FIG.14H shows quantification of the minimum Feret’s diameter of MyGa in intestinal regions. Box plots are reported as mean ± 95% CI. n = 24 ganglia / segment. FIG.14I shows a schematic overview of MyGa enrichment from the enzymatically digested bowel using counter filtration. FIG.14J shows a Representative image of the MyGa-enriched fraction after digestion from the gut of BAF53b::tdT; Plp1-EGFP mice. Dotted inset shows a high magnification view of a MyGa within the digested material. Scale bar = 500µm. FIG.14K shows a quantitative PCR of Tubb3, Phox2b, Chat, Nos1, Ngfr, Pdgfra, and Col1a1 in digested single cell suspensions and the MyGa-enriched fractions. One-sample t-test to LogFC of 0, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 6-7 mice per group. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG 15A – 15H shows that neurospheres generated from the MyGa-enriched fraction have a higher purity of enteric neurons, EGCs, and progenitor cells. FIG 15A shows generation of neurospheres from MyGa and the single cell flow through. FIG 15B shows representative images of neurospheres generated from single cell suspensions (IM-SCs) and the MyGa enriched fractions from the small intestine of BAF53b::tdT; Plp1-EGFP mice. Scale bar = 250µm. FIG 15C shows quantification of the number of neurospheres generated from single cell suspensions and the MyGa-enriched fractions. Data are mean ± SEM. Unpaired t-test, * p < 0.05, n = 3 mice per group. FIG.15D shows quantitative PCR of Tubb3, Phox2b, Chat, Ngfr, Pdgfra, and Col1a1 in neurospheres generated from IM-SCs and the MyGa-enriched fractions. One-sample t-test to LogFC of 0, * p < 0.05, ** p < 0.01, n = 6-7 mice per group. FIG.15E – FIG.15E’ show cells from neurospheres after one week of migration forming monolayers on fibronectin. Samples were originally generated from IM-SCs and the MyGa-enriched fractions as above. Scale bars =1000µm (FIG.2E) and 500µm (FIG.2E’). FIG.15F shows flow cytometry data for EGCs / progenitors (Plp1-EGFP) and enteric neurons (BAF53b::tdT) derived from monolayer cultures. FIG.15G shows quantification of the number of EGCs / progenitors (Plp1-EGFP), enteric neurons (BAF53b::tdT), and double negative cells from flow cytometry on monolayer cultures originally derived from IM-SCs and the MyGa-enriched fractions. Data are mean ± SEM. Unpaired t-test, *** p < 0.001, **** p < 0.0001, n = 3 mice per group. FIG.15H shows a pie chart representation of the proportions of EGCs / progenitors (Plp1-EGFP), enteric neurons (BAF53b::tdT), and double negative cells from flow cytometric analysis. FIG.16A – FIG.16S show neurospheres generated from MyGa retain neuronal subtypes important for the recapitulation of the ENS and MyGa-derived ENSCs exhibit higher rates of enteric neurogenesis. FIG.16A - FIG.16Hʹ show representative images of monolayer cultures originally derived from intramuscular single cells (IM-SCs) or the MyGa enriched fractions immunohistochemically labelled for calretinin (FIG.16A - FIG.16Bʹ), nNOS (FIG.16C - FIG.16D’), GFAP (FIG.16E - FIG.16Fʹ) and P75 (FIG.16G - FIG.16Hʹ). FIG.16A – FIG.16H, Scale bars = 1000µm. FIG.16A – 16H’, Scale bars = 250 µm. FIG.16I shows quantification of the percentage of EGCs / progenitors (Plp1-EGFP) and enteric neurons (BAF53b::tdT) in monolayer cultures determined by fluorescent imaging. FIG.16J shows EGC to neuron ratios in the same samples determined by Plp1-EGFP and BAF53b::tdT fluorescence. Data are mean ± SEM. Unpaired t-test, **** p < 0.0001, n = 19-20 independent cultures per group. FIG.16K - FIG.16N shows quantification of the percentage of neurons (BAF53b::tdT) immunoreactive (IR) for calretinin (k) and nNOS (l), as well as the percentage of EGCs / progenitors (Plp1- GFP) IR for GFAP (m) and P75 (n). Data are mean ± SEM. Unpaired t-test, *** p < 0.001, n = 5-7 independent cultures per group. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.16O shows Plp1-EGFP cells immediately after purification by fluorescence activated cell sorting of cultures derived from IM-SCs or MyGa enriched fractions yielding IM-ENSCs and MyGa- ENSCs. Scale bars = 250µm. FIG.16P shows expression of the neuronal marker BAF53b::tdT in neurospheres generated from IM-ENSCs and MyGa-ENSCs after 6 days in culture. Scale bars = 100µm. FIG.16Q shows IM-ENSC and MyGa-ENSC cultures after migration for 4 days on fibronectin. Scale bars = 200µm. FIG.16R – FIG.16S shows EGC to neuron ratios (FIG.16R) and the percentage of neurons to the total number of cells (FIG.16S) in the same samples determined by Plp1-EGFP and BAF53b::tdT fluorescence. Data are mean ± SEM. Mann-Whitney, ** p < 0.01, n = 6 independent cultures per group. FIG.17A – FIG.17P show MyGa-derived cells generate calcium transients in vitro and show functional competency following transplantation to the intestine in vivo. FIG.17A - FIG.17Aʹ show enteric neurons derived from MyGa cultures generated from BAF53b::tdT-GCaMP mice. Expression of tdT in enteric neurons (FIG.17A) and calcium transients in response to ACh (FIG.17A’).. Scale bar = 300µm FIG.17B shows representative traces of calcium transients (ΔF / F0) in response to ACh stimulation. FIG.17C - FIG.17C’ show enteric neurons derived from MyGa cultures from BAF53b::tdT- GCaMP mice as above with BAF53b::tdT expression (FIG.17C) and calcium transients in response to electric field stimulation (EFS) (FIG.17C’). Scale bar = 100µm FIG.17D shows representative traces of calcium transients (ΔF / F0) in response to EFS stimulation. FIG.17E shows MyGa-derived neurospheres from BAF53b::tdT; Plp1-EGFP mice transplanted to the colonic muscularis propria of a colorless recipient. Scale bar = 500µm. FIG.17E’ - FIG.17E’’ show high magnification images of the cell transplantation site (FIG.17Eʹ) and the extension of nerve fiber processes from the transplant (FIG.17E’’). Scale bars = 250µm. FIG.17F shows integration of transplanted BAF53b::tdT; Plp1-EGFP cells with the endogenous myenteric plexus of the recipient labeled by Tuj1. Scale bars = 100µm. FIG.17G shows BAF53b::tdT; Plp1-EGFP cell transplantations expressing the progenitor marker P75. Scale bars = 50µm. FIG.17G’ shows interconnected network formed between transplanted cells. Scale bar = 25µm. FIG.17H shows neurospheres generated from IM-SCs and the MyGa enriched fractions from Chat-tdT-ChR2 mice. Scale bars = 200µm. FIG.17I shows Chat-tdT expression in Hu immunoreactive enteric neurons in cultures of MyGa- derived cells. Scale bars = 200µm. FIG.17J shows the percentage of neurospheres from IM-SCs and MyGa enriched fractions containing Chat-tdT neurons. Data are mean ± SEM. Unpaired t-test, ** p < 0.01, n = 3 independent cultures per group. FIG.17K shows Chat-tdT cells from MyGa-derived neurospheres transplanted into the muscularis propria of a Plp1-EGFP recipient mouse. Scale bar = 200µm. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.17L shows an Experimental setup for simultaneous recordings of luminal pressure and electromyography (EMG) in vivo. FIG.17M shows an in vivo EMG in the smooth muscle and intraluminal pressure of the colon in response to blue light stimulation (BLS) of transplanted Chat-tdT-ChR2 cells. FIG.17N shows an experimental setup for colonic ring force contraction recordings ex vivo. FIG.17O shows force of contraction produced by colonic smooth muscle in response to BLS of transplanted Chat-tdT-ChR2 cells in ex vivo organ bath experiments. FIG.17P shows a trace of the above with BLS of transplanted BAF53b::tdT-ChR2 cells and TTX inhibition of neural activity. FIG.18A – FIG.18F show intact MyGa can be isolated from the intestinal muscularis propria in resected human specimens. FIG.18A shows representative images of the myenteric plexus in the muscularis propria from a resected colon immunohistochemically labeled with the pan-neuronal markers PGP9.5 and HuC / D. Scale bars = 100µm. FIG.18B shows an average minimum Feret’s diameter of MyGa from subjects with a normal enteric nervous system and the ganglionated segment of those with Hirschsprung disease (enteric neurocristopathy). Data shown as mean ± 95% CI, n = 3-10 ganglia per sample. FIG.18C shows immunohistochemical labelling of TUBB3 in wholemount preparations of the muscularis propria and high magnification image of the MyGa (boxed area). Scale bar = 1mm. FIG.18D shows labelling of TUBB3 after enzymatic digestion of the muscularis propria. Scale bars = 500µm. Boxed area shows higher magnification of a MyGa. Scale bars = 100µm. FIG.18E shows quantitative PCR of PLP1, NGFR, PHOX2B and ELAVL4 in digested single cell suspensions (IM-SCs) and the MyGa-enriched fractions of resected specimens. Data shown as mean ± SEM. Two-way ANOVA with Holm-Sidak posthoc test, * p < 0.05, ** p < 0.01, **** p < 0.0001, n = 4 individuals per group. FIG.18F shows representative images of neurosphere formation from manually selected fragment of myenteric ganglia. Scale bars = 500µm. FIG.19A – FIG.19T show neurospheres generated from human MyGa are highly neurogenic and contain ENSC subpopulations with distinct transcriptional profiles. FIG.19A - FIG.19Aʹ show free floating neurospheres generated from the MyGa-enriched fraction (counter filtered) of human specimens. FIG.19A, Scale bar = 500µm. FIG.19A ʹ, scale bar = 100µm. FIG.19B shows population doubling levels of cultures generated from intramuscular single cells (IM-SCs) and counter-filtered fragments enriched for MyGa at the first (P1) and second (P2) passage. n = 5-10 individuals per group. FIG.19C shows total cell counts before cells were passaged (P0) and estimated total yield of cells from proliferation assays at the first and second passages normalized per gram of starting tissue. n = 4-12 individuals per group. FIG.19D shows quantitative PCR of PHOX2B, TUBB3, PLP1 and NGFR, prior to passaging, and at the first and second passage in neurospheres generated from IM-SCs and the MyGa-enriched fractions of resected specimens. Two-way ANOVA with Holm-Sidak posthoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3-9 individuals per group. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.19E shows quantitative PCR in neurospheres at the first passage generated from IM-SCs, the MyGa-enriched fractions (MyGa), and pure hand-picked MyGa (picked MyGa). One-way ANOVA with Holm-Sidak posthoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3-9 individuals per group. FIG.19F - FIG.19G show UMAP representation of cells from cultures of IM-SCs, the MyGa- enriched fractions (MyGa), and pure hand-picked MyGa (picked MyGa) (FIG.19F) and unsupervised clustering (FIG.19G) of cell populations. FIG.19H shows the proportion of cell populations in cells generated from IM-SCs, the MyGa- enriched fractions (MyGa), and pure hand-picked MyGa (picked MyGa) defined by unsupervised clustering. FIG.19I shows UMAP visualization of bona fide markers for EGC / ENSCs (SOX10 and PLP1), enteric neurons (ELAVL4) and enteric mesenchymal cells (PDGFRA). FIG.19J shows a heatmap visualization of the top 10 markers by LogFC (>60% of cells) for each cell population. Data are presented as z-scores. FIG.19K - FIG.19L show UMAP representation of EGC / ENSC subpopulations from cultures of IM-SCs, the MyGa-enriched fractions (MyGa), and pure hand-picked MyGa (picked MyGa) (FIG.19K) and unsupervised clustering (FIG.19L) of populations. FIG.19M shows UMAP visualization of gene expression markers for human EGC / ENSCs and their subpopulations. FIG.19N shows a ridgeplot of the expression of neuronal markers and pro-neurogenic factors in EGC / ENSCs and enteric neurons. FIG.19O - FIG.19Q shows representative images of immunocytochemical labelling of ITGA6, NGFR (FIG.19O - FIG.19P) and TUBB3 (FIG.19Q) in monolayer cultures. Scale bars = 200µm. FIG.19R shows quantification of the proportion of cells expressing ITGA6, NGFR and TUBB3. n = 5 subjects per group, Ratio paired t-test, * p < 0.05, *** p < 0.001, n = 5 individuals per group. FIG.19S shows clustering of cells from cultures of mouse neurospheres. FIG.19T shows expression module scores for human enteric neuron and EGC / ENSC 1 and 2 population markers in mouse neurosphere cell populations. FIG.20A – FIG.20H shows human MyGa-derived cells demonstrate functional competency and have utility for cell therapy applications. FIG.20A shows maximum intensity projections of calcium responses over time using the Fluo-4 calcium indicator in human MyGa derived cultures stimulated with ACh, or with ACh stimulation following pretreatment with tetrodotoxin (TTX). Scale bars = 500µm. FIG.20B shows still images of calcium transients using the Fluo-4 calcium indicator following the application of ACh (T0) after 10 and 20 seconds. Scale bars = 500µm. FIG.20B’ shows representative trace of a calcium response (ΔF / F0) to ACh in an individual cell. FIG.20C shows a heatmap of calcium transients (ΔF / F0) with the y-axis representing individual cells and the x-axis representing time. FIG.20D shows expression of the GFP reporter after transduction of MyGa-derived cells with AAV6-ChR2-GFP in monolayer cultures on fibronectin. Scale bar = 500µm. Dotted inset scale bar = 100µm. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.20E shows expression of GFP in AAV6-ChR2-GFP transduced cells after reformation of neurospheres in free floating conditions. Scale bar = 500µm. FIG.20F shows a representative image of transplanted MyGa-derived cells to the muscularis propria of NOD-scid IL2Rgammanull(NSG) mice after 3 weeks of engraftment. Samples were labeled for the neuronal marker Tuj1 and stained with DAPI. Scale bar = 200µm. FIG.20G shows high magnification images of transplanted cells defined by GFP expression and overlapping expression of Tuj1. Scale bar = 100µm. FIG.20H shows force of contraction produced by colonic smooth muscle in response to blue light stimulation (BLS) of transplanted cells expressing AAV6-ChR2-GFP in ex vivo organ bath experiments. FIG 20I shows quantification of contractile force in colonic smooth muscle in response to blue light stimulation (BLS) compared to baseline measurements in the same sample. Paired t-test, * p < 0.05, n = 3 mice per group. FIG.20J shows representative traces of smooth muscle contractions in the presence of TTX. FIG.21A- FIG.21F shows additional scRNA-seq analysis. Related to FIG.19. FIG.21A shows dotplot visualization of markers for EGC / ENSCs, muscularis EGCs and enteric neurons in human cultures. FIG.21B shows UMAP visualization of gene expression markers for human (left) and mouse (right) EGC / ENSC 1 and 2 subpopulations. FIG.21C shows UMAP visualization of glia clusters from human intestine defined by Drokhlyansky et al.2020 (Cell 182, 1606-1622.e23). In particular, in Drokhlyansky, cells were clustered at two stages of the analysis: first, to initially partition the cells into neuron, glia, and “other” compartments, and second, to sub-cluster neurons and glia into different subsets. In all cases, we ran low-rank PCA on the variable genes of the batch-corrected log2(TP10K+1) expression matrix. We then applied Leiden clustering (Traag et al. (2019). Sci. Rep.9, 5233) or Phenograph (Levine et al. (2015). Cell 162, 184–197) to the k-NN graph defined using the first n PCs and k nearest neighbors, which were separately estimated for each dataset. Leiden was used to cluster nuclei from droplet-based data (due to its speed), while Phenograph was used for clustering of all ENS cells. To estimate n, we calculated the number of “significant” PCs using a permutation test. Because this test may underestimate the number of PCs, we conservatively increased this number (i.e. to 15 or 30; see table below) to ensure that most of the variability in the dataset was captured. Next, to estimate k, we considered a range of clustering solutions with varying values of k, and calculated the marker genes for each set of clusters. We selected k based on inspection of the data. When clustering data from multiple cell types, we tried to select k such that the major cell types (e.g. neurons, glia, and muscle) were split, without fragmenting them into several sub- clusters. When clustering neurons and glia, we tried to select a k yielding the highest granularity clusters that were still biologically distinct, determined by close examination of the marker gene lists. Finally, the Barnes-Hut t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm was run on the selected PCs with perplexity = 20 and for 1,000 iterations to produce two-dimensional embeddings of the data for visualization. FIG.21D – FIG.21E show module scoring of human EGC / ENSC 1 and 2 markers in human glia populations. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.21F show expression of intraganglionic EGC markers defined by Guyer et al.2023 (Cell Reports 42, 112194.) in human and mouse EGC / ENSC cultures. In particular, in Guyer, scRNA-seq and snMulti-seq data was analyzed with the open-source Seurat and Signac packages implemented in the R computing environment. For the postnatal glial scRNA-seq dataset and neurosphere datasets, cells more than one standard deviation away from the mean number of genes detected were filtered, as were cells with greater than 10% mitochondrial genes. Datasets were integrated using the SCTransform workflow in Seurat (Hafemeister et al. (2019). Genome Biol 20, 296; Choudhary et al. (2022). Genome Biol 23, 27). After integration, principle component analysis (PCA) was performed. Neighbors were identified and UMAP projection was performed using the first 30 principal components. Clusters were identified using the “FindClusters” command with resolution = 0.5 using the Louvain algorithm. FIG.22A - FIG.22G show a scheme for generating neurospheres from ganglionated bowel of Hirschsprung disease subjects. FIG.22A depicts an image showing the muscularis layer of human colon tissue after separation from the mucosa and removal of residual submucosa and blood vessels. This step ensures clean dissection for downstream digestion and cell isolation. FIG.22B depicts an image showing the minced tissue fragments (3–5^mm pieces) prior to enzymatic digestion. This step increases surface area for efficient enzymatic action. FIG.22C depicts an image showing the resulting cell / tissue suspension following sequential enzymatic and mechanical dissociation. The uniformity of the suspension indicates successful digestion and trituration. FIG.22D depicts an image of a sample after a 1000^μm filter was used to remove large tissue fragments from the suspension. This is an initial filtration step to clean up the suspension. FIG.22E depicts an image showing the sample after the 40^μm counter filtration step. Clarified fragment suspension is enriched for ENSC-containing ganglionic fragments. FIG.22F shows a microscopic image highlighting visible ganglionic fragments within the suspension, confirming successful isolation of target ENSC-containing tissue fragments. FIG.22G shows an image of neurospheres formed in ultra-low attachment culture by Day 10. These neurospheres represent aggregations of proliferating enteric neural stem cells derived from human colon. FIG.23A – FIG.23C show an analysis verifying the presence of ENSCs in samples from Hirschsprung disease patients. FIG.23A is a bar graph showing gene expression analysis of neurospheres derived from Hirschsprung disease patient samples. Expression of ENSC-associated genes PLP1 (gliogenic) and PHOX2B (neuronal) was quantified by qPCR and normalized to GAPDH. Data are shown as the relative linear scale normalized expression between two controls: SH-SY5Y neuroblastoma cells (positive control, expression = 1) and cancer-associated fibroblasts (negative control, expression = 0). All HSCR-derived cultures demonstrated positive expression of both markers, indicating the presence of enteric neural stem cells in culture. FIG.23B – FIG.23C show immunocytochemical images of neurospheres derived from HSCR patient samples, cultured as monolayers on fibronectin-coated dishes. Images show expression of TGA6 and NGFR, surface markers enriched in ENSCs, and L1CAM, consistent with committed neural PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 progenitors exhibiting neurite outgrowth. Data in A and B represent two separate donors. These data support the presence of neural progenitor and stem cell populations. FIG.24A – FIG.24F show transplantation and function of ENSCs from Hirschsprung disease subjects to aganglionic Hirschsprung disease tissues. FIG.24A shows images depicting expression of GFP (AAV6-ChR2-GFP) in transduced human cells derived from the ganglionated bowel of Hirschsprung Disease subjects after transplantation to the aganglionic segment in cultured ex vivo for 2 weeks. Expression of neuronal markers TUBB3 and PGP9.5 are shown. Higher magnification image of the inset shown to highlight neurite projections originating from the transplanted cells. FIG.24B – FIG.24C show representative muscle strip myography traces from transplanted aganglionic colon tissues. The traces show normalized contractile force relative to baseline. FIG.24B illustrates a typical smooth muscle response to blue light stimulation of transplanted ChR2-expressing cells. FIG.24C depicts a representative trace of a randomly selected timepoint (random seed) from within the same dataset, used to model spontaneous contractile events and establish pseudo-stimulation behavior. FIG.24D is a bar graph showing the percentage of detected smooth muscle responses during the blue light stimulation and post-stimulation (rebound) periods. Comparisons are made between true stimulation events and random seed control periods to distinguish evoked from spontaneous activity. FIG.24E is a bar graph comparing the latency to response following stimulation across real and pseudo-stimulation conditions (random). Responses in the true stimulation and post-stimulation periods show significantly reduced latency relative to randomly seeded controls. FIG.24F displays bar graphs comparing the negative area under the curve (AUC), relaxation duration, and relative force of smooth muscle relaxation below baseline across conditions. Real stimulation significantly increased all three parameters compared to random seed controls, indicating reliable inhibitory responses mediated by transplanted cells. All bar graphs represent mean ± SEM. FIG.25A – FIG.25E show cryopreservation of ENSCs maintains their neurosphere forming ability and function. FIG.25A shows human enteric neural stem cells, previously cultured and dissociated into single- cell suspensions, were cryopreserved using a controlled-rate freezing protocol and stored in liquid nitrogen. Upon thawing and culture in ultra-low attachment conditions, neurospheres reformed within 48 hours, demonstrating recovery and viability. Notably, direct cryopreservation of intact neurospheres failed to yield viable cultures after thawing, indicating the necessity of single-cell suspension prior to freezing. Data shown are of neurospheres formed post-thaw at 4X and 10X magnification on a Keyence All-in-one imaging platform. FIG.25B shows a representative muscle strip myography trace showing contractile force over time from ex vivo colon preparations of BAF53b-iDTR transgenic mice following diphtheria toxin-induced ablation of endogenous enteric neurons and subsequent transplantation of cryopreserved, AAV6-ChR2- GFP-transduced human ENSCs. Blue light stimulation elicited a robust contractile response during and after stimulation. FIG.25C illustrates a control trace showing contractile activity from the same experimental PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 dataset during a randomly selected 30-second time window, used to model spontaneous contractile behavior and establish pseudo-stimulation responses. FIG.25D shows quantification of the percentage of samples exhibiting detectable contractile responses during the stimulation and post-stimulation (rebound) periods versus random seed control periods. ENSC-derived contractions were significantly more frequent than expected by chance, supporting functional integration of transplanted cells. FIG.25E shows bar graphs comparing contractile force metrics between real blue light stimulation periods and random seed control conditions across the stimulus and post-stimulus period. Data are mean contractile force in the stimulus period, mean contractile force in the post-stimulus period, and mean contractile force area under the curve [AUC] in the post-stimulus period in 3 separate preparations. Blue light stimulation elicited significantly greater mean contractile force and rebound activity (mean force and area under the curve [AUC]), demonstrating preserved neuromuscular function of transplanted ENSCs after cryopreservation. All data are shown as mean^±^SEM. FIG.26A – FIG.26D show treating gastroparesis in a neuronal ablation mouse model. FIG.26A shows a neuronal ablation model of gastroparesis generated by injecting diphtheria toxin (DT) into the gastric corpus of BAF53b-iDTR transgenic mice, which express the diphtheria toxin receptor selectively in neurons under control of the BAF53b promoter. Two weeks after DT administration, gastric emptying was assessed by intragastric gavage of barium sulfate and steel beads. Images show X-ray imaging performed 90 minutes post-gavage and revealed retention of contrast and beads within the stomach in ablated animals. FIG.26B – FIG.26C show a quantification of gastric emptying for solids (FIG.26B, % steel beads passed from the stomach) and liquids (FIG.26C, integrated pixel intensity of barium contrast outside the stomach normalized to the entire GI tract). Neuron-ablated mice exhibited impaired emptying of both solids and liquids compared to untreated controls, confirming a gastroparesis phenotype. In two trails utilizing ENSCs isolated using our specialized methods of making improved solid and liquid emptying was observed compared to gastroparetic controls. All data are presented as mean ± SEM. FIG.26D shows fluorescent imaging confirming engraftment of transplanted cells traced via transgenic BAF53b promoter driven tdTomato+ expression in the gastric walls of recipient mice. Images of BAF53b::tdTomato expression highlight successful neuronal differentiation and neurite network formation from transplanted cells within the gastric wall. FIG.27A – FIG.27F show neurogenesis declines in enteric neuropsheres with increasing age of mice. FIG.27A shows a schematic overview of neurosphere cultures from BAF53b::tdT; Plp1-EGFP dual-reporter mice at a range of ages. FIG.27B shows cells isolated from BAF53b::tdT; Plp1-EGFP dual-reporter mice at 2 weeks, 2 months, 4 months, 8 months, and 1 year of age give rise to GFP+ and tdT+ neurospheres in culture, with Plp1-EGFP serving as a EG / NP marker and BAF53b::tdT as a neuronal marker. Images are representative of whole wells in culture plates. Scale bars = 5mm. FIG.27C shows a comparison of neurospheres cultured from 2-week-old mice vs.1-year-old mice. Images highlight GFP and tdT channel overlays. Scale bars = 1000 um. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.27D shows a quantification of PCR data from neurospheres of 2-week-old mice vs.1-year- old mice. Data are shown as mean ± SEM. Unpaired t-test,∗p < 0.05; n = 3 mice per group. Scale bars = 1000 um. FIG.27E Process of GFP fluorescence binarization, followed by highlighting of binarized regions of interest (ROI), and tdT mean fluorescence intensity (MFI) quantification in the GFP-binarized ROIs. FIG.27F Quantification of MFI of tdT in GFP-binarized ROI. Kruskal-Wallis ANOVA with Dunn’s posthoc test,∗∗∗∗p < 0.0001; n = 51-224 neurospheres per group; individual data points from all wells combined for each age group. Scale bars = 1000 um. FIG.28A – FIG.28F show culture format influences cell composition and assay sensitivity in enteric neural cultures. FIG.28A shows a schematic representation of neurosphere cultures grown under monolayer and free-floating conditions (3D culture) from BAF53b::tdT; Plp1-EGFP mice. FIG.28B shows representative images of neurospheres generated from BAF53b::tdT; Plp1-EGFP dual-reporter mice, in the free-floating group at the 3-week timepoint of culture. Scale bars = 500um. FIG.28C shows representative images of neurospheres generated from BAF53b::tdT; Plp1- EGFP dual-reporter mice, in the monolayer group at the 3-week timepoint of culture. Scale bars = 500um (FIG.28C top), 250um (FIG.28C bottom). FIG.28D shows a quantitative PCR of Plp1, Gfap, Ngfr, Phox2b, and Elavl4 in neurospheres from the free-floating and monolayer groups. Data are shown as mean ± SEM. Two-way ANOVA with Holm-Sidak posthoc test,∗p < 0.05,∗∗p < 0.01; n = 3 mice per group. FIG.28E shows a gating for flow cytometry of EG / NPs (Plp1-EGFP), ENs (BAF53b::tdT), and EMCs (double-negative cells) from BAF53b::tdT; Plp1-EGFP mice. FIG.28F shows a quantification of flow cytometry for EG / NPs (Plp1-EGFP), ENs (BAF53b::tdT), and EMCs (double-negative cells) from free-floating and monolayer groups. Data are shown as mean ± SEM. One-way ANOVA with Holm-Sidak posthoc test,∗∗p < 0.01,∗∗∗p < 0.001,∗∗∗∗p < 0.0001; n = 3-4 wells per group. FIG.29A – FIG.29P show bFGF promotes enteric glial / neural progenitor expansion while RA and GDNF synergize for neuronal differentiation in adult-derived enteric neurospheres. FIG.29A – FIG.29D show a representative GFP-channel images of neurospheres generated from 3-month-old BAF53b::tdT; Plp1-EGFP dual-reporter mice and cultured in negative control media (- CM), positive control media (+CM), or bFGF media conditions. Representation of GFP fluorescence binarization and tdT fluorescence for neurospheres in the -CM (FIG.29B), +CM (FIG.29C), or bFGF (FIG.29D) media conditions. Scale bars = 2mm. FIG.29E shows a quantification of area (um2) per well covered by neurospheres in the -CM, +CM, and bFGF media conditions, based on binarized GFP expression. Data are shown as mean ± SEM. One-way ANOVA with Holm-Sidak posthoc test,∗∗p < 0.01,∗∗∗p < 0.001,∗∗∗∗p < 0.0001; n = 4 independent cultures per group. FIG.29F shows a quantification of tdT MFI in GFP-binarized neurospheres cultured in +CM and bFGF media conditions. Mann-Whitney test,∗∗∗∗p < 0.0001; n = 639 vs 443 neurospheres. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.29G shows a quantification of flow cytometry for EG / NPs (Plp1-EGFP) in neurospheres cultured in the -CM, +CM, and bFGF media conditions. Data are shown as mean ± SEM. One-way ANOVA with Holm-Sidak posthoc test,∗∗∗∗p < 0.0001; n = 4 independent cultures per group. FIG.29H shows a quantification of flow cytometry for ENs (BAF53b::tdT) in neurospheres cultured in the -CM, +CM, and bFGF conditions. Data are shown as mean ± SEM. One-way ANOVA with Holm-Sidak posthoc test; n = 4 independent cultures per group. FIG.29I – FIG.29L show a representative GFP-channel images of neurospheres generated from BAF53b::tdT; Plp1-EGFP dual-reporter mice and cultured in GDNF, RA, or GDNF + RA (G+R) media conditions. J-L) Representation of GFP fluorescence binarization and tdT fluorescence for neurospheres in the GDNF (FIG.29J), RA (FIG.29K), or G+R (FIG.29L) media conditions. Scale bars = 2mm. FIG.29M shows a quantification of area (um2) per well covered by neurospheres in the -CM, +CM, GDNF, RA, and G+R media conditions, based on binarized GFP expression. Data are shown as mean ± SEM. One-way ANOVA with Holm-Sidak posthoc test,∗∗∗p < 0.001,∗∗∗∗p < 0.0001; n = 4 independent cultures per group. FIG.29N shows a quantification of tdT MFI in GFP-binarized neurospheres cultured in G+R and +CM media conditions. Data are shown as mean ± SEM. Mann-Whitney test,∗∗∗∗p < 0.0001; n = 639 vs 248 neurospheres. FIG.29O shows quantification of flow cytometry for EG / NPs (Plp1-EGFP) in neurospheres cultured in the -CM, +CM, GDNF, RA, and G+R media conditions. Data are shown as mean ± SEM. One- way ANOVA with Holm-Sidak posthoc test,∗∗p < 0.01,∗∗∗p < 0.001,∗∗∗∗p < 0.0001; n = 4 independent cultures per group. FIG.29P shows a quantification of flow cytometry for ENs (BAF53b::tdT) in neurospheres cultured in the -CM, +CM, GDNF, RA, and G+R media conditions. Data are shown as mean ± SEM. One- way ANOVA with Holm-Sidak posthoc test,∗∗∗p < 0.001; n = 4 independent cultures per group. FIG.29N - FIG.29P) -CM, +CM data from FIG.29E – FIG.29H) are included as a basis of comparison. FIG.30A – FIG.30H show GDNF, RA and bFGF (GRF) improves enteric neuronal composition while maintaining enteric glial / neural progenitor expansion in adult-derived neurosphere cultures. FIG.30A shows a schematic representation of neurosphere cultures grown in positive control media (+CM), bFGF, and GDNF + RA + bFGF (GRF) media. FIG.30B shows representative images of neurospheres generated from BAF53b::tdT; Plp1-EGFP dual-reporter mice, cultured in the +CM media. FIG.30C shows representative images of neurospheres generated from BAF53b::tdT; Plp1- EGFP dual-reporter mice, cultured in the GDNF + RA + bFGF (GRF) media. Scale bars = 2mm. FIG.30D shows a quantitative PCR of Plp1, Gfap, Ngfr, Pdgfra, Phox2b, And Elavl4 in neurospheres from the +CM and GRF media groups. Data are shown as mean ± SEM. Unpaired t-test,∗p < 0.05,∗∗∗p < 0.001; n = 6 independent cultures per group. FIG.30E – FIG.30F Representative images of monolayer cultures in +CM (FIG.30E) and GRF (FIG.30F) media. Scale bars = 500um. FIG.30G shows a representation of cells in the +CM and GRF media groups falling into the gating thresholds set for EG / NPs (Plp1-EGFP) and ENs (BAF53b::tdT) from BAF53b::tdT; Plp1-EGFP mice. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 FIG.30H shows a quantification of flow cytometry for EG / NPs (Plp1-EGFP) and ENs (BAF53b::tdT) in neurospheres cultured in the +CM and GRF media groups. Data are shown as mean ± SEM. Unpaired t-test,∗p < 0.05,∗∗p < 0.01; n = 10 independent cultures per group. FIG.31A – FIG.31O show GDNF, RA and bFGF (GRF) promote enteric neurogenesis directly from enteric glial / neural progenitors. FIG.31A shows a schematic illustration of ENs (BAF53b::tdT) sorted from BAF53b::tdT; Plp1- EGFP dual-reporter mice, used for survival and neurogenesis assays performed on neurospheres isolated directly from BAF53b::tdT; Plp1-EGFP mice. FIG.31B shows Representative tdT-channel images of ENs (BAF53b::tdT) sorted from BAF53b::tdT; Plp1-EGFP dual-reporter mice, then cultured in negative control (-CM), positive control (+CM), or GDNF + RA + bFGF (GRF) media conditions, at the 2-week timepoint of culture. Scale bars = 500um. FIG.31C shows a quantification of neurons per well in ENs (BAF53b::tdT) as above. Data are shown as mean ± SEM. One-way ANOVA with Holm-Sidak posthoc test,∗p < 0.05,∗∗p < 0.01; n = 3 independent cultures per group. FIG.31D shows representative images of neurosphere formation from EG / NPs (Plp1-EGFP), sorted from BAF53b::tdT; Plp1-EGFP dual-reporter mice, at the 2-week timepoint of culture in -CM, +CM, bFGF, GDNF + RA (G+R), or GRF media conditions. All images were taken at the same exposure for comparative purposes. Scale bars = 200um. FIG.31E shows a quantification of tdT MFI in GFP-binarized neurospheres as above. Data are shown as mean ± SEM. One-way ANOVA with Holm-Sidak posthoc test,∗p < 0.05,∗∗∗∗p < 0.0001; n = 4 independent cultures per group. FIG.31F – FIG.31O show representative images of EG / NPs (Plp1-EGFP) and ENs (BAF53b::tdT) in sections of neurospheres cultured in the bFGF (FIG.31F) and GRF media (FIG.31K). Sectioned neurospheres cultured in the bFGF (FIG.31G – FIG.31J) and GRF media (FIG.31L – FIG. 31O) and immunocytochemically labeled for SOX10 (FIG.31G, FIG.31L), GFAP (FIG.31 H, FIG.31M), S100B (FIG.31I, FIG.31N), and HuC / D (FIG.31J, FIG.31O). Scale bars = 50um . FIG.32A – FIG.32L show neuronal and enteric glial / neural progenitor composition in vitro parallels graft-derived network formation after transplantation. FIG.32A shows a schematic illustration showing transplanted neurospheres grown in bFGF and GRF media prior to transplantation. FIG.32B shows representative images of BAF53b::tdT; Plp1-EGFP neurospheres grown in GRF media immediately after transplantation to the distal colon. FIG.32C – FIG.32D show representative images of transplants derived from neurospheres grown in bFGF (FIG.32C) and GRF (FIG.32D) media 12 weeks after implantation. Scale bars = 1mm. FIG.32E – FIG.32F show higher magnification images of individual Plp1-EGFP+EGCs cells and BAF53b::tdT+ENs derived from neurospheres grown in bFGF (FIG.32E) and GRF (FIG.32F) media. Scale bars = 100um. FIG.32G – FIG.32L show quantification of BAF53b::tdT / Plp1-EGFP cell ratios (FIG.32G), number of tdT+ neurite branches (FIG.32H), number of neurite junctions (FIG.32I), maximum neurite branch length (FIG.32J), mean neurite branch length (FIG.32K) and sum of neurite branch lengths (FIG. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 32L) between bFGF and GRF neurosphere transplants. Data are shown as mean ± SEM. Mann-Whitney test,∗p < 0.05. n = 4 transplants per group. FIG.33A - FIG.33I’ show GDNF, RA and bFGF (GRF) media promotes neuronal composition in human cultures. FIG.33A shows a schematic representation of neurospheres generated from human intestinal tissue and grown in positive control media (+CM) and GDNF + RA + bFGF (GRF) media. FIG.33B shows a representative image of tissue after dissection and isolation of the muscularis propria. Scale bars = 1 cm. FIG.33C shows representative images of neurospheres grown in +CM and GRF media. Scale bars = 500um. FIG.33D shows an expression of EG / NP markers PLP1, NGFR and the neuronal marker PHOX2B in neurospheres after 10-14 days of free-floating culture. Data are shown as mean ± SEM. Two- way ANOVA with Holm-Sidak posthoc test,∗∗p < 0.01,∗∗∗p < 0.001; n = 3-4 subjects. FIG.33E shows a representative image of neurospheres transferred to monolayer conditions in GRF media. Scale bars = 500um. FIG.33F shows a cell yield divided by initial weight of resected tissue (mg). Data are shown as mean ± SEM. Ratio paired t-test,∗p < 0.05; n = 4 subjects. FIG.33G shows a quantification of the percentage of cells expressing ITGA6, NFGR and TUBB3. Data are shown as mean ± SEM. Two-way ANOVA,∗∗p < 0.01; n = 3 subjects. FIG.33H – FIG.33I Representative images from samples grown in +CM (FIG.33H) and GRF media (FIG.33I and FIG.33I’) and immunohistochemically labeled for ITGA6, NFGR and TUBB3. Scale bars = 500um. DETAILED DESCRIPTION OF THE INVENTION Disclosed are compositions including a population of human enteric neural stem cells (ENSCs), where the population includes at least one of a cell expressing nerve growth factor receptor (NGFR), a cell expressing integrin alpha 6 (ITGA6), a cell expressing L1 cell adhesion molecule (L1CAM), a cell that expresses proteolipid protein 1 (PLP1), and / or a cell that expresses paired-like homeobox 2B (PHOX2B). Also disclosed are methods of obtaining such ENSCs and tissue transplants (e.g., neurospheres) including such ENSCs, as well as their uses in treating neurointestinal diseases, including Hirschsprung disease and gastroparesis. Markers for ENSCs The human ENSCs of the disclosure express NGFR, ITGA6, L1CAM, PLP1, and / or PHOX2B. In one embodiment, the human ENSCs express each of NGFR, ITGA6, and L1CAM at the protein level. In another embodiment the human ENSCs further express PLP1 and PHOX2B at the mRNA level. In one embodiment, the ENSCs are autologous to a patient being treated. PLP1 is a glial / neuronal progenitor biomarker known as proteolipid protein 1, which is the primary constituent of myelin in the central nervous system. The human gene encoding Plp encodes a 276-amino acid polypeptide with 5 strongly hydrophobic domains that interact with the lipid bilayer as trans- and cis- membrane segments. Diehl, Schaich, Budzinski, and Stoffel (1986) determined that the human Plp gene PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 contains 7 exons and spans approximately 17 kb. Quantification of Plp1 expressing cells can be done via flow cytometry and immunohistochemistry. Expression of the human Plp1 can be assayed by PCR using, for example, the RNeasy Micro Kit (Qiagen). PCR can be used to validate the samples as it detects expression at the gene (mRNA) level in cell lysates. The human PLP1 protein can also be assayed using enzyme-linked immunosorbent assay (ELISA), for example, the “human proteolipid protein 1, myelin (PLP1) ELISA kit”. Such assays generally have high sensitivity and excellent specificity for protein detection. Typically, ELISAs are used to quantify secreted proteins. They can be used to measure proteins like PLP1 in a cell lysate. mRNA expression can be assessed, for example, by qPCR or by RNA sequencing. Neuronal biomarker nerve growth factor receptor (NGFR) is also referred to as p75 neurotrophin receptor (P75(NTR)) because of its molecular mass and its ability to bind at low affinity to not only NGF, but also other neurotrophins such as neurotrophin-3, neurotrophin-4, and brain-derived neurotrophic factor. It is a low affinity nerve growth factor receptor and is important for developing neurons, refinement of neuronal connections, neuronal survival, and death. Human sequence information for P75 is known in the art (Johnson et al., 1986; Ota et al., 2004). Quantification of P75 expressing cells can be done via immunohistochemistry, via PCR, for example, using the RNeasy Micro Kit (Qiagen), to measure expression at the gene (mRNA) level in cell lysates or via using ELISA kits such as the Biosensis NGFR / p75ECDELISA kit on cell lysates. Integrin alpha 6 (ITGA6) is a heterodimeric integral membrane protein composed of an alpha chain and a beta chain that function in cell surface adhesion and signaling. The gene encodes preproprotein is proteolytically processed to generate light and heavy chains that comprise the alpha 6 subunit. ITGA6 is widely expressed, including in the colon. ITGA6 is a marker for EGC / ENSC cells and expression may be detected, for example, using an immunohistochemistry assay, flow cytometry, or Western blotting. L1 cell adhesion molecule (L1CAM) is a neural cell adhesion molecule that is a canonical marker of developing enteric neurons. Expression may be detected, for example, using an immunohistochemistry assay, flow cytometry, or Western blotting. Paired-like homeobox 2B (PHOX2B) is a pro-neurogenic gene and a marker of ENSCs. mRNA expression can be assessed, for example, by quantitative polymerase chain reaction (qPCR) or RNA sequencing. Methods of treating Hirschsprung disease Hirschsprung disease (HSCR) is a condition that affects the large intestine (colon) and causesproblems with passing stool. The condition is present at birth (congenital) as a result of missing nerve cells in the muscles of the infant's colon. Without these nerve cells stimulating gut muscles to help movecontents through the colon, the contents can back up and cause blockages in the bowel. A newborn whohas Hirschsprung's disease usually cannot have a bowel movement in the days after birth. In mild cases, the condition might not be detected until later in childhood. Uncommonly, Hirschsprung's disease is first diagnosed in adults. Currently the standard treatment is surgery to bypass or remove the diseased part of the colon is the treatment. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 In one embodiment, the disclosure provides a method of treating HSCR in a patient in need thereof (e.g., a pediatric patient such as an infant). This method includes administering a composition or a tissue transplant including a population of human ENSCs, where the population comprises at least one of a cell expressing NGFR, a cell expressing ITGA6, a cell expressing L1CAM, a cell expressing PLP1, and a cell expressing PHOX2B. In one embodiment, the ENSCs are autologous to the patient. In one embodiment at least one neurosphere including an ENSC described herein is administered to the patient. Methods of treating gastroparesis Gastroparesis is a condition in which the muscles in the stomach do not move food as they should for it to be digested. Normally, muscles contract to send food through the digestive tract. But with gastroparesis, the stomach's movement, called motility, slows or does not work at all. This keeps the stomach from emptying well. In one embodiment, the disclosure provides a method of treating gastroparesis in a patient in need thereof. This method includes administering a composition or a tissue transplant including a population of human ENSCs, where the population comprises at least one of a cell expressing NGFR, a cell expressing ITGA6, a cell expressing L1CAM, a cell expressing PLP1, and a cell expressing PHOX2B. In one embodiment, the ENSCs are autologous to the patient. In one embodiment at least one neurosphere including an ENSC described herein is administered to the patient. Methods of producing ENSCs and neurospheres To produce human ENSCs, intestinal tissue can be dissected to remove the muscularis propria from the mucosa, submucosal, and serosal layers by mechanical dissection with fine forceps and blunt dissection with microdissection scissors. A prewarmed enzymatic solution can be used to disassociate the tissue. For example, collagenase type XI (e.g., at a concentration of 1 mg mL−1) and dispase (e.g., at a concentration of 0.6U mL−1) in a culture medium such as Dulbecco's Modified Eagle Medium (DMEM) / F12 can be added to tissues, for example, at 1 mL of enzymatic solution added per 200mg of tissue. Tissues can then be minced into approximately 5mm pieces with sterile microdissection scissors and incubated in a shaker at 37˚C. For example, the tissues can be incubated for 2 to 6 hours (e.g., 4 hours). Samples can then be triturated using glass serological pipettes with progressively smaller bore diameters (e.g., 3 mm, 2 mm, and 1 mm) until the sample is liquefied. Large undigested fragments of tissue can be filtered out of the solution using a 1000 µm cell strainer. For tissue obtained from an adult, samples can then be passed through a 100 µm cell strainer to collect the single cell suspension (<100 µm), and the remaining contents that failed to pass through (100-1000 µm) can be collected to isolate the human MyGa enriched fraction. For intestinal tissue obtained from a pediatric patient, after passing the samples through the 1000 µm cell strainer, samples can be passed through a 40 µm cell strainer to collect the single cell suspension (<40 µm), and the remaining contents that failed to pass through (40-1000 µm) can be collected to isolate the human MyGa enriched fraction. To obtain human ENSCs, the MyGa enriched fraction can be cultured in growth media consisting of the following: DMEM, high glucose, pyruvate, Gibco N-2 Supplement 100X, Gibco B-27 Supplement, minus Vitamin A (Cat# 12587001), Human Recombinant GDNF (50ng / mL final concentration in media), PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Retinoic acid (75ng / mL final concentration in media) and Human Recombinant bFGF (20ng / mL final concentration in media). The ENSCs, once harvested, can be cryopreserved as described herein or can be further cultured in ultra-low attachment culture dishes to form neurospheres. Cryopreservation In some embodiments, human enteric neural stem cells (ENSCs) described herein, as a single cell suspension, are cryopreserved. In one example, single cell suspensions are harvested. Cells are typically then counted according to standard methods, for example, using a hemacytometer prior to being pelleting (for example using a centrifugation (500 g, 5 min)). Cell pellets are then resuspended in a cryopreservation media such that there are between 0.5-2x106 cells per mL of solution. Cryoprotectants include, for example, dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), 1,2-propanediol (1,2-PD), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol (DPG), and glycerin. Commercially available cryopreservation solutions including dimethyl sulfoxide (DMSO), glycerin, and / or propylene glycol as a major component can be used. Preferably, cells are cryopreserved in a serum-free cryopreservation medium such as the commercially available Bambanker Cell Freezing Medium (Bulldog Bio, Cat. No. BB05). Aliquots of 1mL are transferred to cryopreservation tubes and placed into an insulated cooling container which is then transferred to a freezer set at -80°C to achieve a cooling rate of approximately - 1°C. Cryopreservation tubes are then transferred to a liquid nitrogen storage container for long term preservation. The preservation period of the “long-term preservation” is not limited, and for example, one week or more, one month or more, six months or more, one year or more, three years or more, or five years or more. There is no upper limit for the preservation period of the long-term preservation, and the preservation period includes, for example, 1 year, 5 years, 10 years, 15 years or 20 years or more. The frozen cells can be thawed and used as appropriate. The thawing method is not particularly limited, but it is desirable to perform the thawing at about body temperature in a short period from the viewpoint of the function, activity, and viability of the cells. Specifically, it is desirable to perform the thawing at 30° C to 40° C, preferably at 35° C to 38° C, and more preferably at a temperature around human body temperature, for example, at about 37° C. The cell suspension which is frozen as described above can maintain properties equivalent to those of an unfrozen cells. For example, the frozen cells are subjected to recovery culture for 2-7 days after thawing has a marker expression rate equivalent to that of the cell aggregate before freezing. The equivalent marker expression rate means that the difference in numerical values of the percentages of the cells expressing a maker to the total cells between before freezing and after thawing, or after culturing for 7 days after thawing is about 10% or less. Neurospheres are typically observed to reform from the previously cryopreserved single cell suspensions as is described herein. EXAMPLES PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 The following examples are provided as a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and is not intended to limit the scope of what the inventors regard as their invention. Example 1: Mature Enteric Neurons have the Capacity to Reinnervate the Intestine with Glial Cells as their Guide This example shows that plasticity exists within the postnatal enteric nervous system by demonstrating the reinnervation potential of post-mitotic enteric neurons (ENs). Employing BAF53b-Cre mice for selective neuronal tracing, the reinnervation capabilities of mature postnatal ENs are shown across multiple model systems. Isolated ENs regenerate neurites in vitro, with neurite complexity and direction influenced by contact with enteric glial cells (EGCs). Nerve fibers from transplanted ENs exclusively interface and travel along EGCs within the muscularis propria. Resident EGCs persist after Cre-dependent ablation of ENs and govern the architecture of the myenteric plexus for reinnervating ENs as shown by nerve fiber projection tracing. Transplantation and optogenetic experiments in vivo highlight the rapid reinnervation potential of post-mitotic neurons, leading to restored gut muscle contractile activity within two weeks. This example illustrates the structural and functional reinnervation capacity of post- mitotic ENs and the critical role of EGCs in guiding and patterning their trajectories. Further, described below are methods of cell isolation and culture, neuron ablation, nerve fiber tracing, stimulation, and transplantation facilitated by a post-mitotic neuronal Cre driver mouse line (BAF53b-Cre) to uncover the ability of post-mitotic ENs to undergo neurogenesis and reinnervate the intestine in near physiological conditions with an intact EGC population. These results revealed that mature post-mitotic ENs within the intestinal wall can exhibit plasticity in their nerve fiber projections and are capable of reforming functional neural networks guided by EGCs in a postnatal environment. Results BAF53b-Cre selectively targets post-mitotic enteric neurons (ENs) in the intestinal wall. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 To selectively label ENs, transgenic BAF53b-Cre driver mice were crossed with Rosa26- tdTomato (R26-tdT) reporter mice to generate BAF53b-Cre::R26-tdT mice (abbreviated as BAF53b::tdT). For all experiments, mice were utilized after 3 months of age when they had reached adulthood (Flurkey et al. (2007), In The Mouse in Biomedical Research (Second Edition), J.G. Fox, M.T. Davisson, F.W. Quimby, S.W. Barthold, C.E. Newcomer, and A.L. Smith, eds. (Academic Press), pp.637-672.). BAF53b promoter-driven fluorescence was observed in reticular networks in the muscularis propria of the intestine, which is a distinguishing morphological feature of the myenteric plexus of the ENS (FIG.1A – FIG.1A’), with fluorescent labelling observed in both the ganglia and interconnected nerve fibers (FIG.1A – FIG.1A’). In wholemount preparations of the entire colonic muscularis propria, this labeling pattern was observed throughout the gut (FIG.1B). Further discernable features of murine ENS anatomy include the hypoganglionated region of the proximal colon that occurs between mucosal folds on the anti-mesenteric side (Zaitoun et al. (2013), Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society 25, e233-244.)(FIG.1B). The reported specificity of BAF53b transgene expression to ENs was confirmed in both the neuronal soma and nerve fiber processes by immunolabelling with HuC / D and TUBB3, respectively (FIG.1C – FIG.1D) (Morarach et al. (2021), Nature neuroscience 24, 34-46.). Neuronal expression of BAF53b was further validated by generating BAF53b::tdT mice with transgenic expression of GFP under the glia-specific Plp1 promoter (BAF53b- Cre::R26-tdT; Plp1-EGFP mice abbreviated as BAF53b::tdT; Plp1-EGFP). In the myenteric plexus of these mice, Plp1 expressing EGCs were observed adjacent to BAF53b expressing ENs in the myenteric ganglia and in close proximity to intramuscular nerve fibers (FIG.1D). Analyses were also performed on single cell RNA-sequencing (scRNA-seq) data originally generated by Drokhlyansky et al. (2020) Cell 182, 1606-1622.e1623 from the colons of C57Bl / 6 mice (FIG.1E). The BAF53b gene, Actl6b, was found to be specifically expressed in ENs, with this population being confirmed by the expression of the pan- neuronal markers Elavl4 (Hu) and Uchl1 (PGP9.5). Likewise, EGCs specifically expressed Plp1, thus validating the use of these neuronal and EGC transgenic reporter systems. To evaluate whether BAF53b::tdT is a marker of mature ENs that are post-mitotic and terminally differentiated, scRNA-seq was conducted on postnatally-derived enteric neurospheres, which exhibit high rates of cell cycling and neurogenesis (FIG.1F). Cycling cell states and the proliferation marker Mki67 were only observed in EGCs (Plp1) and enteric mesenchymal cells (EMCs, Col6a2), and not in BAF35b-expressing neurons. Expression of Actl6b appeared to become expressed after the neuroblast marker Ascl1 and the neuronal marker Elavl4. The expression of the cell proliferation marker Ki-67 was assessed in highly proliferative in vitro cultures derived from neurospheres of BAF53b::tdT; Plp1-EGFP mice (FIG.1G). While over half of Plp1-EGFP+cells were actively proliferative in these conditions, no evidence for the proliferation of BAF53b::tdT+neurons was observed, confirming BAF53b as a specific marker of post-mitotic ENs (FIG. 1H). Enteric glial cells (EGCs) govern the architecture of neural projections of post-mitotic enteric neurons (ENs) in a contact-dependent manner. Since BAF53b::tdT is highly specific to mature ENs and their nerve fiber projections, we utilized this model to investigate the ability of post-mitotic ENs to reform physical networks. Previously it has been shown that enteric neural progenitor cells can reform neural networks in vitro (Kruger et al. (2002) Neuron PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 35, 657-669.), however it is unclear whether mature terminally differentiated ENs possess a similar capability. The muscularis propria of the colon, which includes the myenteric plexus, was digested and cell suspensions were cultured in vitro to assess the morphological properties of BAF53b::tdT neurons temporally. The digestion process resulted in denervation of neurons which lacked neurite fiber extensions after 1 day in culture (FIG.2A). ENs had projected neurites after 4 days and formed an extensive network by day 7, suggesting that mature ENs are capable of reforming neural projections (FIG.2A). When these experiments were repeated in BAF53b::tdT; Plp1-EGFP dual reporter mice, the projections of neurites failed to extend beyond the migrating and expanding population of EGCs, leading us to hypothesize that neurite extensions from post-mitotic ENs require communication with EGCs to reform networks. To elucidate the role of ECGs in neurite formation, a purified population of primary ENs was cultured alone, or in the presence of EGCs or enteric mesenchymal cells (EMCs) serving as a cellular control (FIG.2B). EMCs were characterized as an intestinal fibroblast population expressing Collagen VI and / or PDGFRɑ (Roulis et al. (2020) Nature 580, 524-529.)(FIG.8A – FIG.8B). The mature post-mitotic ENs (BAF53b::tdT) were purified, and then cultured with EGCs from Plp1-EGFP expressing mice to avoid neurite observations in the newborn EGC-derived neurons of BAF53b::tdT; Plp1-EGFP dual reporter mice (FIG.2B – FIG.2C). ENs did not proliferate like the EGCs within the same culture (FIG. 2D), supporting the idea that they are post-mitotic. The presence of both EGCs and EMCs enhanced the ability of ENs to project neurites in vitro (FIG.8C, FIG.9A – FIG.9B). When the properties of the neurites projected by ENs were compared between co-cultures with EGCs versus EMCs, no differences in the sum of neurite lengths were observed (FIG.9C), but co-cultures with EGCs exhibited neurites with higher order branching (FIG.9D) and more numerous neurite branches (FIG.9E). The additional number of neurite branches in co-cultures with EGCs appeared to be driven by those above the third order of branching (FIG.2E), indicating that EGCs foster greater complexity in the projections of ENs compared to the EMCs. In timelapse videos, neurite projections were observed to acquire increasing complexity once the neurite made physical contact with EGCs, regardless of whether contacts were made early (day 7) or late (day 14) in co-culture experiments. Although physical contact with EGCs was not necessary for ENs to project neurites, pre-existing projections became more convoluted and new branches were formed once neurite projections did make contact. These projections advanced along and in between colonies of EGCs with a pattern that appeared to mirror EGC morphology. To determine whether direct contact with EGCs was necessary for their role in neurite formation and branching, ENs were subclassified as either having projections in direct physical contact with a Plp1- EGFP expressing EGC (FIG.2F – FIG.2H, FIG.9F – FIG.9I) or having no physical contact (FIG.2F, FIG.2H, FIG.9F – FIG.9I). Direct contact did not enhance the sum of the length of neurites (FIG.9G), but did cause an increase in higher order neurites (FIG.9H) and yielded more neurite branches (FIG.9I) that predominately occurred above the third order (FIG.2F). Further experiments in different in vitro conditions were conducted to elucidate the contact dependent and paracrine roles of EGCs on neurite formation from mature ENs. Time-series comparisons between EGCs in a transwell system and direct co- culture with ENs were conducted over 2 weeks. These studies showed that EGCs can promote the ability of ENs to project neurites in a paracrine manner. However, this effect was significantly increased by direct PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 EGC contact, which encouraged neurons to begin to produce neurites faster. Both paracrine and direct contact improved the survival of neurons and their new networks post-neurite formation (FIG.2I). The properties of neurites were compared between the direct EGC contact and transwell groups and reinforced the observation that direct EGC contact increases the branching and complexity of enteric neurites (FIG.10). Additional experiments utilizing dual chamber culture channels loaded with ENs and EGCs together (contact) or at opposing ends and sharing the media (paracrine) yielded similar observations of intricate high order neurite formation in the direct EGC culture group (FIG.11A – FIG. 11B). In this experimental setup paracrine secretion did not appear to promote neurite extensions in the direction of the EGCs (FIG.11C). The neurite projections in these cultures were visualized at high magnification, showing that neurites formed during EGC contact are highly filamentous with puncta reminiscent of ENs in vivo, while those formed in paracrine conditions were typically thickened and lacked higher order branching (FIG.2J – FIG.2K). Neurite direction and branchpoints coincided with the cellular processes of EGCs, with neurites often running along the processes of EGCs before splitting in direction between two EGCs. Together these data confirm that (1) mature post-mitotic ENs can reproject neurites and (2) EGCs promote neurite formation and guide the trajectory of their complex projections via a contact-dependent mechanism. Neural projections of post-mitotic ENs are guided along the myenteric plexus and maintained by EGCs. To explore whether EGCs similarly govern nerve fiber projections in vivo, neurospheres were generated from BAF53b::tdT; Plp1-EGFP mice and delivered to the muscularis propria of wildtype mice, which have a fully formed myenteric plexus (FIG.3A), or to the aganglionic region of Ednrb knock out (KO) mice, which lack an intrinsic ENS but contain EGCs that reside on extrinsic nerve fibers in the aganglionic segment (FIG.3B). In these models, the projection of new neurites from transplanted neurons (BAF53b::tdT) could be visualized in relation to transplanted EGCs (Plp1-EGFP and S100B), while the endogenous EGCs of the recipient mice express S100B but not Plp1-EGFP. In wildtype mice thin nerve fibers projecting from transplanted ENs were observed exclusively in contact with EGCs. This included neurites within the recipient myenteric ganglia, the EGCs forming interconnections between the ganglia, and along other EGCs originating from the transplant (FIG.3A). Similar observations were made in aganglionic mice, which lack endogenous ENs, with nerve fibers extending from transplanted neurons along other transplanted EGCs or along the hypertrophic nerve fiber bundles which possess endogenous EGCs (FIG.3B). The morphologies of the neuronal fibers that followed the architecture of the recipient myenteric plexus were filamentous with several branch points similar to that observed in the in vitro co- cultures where EGCs and neurites were in contact. Other neural projections traversed linearly through the intramuscular space along bipolar EGCs with limited branching, which was more prominent in Ednrb KO mice lacking a myenteric plexus. This further suggested that the structure of newly formed neuronal fibers strongly coincides with the architecture of EGCs. To temporally track neurites as they formed within the muscularis from post-mitotic ENs, grafts of the myenteric plexus from BAF53b::tdT mice were transplanted to Plp1-EGFP recipients ex vivo (FIG. 3C). Daily imaging confirmed that the newly forming nerve fiber projections extended within the recipient myenteric plexus with a trajectory that followed the pre-existing architecture that was highlighted by Plp1- PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 EGFP expression (FIG.3D). In further experiments, specific ablation of EGCs was performed in Plp1- CreERT::iDTR with local administration of diphtheria toxin. Although neuronal cell bodies were observed the nerve fiber network was greatly disrupted after EGC ablation (FIG.3E). Together these data indicate that the architecture of the myenteric plexus, and the EGCs in particular, is critical for guiding the trajectory of newly forming neuronal projections and for maintaining the fidelity of the reticular myenteric neural networks. BAF53b-Cre facilitates a novel diphtheria toxin-induced model of localized aneural intestine that retains the gross architecture of EGC networks. The previous results indicate that ENs are heavily influenced by the supporting cells and microenvironment, with EGCs and surrounding mesenchyme likely to be critical for reinnervation of the intestine. The majority of previous models employed to assess neuroregeneration involve ablation of the entire ENS, including EGCs and neural progenitors, or induction of inflammation, and are therefore largely unsuitable to address the capabilities of postnatal neurogenesis and reinnervation by ENs. To study the ability of postnatal neurons to reinnervate the intestine, we developed a mouse model of localized neuronal ablation. The ENS is critical to intestinal motility and necessary for survival, however mice are capable of surviving when small regions of ENS are ablated (Bhave et al. (2019) Sci Rep 9, 18756.). DT was administered locally by microinjection to the muscularis propria of the colon of mice with ROSA26-inducible diphtheria toxin receptor (R26-iDTR) expression driven by BAF53b-Cre (BAF53b- Cre::R26-iDTR mice abbreviated as BAF53b::iDTR) to induce neuron specific ablation (FIG.4A). Mice sacrificed 1 week after DT injection exhibited loss of the neuronal marker TUBB3 at the injection site (FIG.4B). In BAF53b-Cre::R26-iDTR; Plp1-EGFP (BAF53b::iDTR; Plp1-EGFP) mice, ablation of neurons did not affect the gross morphology of EGCs (FIG.4B) after 1 week. No ablation of ENs was observed after local DT injection to the colon of littermate R26-iDTR; Plp1-EGFP mice, lacking BAF53b-Cre expression, as indicated by staining patterns of TUBB3 nerve fibers and the pan neuronal body marker Hu (FIG.4C). However, we observed a complete loss of Hu and TUBB3 immunoreactivity at the injection site in BAF53b::iDTR; Plp1-EGFP mice after 2 weeks (FIG.4C). Quantification of Hu immunoreactivity in BAF53b::iDTR mice indicated a gradual loss of ENs up until 2 weeks leading to a near complete ablation (FIG.4D – FIG.4E, FIG.12A). While neurons were virtually completely ablated at the injection site, administration of DT created a spatial gradient of neuronal loss with reduced neuronal numbers in the ganglia 10-15 mm adjacent to the injection site, coinciding with Hu translocation to the nucleus, an indicator of EN injury and non-apoptotic death (Desmet et al. (2014) Neurogastroenterology & Motility 26, 1131-1143.) (FIG.4C, FIG.12B). To confirm the specificity of the BAF53b::iDTR neuron ablation strategy, RNA-seq was performed on the DT-injected region of colons from BAF53b::iDTR and R26-iDTR littermate control mice (FIG.12C). Analysis of differentially expressed genes (DEGs) identified 57 genes upregulated and 249 genes downregulated (FDR 0.05) in BAF53b::iDTR mice (FIG.4F). We analyzed the scRNA-seq data from the colon, which contained 29 different cell types or cell states, including ENs and EGCs (FIG.4G). The gene signature of downregulated DEGs in BAF53b::iDTR mice was highly associated with cells in EN clusters (FIG.4G), suggesting that DT ablation was specific to ENs without causing cell death in other cell types including EGCs, and was consistent with our immunohistochemical data. No overt off target effects were PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 detected at the gene expression level after neuronal ablation, including inflammatory cytokine expression, or loss of smooth muscle or EGC marker expression (FIG.12D). In BAF53b::iDTR mice, pan-neuronal markers such as Tubb3, Elavl4 (Hu) and Uchl1 (PGP9.5) were downregulated, further indicating successful neuronal ablation (FIG.4H). Our ablation strategy resulted in indiscriminate neuronal loss, with a downregulation of neurochemical coding markers for all major classes of neurons, including cholinergic (Slc18a3, VAChT), nitrergic (Nos1), dopaminergic (Dbh), sensory (Calcb, CGRP), and various other neuropeptides (Vip, Nmu, Sst, Tac1, Grp) and neurotransmitter receptors expressed by diverse neuron classes (FIG.4H). Over-representation analysis of the genes that are downregulated in BAF53b::iDTR compared to controls produced a strong enrichment for gene ontologies associated with the nervous system, including those related to ion channels, synaptic communication, and neuronal structures (Top 5 by P value shown) (FIG.4I). Regeneration of ENs and intrinsic innervation in the postnatal intestine. Given our hypothesis that EGCs are required for neural regeneration and nerve fiber projections, the BAF53b::iDTR model was used to selectively ablate neurons and sacrificed the mice between 2 weeks to 3 months after DT administration to assess levels of neurogenesis and reinnervation. Immunohistochemical visualization revealed the presence of ENs in the previously aneural region as indicated by labeling of neuronal soma (Hu) at the DT injection site with the numbers of ENs gradually increasing from 4 weeks to 3 months after DT administration (FIG.5A – FIG.5B). At 3 months, the DT administered region remained sparsely populated with neuronal cell bodies, despite strong nerve fiber labelling with TUBB3 and Calretinin. Similarly, neuronal densities in the region adjacent to DT injection remained decreased (FIG.5C – FIG.5D). To assess the ability of the regenerated neurons to regulate smooth muscle contractility, organ bath experiments were conducted utilizing electric field stimulation (EFS) to stimulate ENs and record force contractions evoked in isolated rings of smooth muscle (FIG. 5E). At 2 weeks after DT injection, no contractile smooth muscle responses to EFS were detected. Responses to EFS were still limited 3 months after neural ablation compared to wildtype mice, however, evidence for the ability of the repopulating ENs to partly restore muscle function was observed (FIG.5E). To determine whether intrinsic innervation has the capacity to regenerate in the postnatal intestine, experiments were conducted to locally label ENs just proximal to a region of ablated neurons in the distal colonic segment of BAF53b::iDTR mice, enabling the tracking of reinnervation (FIG.5F – FIG. 5G). AAV1-FLEX-mCherry was injected locally to the colonic wall after laparotomy to label BAF53b-Cre expressing post-mitotic ENs including both their soma and distally projecting nerve fiber processes (FIG 5F). DT was administered simultaneously 5mm distally to the colons of BAF53b::iDTR mice to induce neural ablation. The appearance of mCherry-labeled nerve fibers from the proximal segment were evaluated in the ablated region 2 and 8 weeks after ablation (FIG 5G). Nerve fiber projections traced with mCherry extended from the proximal ENs into the ablated area, with the density of neuronal fibers in the myenteric plexus increasing over time (FIG 5H – FIG.5I). The mCherry projections extending into the ablation region resided among the pre-existing architecture of the EGC network. This included nerve fibers intertwining with EGCs within the myenteric plexus and linear intramuscular projections in contact with bipolar EGCs (FIG.13). These data indicate that post-mitotic ENs have plasticity for reinnervation of the intestine, a process which also appears to be guided by the EGC architecture. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Transplantation of postnatal primary enteric ganglia demonstrate functional reinnervation of aneural intestine. In order to confirm that the mature post-mitotic ENs are capable of projecting nerve fibers and reinnervating the gut, intact enteric ganglia were isolated from adult BAF53b::tdT mouse intestine and immediately transplanted to the muscularis layer of the colon of recipient mice to trace their projections (FIG.6A – FIG.6Bʹ). After 2 weeks, ENs engrafted into the muscularis propria, extended nerve fibers radially and formed networks in the wildtype ganglionated colon (FIG.6C). Elongated linear projections could also be observed in between muscle fiber striations (FIG.6D – FIG.6Dʹ). When neurons from BAF53b::tdT mice were transplanted to Plp1-EGFP mice, neurites integrated with the recipient EGCs of the myenteric plexus (FIG.6E). Ganglia were also isolated from the BAF53b::tdT; Plp1-EGFP dual reporter mice (FIG.6F). As enteric ganglia were transplanted to wildtype recipients with an intact ENS and EGC population, we observed that the nerve fiber projections (BAF53b::tdT+) extended further from the injection site than Plp1-EGFP-expressing EGCs originating from the transplant (FIG.6G), consistent with our observations after neurosphere transplantation that neurites progress further along the endogenous EGCs of the recipient (FIG.3A). To examine the ability of postnatal neurons to reinnervate an aneural region of the colon, enteric ganglia from BAF53b::tdT mice were transplanted 1 week after DT-mediated ablation of ENs in recipient BAF53b::iDTR mice. Similar to the observations in the ganglionated recipient, ENs successfully engrafted in the aneural colon and extended nerve fibers in the muscularis after 2 weeks (FIG.6H). The transplanted cells organized into reticular plexus-like structures with interconnected fibers between transplanted neurons (FIG.6I – FIG.6J). In BAF53b::iDTR mice, injection of DT induced a delay in fecal pellet production (constipation) as measured by scoring the time taken for the first spontaneous pellet excretion (constipation score = +1 for every 20min until 120min) (FIG.6K). Constipation in BAF53b::iDTR mice was reversed by transplantation with enteric ganglia isolated from large bowel or small bowel (FIG. 6K), whereas no differences were observed in fecal water content (FIG.6L). These results suggest that constipation was a result of delayed motility due to deletion of ENs, which was improved by enteric ganglia transplantation. The functional effects of transplantation were further examined in organ bath experiments using 5mm colonic rings isolated from the DT-injected region (FIG.6M). The activity of smooth muscle contraction in colonic rings was recorded in response to electrical field stimulation (EFS) with parameters specific to the activation of ENs (FIG.6M). Samples obtained from mice following neuronal ablation did not respond to EFS stimulation, but the response was restored in mice that received enteric ganglia from either the small bowel or large bowel (FIG.6M – FIG.6N). These data indicate that post-mitotic ENs can engraft, reinnervate, and influence muscle contractions within 2 weeks after their transplantation to aneural gut. Post-mitotic ENs innervate and regulate smooth muscle activity in a time dependent manner in the postnatal environment. To further elucidate the ability of post-mitotic ENs to reinnervate and exert functional control over smooth muscle cells, myenteric ganglia were isolated from BAF53b-Cre mice crossed with ROSA26- tdTomato-channelrhodopsin-2 (R26-tdT-ChR2) mice (BAF53b-Cre::R26-tdT-ChR2 abbreviated as PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 BAF53b::tdT-ChR2) and transplanted to the colons of wildtype recipients (FIG.7A). This model system allows specific stimulation of transplanted neurons by blue light to assess the time taken for functional reinnervation of colonic smooth muscle at days 3, 7 and 14 post-transplantation. Smooth muscle activity was recorded in vivo by simultaneous electromyography (EMG) of the muscularis propria and intraluminal pressure measurement in response to blue light stimulation at the transplantation site (FIG.7B). Functional responses to blue light stimulation were negligible after 3 days post-transplantation but increased over time, with the highest electrical activity (FIG.7C) and increased intraluminal pressure responses (FIG.7D) observed at 2 weeks. Similar results were observed when measuring contractile responses in ex vivo organ bath experiments with blue light stimulation of transplanted neurons in colonic rings (FIG.7E). In these experiments relaxation responses increased at day 7 after transplantation as compared to day 3, and remained stable at 2 weeks (FIG.7F), whereas force contraction responses increased over time, peaking at the 2-week timepoint (FIG.7G). Discussion This example illustrates the structural plasticity of mature ENs during ENS regeneration and establishes the fundamental importance of EGC architecture to guide neurites from ENs in physiology, disease, and therapy. In this example, transgenic models were utilized to characterize the ability of ENs to innervate the postnatal intestinal environment. In vitro / ex vivo culture and in vivo transplantation studies using adult BAF53b::tdT reporter mice demonstrated that ENs can reinnervate the intestinal wall of mice postnatally. The guidance of these projections is dependent on physical contact with EGCs, which appear to govern the gross structure of the ENS. By utilizing models that allow specific ablation of ENs whilst maintaining EGCs, physiological neurogenesis was confirmed in adult mice, albeit at a slow rate of approximately 0.5% (1-2 neurons per mm2) of the normal neuronal density per week. However, the ablated regions were heavily reinnervated by nerve fiber processes extending from ENs adjacent to the ablated zone. Mature ENs were found to rapidly reinnervate the adult intestine and are functionally competent in eliciting smooth muscle contractility within 1 to 2 weeks. The results from this example demonstrate that in an environment that includes EGCs, postnatal ENs can also innervate and exert control over the smooth muscle in a 1-2 week time frame, similar to the embryo. The reinnervating postnatal ENs exhibited inhibitory activity over the smooth muscle before excitation responses. These results also indicate that the initial neural activity by reinnervating postnatal neurons adheres to an analogous developmental sequence present in emerging prenatal neural networks. The data from this example revealed that physical contact with EGCs is important for the guidance of neurites and reinnervation by post-mitotic ENs. These conclusions are drawn from: 1) in vitro co-cultures showing that EGCs promote neurite extensions, branching, complexity and guide direction, 2) in vivo tracing of neurites from transplanted neurons showing exclusive association with EGCs, 3) timelapse imaging showing new nerve fiber projections extending within the architecture of the myenteric plexus, and 4) dense innervation of the myenteric plexus by proximally traced intrinsic nerve fibers after neuronal ablation in mice with intact EGCs. In this example, EGCs have been tracked by their expression of Gfap, Sox10, Sox2 and Plp1, with acquisition of neuronal markers indicating neurogenesis (Joseph et al. (2011) J Clin Invest 121, PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 3398-3411; Laranjeira et al. (2011) J Clin Invest 121, 3412-3424; Belkind-Gerson et al. (2017) Scientific reports 7, 1-15; Guyer et al. (2023) Cell Rep 42, 112194.). This example demonstrates the potential of mature ENs to project nerve fibers and reinnervate the postnatal intestine. This process relies on the presence of direct contact with EGCs to guide complex nerve fiber projections, which are capable of forming fully functional neuromuscular connections within 2 weeks. As several neurointestinal diseases, including Hirschsprung disease, esophageal achalasia, and gastroparesis are associated with deficient or abnormal innervation, further studies should seek to clarify the molecular mechanisms of postnatal neural reinnervation in order to leverage this knowledge to develop novel regenerative approaches to treating these conditions. The following sections provide details on the methods used to obtain the above-described results. EXPERIMENTAL MODEL AND SUBJECT DETAILS Ethical Statement This study was performed according to experimental protocols approved by the Institutional Animal Care and Use Committees IACUC (2009N000239) of Massachusetts General Hospital. Mice All mice were purchased from Jackson laboratory (Bar Harbor, ME) with the exception of and Plp1-EGFP mice which were kindly donated by Wendy Macklin PhD, University of Colorado (Mallon et al. (2002) Journal of Neuroscience 22, 876-885.). Details are provided in Table 1. All mice were 12-14 weeks of age at time of experiment. All mice were housed and bred under specific-pathogen-free conditions at the Center for Comparative Medicine animal facility at Massachusetts General Hospital (MGH). Rodents were housed in Allentown Inc rectangular caging (160 cages per individually ventilated cage racks; which uses blower at 60 air changes per hour) under a 12h:12h light:dark cycle from 7 am – 7 pm. Bedding consisted of Hardwood Sanichip; with Carefresh nesting material and mice had access to Prolab Isopro RMH 3000 chow mix (ScottPharma) ad libitum. METHOD DETAILS Immunohistochemistry of wholemount preparations Wholemount preparations were prepared for immunohistochemistry by cutting the colon along the mesenteric border, pinning the tissue onto silicone-lined petri dishes and removing the mucosa and submucosa under a dissection microscope prior to fixation in 4% paraformaldehyde (PFA) for 4 hours. Tissues were blocked and permeabilized in solution containing 10% donkey serum, 10% bovine serum albumin (BSA) and 1% triton in PBS for 1 hour at room temperature on a rocking platform. Primary antibodies were diluted in the same buffer and incubated overnight at 4˚C before washing tissues in PBS. Corresponding secondary antibodies were applied for 3 hours and nuclei were stained with DAPI before washing tissues in PBS and mounting onto glass slides with AquaPolymount (Polysciences). Primary antibodies utilized in this study included anti-Tubulin β3 (TUBB3, 1:200, TUJ1 clone conjugated to Alexa PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Fluor 647 or Alexa Fluor 488; Biolegend), human anti-Hu (ANNA1) positive serum (1:16,000, gifted from Dr. Vanda Lennon; Mayo Clinic), rabbit anti-S100B (1:100, clone EP1576Y, ab52642; Abcam) and rabbit anti-Calretinin (1:200, Invitrogen, Thermo Fisher Scientific, 18-0211). Secondaries were utilized at 1:500 for donkey anti-rabbit Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, MA) and donkey anti-human DyLight™ 594 (Thermo Fisher Scientific, MA). Imaging was conducted on a Keyence BZX-700 All-In-One Microscopy system (Keyence America Itasca), a Zeiss LSM 800 Airyscan confocal microscope (Carl Zeiss Meditec, Dublin, CA) and a Nikon AXR confocal microscope (Nikon Cambridge, MA). For quantification of neuronal density six images with a total field of view of 9.5mm2were assessed per mouse. These images were acquired on the Keyence BZX-700 microscope and cells were quantified using the cell counter plugin of ImageJ software after applying the Top Hat function for background equalization. Single cell RNA-seq of neurospheres Profiling of postnatal enteric neurospheres by scRNA-seq was conducted in Guyer et al.2023 (Cell Rep 42, 112194.) with ancillary experiments reported here to profile the non-EGC cell population. Briefly, the small intestine was excised from 3-month-old Plp1-EGFP mice and the muscularis was obtained by microdissection. Tissues were digested for 60 minutes at 37°C in dispase (250 mg / ml; STEMCELL Technologies, Vancouver, BC) and collagenase XI (1mg / ml; Sigma-Aldrich, St. Louis, MO) and filtered using a 40µm cell strainer to obtain single cell suspensions. Cells were plated at a density of 1×105cells / mL in enteric neurosphere proliferation media comprised of a 1:1 mixture of DMEM (Thermo Fisher, Waltham, MA) and NeuroCult Basal Media (STEMCELL Technologies, Vancouver, BC) supplemented with 20 ng / mL FGF, 20 ng / mL IGF1, 2% B27 supplement, 1% N2 supplement, 50 mM b- mercaptoethanol, and 75 ng / mL retinoic acid. After 10 days of culture in standard humidified cell culture conditions (37˚C, 5% CO2 and atmospheric O2), neurospheres were digested with Accutase (STEMCELL Technologies, Vancouver, BC) for 45 minutes before Plp1-EGFP positive and Plp1-EGFP negative cell populations were collected by fluorescence activated cell sorting (FACS) using a BD FACSAria cell sorter (BD Biosciences, Franklin Lakes, New Jersey). Libraries were prepared using the Chromium Single Cell 3' Reagent Kit v3.1 (10X Genomics, CA) using the standard 10X Genomics workflow according to manufacturer’s instructions and sequenced on an Illumina NovaSeq platform (Illumina, Inc., CA) at the Harvard University Bauer Core Facility. Feature-barcode matrices were generated using the 10X Genetics Cell Ranger software pipeline and data were analyzed using the Seurat package (v4.0.2) in the RStudio integrated development environment. (Tirosh et al. (2016) Science (New York, N.Y.) 352, 189- 196). Data from Plp1-EGFP positive and Plp1-EGFP negative cell populations were merged and preprocessed by removing ribosomal, heat shock protein, immediate early, and sex specific genes. High quality cells were included with between 1000-6000 unique features and less than 10% of mitochondrial RNA before being processed using the standard Seurat workflow by running the NormalizeData, ScaleData, FindVariableFeatures, RunPCA, FindNeighbors(dims = 1:10) followed by the RunUMAP(dims = 1:10) commands for dimensionality reduction and visualization. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 In vitro Neurite Outgrowth Assays For in vitro cell experiments tracing neurites cell were isolated from colons collected from euthanized BAF53b::tdT or BAF53b::tdT; Plp1-EGFP mice. Colons were cut along the mesenteric border and pinned flat in silicone-lined petri dishes to remove the mucosa and submucosa by microdissection leaving the muscularis propria. Tissues were transferred to a 15mL tube, where the muscularis propria was minced and digested in 2mL of collagenase XI (1 mg mL−1; Sigma Aldrich, St. Louis, Missouri) and dispase (250 μg mL−1; STEMCELL Technologies, Vancouver, Canada) solution at 37˚C for 1 hour before filtering through a 70µm nylon mesh. Cells were plated at a density of 5×104cells / cm2in a 24-well cell culture plate that had previously been coated with fibronectin (Sigma, #F1141, 1:100 for 1h) in DMEM / F12 media (ThermoFisher, Gibco) containing 10% FBS (#10438-018, ThermoFisher, Gibco) and 1% penicillin-streptomycin (ThermoFisher, Gibco, 15140122). A standard humidified cell culture incubator was utilized for all experiments (37˚C, 5% CO2 and atmospheric O2). For co-culture experiments, the muscularis propria was separated from the small intestine and cultured in the same conditions as above from Plp1-EGFP or BAF53b::tdT; Plp1-EGFP mice. Cells were plated on fibronectin coated dishes and cultured for 2 days as above prior to trypsinization and FACS using a BD FACSAria cell sorter (BD Biosciences, Franklin Lakes, New Jersey). Data from FACS experiments were presented using FlowJo software (FlowJo, LLC, OR). Post-mitotic ENs and EMCs were collected from BAF53b::tdT; Plp1-EGFP mice and EGCs were collected from Plp1-EGFP mice to eliminate analysis in immature neurons as we have previously shown that EGCs transdifferentiate into ENs in vitro in the BAF53b::tdT; Plp1-EGFPmodel (Guyer et al. (2023) Cell Rep 42, 112194). Cells were co-cultured at a ratio of 1:1 with a total of2×103cells seeded per well of a fibronectin-coated 48-well culture plate in DMEM / F12 media supplemented with 2% FBS and 1% penicillin-streptomycin. Timelapse images were taken on a Keyence BZX-700 All-In-One Microscopy system (Keyence America Itasca) with a total field of view of 28mm2per independent culture. Data on the characteristics of neurites were acquired using Neurolucida software (MBF Bioscience, Williston, Vermont). Neurites >5µm were considered for analysis with branches ordered by the centrifugal scheme. For transwell experiments the procedures were repeated as above to purify BAF53b::tdT+ENs and Plp1-EGFP+EGCs. ENs were seeded into a fibronectin-coated 24-well culture plate in DMEM / F12 media supplemented with 10% FBS and 1% penicillin-streptomycin at a density of 1×103cells per well. EGCs were either seeded directly with the ENs, or were seeded in semipermeable transwell inserts (0.4 μm pore size, polyester membrane, Corning, #3470) at a ratio of 1:4. Daily imaging taken with a 10mm2area per sample was performed for 14 days on a Keyence BZX-700 to generate timelapse videos to assess the number of neurons that produce neurites, the time taken to the first neurite production and the rate of cell death after neurite formation over the 14 day period. Neurite properties were analyzed in greater detail at the 10 day time point of culture using the SNT plugin of ImageJ and the A* search pathfinding algorithm for semi-automated neurite tracing (Arshadi et al. (2021) Nature Methods 18, 374- 377). To perform monolayer paracrine explements, µ-Slide VI (ibidi, Cat.No: 80666) cell culture slides were utilized which contain two loading wells on opposing ends of a channel with shared media. Slides were pretreated with fibronectin as of above and 5×102ENs were seeded into one loading well with EGCs seeded in the opposing well at a 1:4 ratio. Direct co-culture was utilized to compare neurite morphology, with ENs and ECGs seeded in the same well, leaving the opposing well empty. Cells were seeded in a PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 volume of 5µL, and allowed to adhere to the fibronectin for 2h in standard culture conditions (37˚C, 5% CO2 and atmospheric O2) before adding the remaining media which was the same as utilized in transwell experiments. Images were taken on a Keyence BZX-700 and the direction of neurite extensions were quantified after 10 days of culture by measuring the vector of the primary neurite extension from where it first protrudes from the soma to its terminal end using ImageJ. Immunocytochemistry and cell proliferation assay Neurospheres were generated from the small intestines of BAF53b::tdT; Plp1-EGFP mice and cultured as above for 10 days before being transferred to 24-well cell culture plates coated with fibronectin (sigma, #F1141, 1:100 for 1h) in neurosphere media supplemented with 5% FBS (#10438- 018, ThermoFisher, Gibco) to promote monolayer formation for immunocytochemical visualization of the cell proliferation marker Ki-67. After 3 days cells were fixed for 30 min with 4% PFA (Electron Microscopy Science, #15710), permeabilized with 0.1% triton in PBS for 20 minutes and blocked in solution containing 10% donkey serum (Sigma, #D9663) for 1 hour all at room temperature. Samples were incubated with rabbit anti-Ki-67 (1:100, Abcam, ab15580) in PBS containing 10% donkey serum at 4˚C overnight before incubation with donkey anti-rabbit Alexa Fluor 647 (1:400, Thermo Fisher Scientific) for 1 hour in the same blocking solution at room temperature and stained with DAPI (Invitrogen, #D1306) for 10 minutes. For quantification of cells expressing Ki-67, BAF53b::tdT and Plp1-EGFP images were acquired on Keyence BZX-700 All-In-One Microscopy system (Keyence America Itasca) with cells counted in a 1.5 mm2area per sample using the cell counter plugin of ImageJ software. For characterization of EMCs, the FACS purified double negative fraction from BAF53b::tdT; Plp1-EGFP mice were processed as above with the primary antibodies rabbit anti-Collagen VI (1:250, Abcam, ab182744) and goat Anti-PDGFRɑ (1:200, R&D systems, FAB1062G) with secondary antibodies donkey anti-rabbit Alexa Fluor 647 (1:400) and donkey anti-goat Alexa Fluor 488 (1:400, Thermo Fisher Scientific). Neuron and ECG ablation To ablate ENs expressing an inducible diphtheria toxin receptor (iDTR), BAF53b::iDTR mice were generated. ECGs were ablated in Plp1::CreERT2-iDTR mice administered with tamoxifen (Sigma, T6648- 1, 100 mg / kg) dissolved in corn oil via intraperitoneal injection daily for 7 days prior to the ablation procedure to ensure sufficient Cre recombination. Diphtheria toxin (DT) (Sigma Aldrich, St. Louise, MO, USA, #D0564) was prepared by dilution in PBS and India ink to a final dose of 0.33ng / μL of DT and 10% India ink at final volume. Mice were anesthetized by isoflurane to perform laparotomy and delivery of 4μL of the DT solution by microinjection to the muscularis propria with the India ink used to confirm the injection site. Injection of the DT solution was performed in littermate mice lacking iDTR expression as experimental controls. Colons were excised from mice sacrificed by CO2 inhalation after 3 days, 1, 2, 4, 8 weeks and 3 months post-procedure. Colons were then cut along the mesenteric border and pinned on silicon-lined petri dishes and fixed for immunohistochemical validation of neuronal ablation and neuronal density quantification. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Cell transplantation To obtain ganglia for cell transplantation the muscularis propria was obtained from the colon or small bowel and was minced and digested in collagenase XI (1 mg mL−1) and dispase (250 μg mL−1) solution at 37˚C for 1 hour with the suspension triturated until all the sample could pass through a 1000μL pipette tip. Samples were then passed through a 40μm filter with the material greater than 40μm retained for further processed through a 70μm filter with the flow through between 40μm to 70μm enriched for myenteric ganglia due to their lack of digestion. Samples were centrifuged at 500G for 5 minutes and resuspended in PBS at a dose of ~100 ganglia / μL. Administration was performed as previously described (Pan et al. Nat Commun 15, 2479). Briefly, mice were anesthetized by isoflurane, and an incision was made along the midline to access the colon for microinjection of 5μL of the ganglia containing solution into the muscularis propria using a NanoFilTM microliter syringe (33 G, NF33BV-2, World Precision Instruments, FL, USA). In experiments involving prior neuronal ablation, mice underwent anesthesia, and the initial laparotomy incision was reopened 1 week post DT injection. Then, 5μl of the suspension was microinjected into the aneural colon, targeting the area marked by India ink. For experiments involving the injection of enteric neurospheres, cultures were prepared as described in the section above ‘Single cell RNA-seq of neurospheres’, with the exception that neurospheres were generated from BAF53b::tdT; Plp1-EGFP mice. Neurospheres were injected to the colorectum using an approach similar to previously described (Hotta et al. (2016) Neurogastroenterol. Motil.28, 498-512). Normal wildtype and Ednrb KO mice with aganglionosis were anesthetized by isoflurane inhalation. A circumanal skin incision was made to expose the colorectum and approximately 100 enteric neurospheres in 5μL of PBS were delivered to the muscularis propria using a NanoFilTM microliter syringe (33 G). Plexus grafting experiments ex vivo. To track neuronal projections in the myenteric plexus ex vivo, fragments of the myenteric plexus of BAF53b::tdT; Plp1-EGFP mice were transplanted to organotypic preparations of the muscularis propria of Plp1-EGFP mice. Organotypic preparations were prepared by microdissection as conducted previously (Stavely et al. (2024) Scientific Reports 14, 6649; Stavely et al. (2021) Stem Cells 39, 1236-1252). To obtain fragments of the myenteric plexus, colons were processed and digested as above in the ‘Cell transplantation’ section and tissue fragments >70μm were collected and visualized under a Leica MZ FLIII fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany) to collect partially digested pieces of myenteric plexus. Using fine forceps, the longitudinal and circular muscle layers were separated in the middle of the Plp1-EGFP colonic preparation to create a pocket to insert the BAF53b::tdT; Plp1- EGFP myenteric plexus fragments. Preparations were cultured in a standard humidified cell culture incubator (37˚C, 5% CO2 and atmospheric O2) in DMEM / F12 media supplemented with 10% FBS and 1% penicillin-streptomycin. Nicardipine was added to the media at a final concentration of 3µM to prevent muscle contractions during daily imaging on a Keyence BZX-700 microscope to track BAF53b::tdT nerve fiber projections using the SNT plugin of ImageJ. Bulk RNA-Seq The transcriptome in the colon was evaluated 2 weeks after neural ablation via local injection of DT. All mice had received laparotomy and injection of DT with India ink as described above, with mice PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 lacking iDTR expression serving as controls. The injection sites were located by staining with India ink and full-thickness specimens of the injection area of approximately 20mm2were dissected (FIG.12C) and immediately frozen in LN2 for storage. Total RNA extraction was performed using Trizol reagent as of the manufacturers user guide (Thermo Fisher Scientific, Waltham, MA, USA). Samples were processed by Genewiz for PolyA purification and library preparation using the NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA). Samples were sequenced on a Illumina HiSeq instrument using a 2x150 Paired End configuration. Quality control and adapter trimming of fastq files was performed using Fastp (Chen et al. (2018) Bioinformatics 34, i884-i890.), reads were aligned to the mm10 reference genome by HISAT and reads were quantified by FeatureCounts (Liao et al. (2014) Bioinformatics 30, 923-930) with data reported as transcripts per million (TPM). Differentially expressed genes (DEGs) were assessed using EdgeR with p<0.05 using the Benjamini-Hochberg correction considered differentially expressed. Heatmaps for DEGs and manually curated list of genes for pan- neuronal markers, neurochemical coding subtypes of ENs and neurotransmitter were visualized using the web-based tool Morpheus and presented as Z-score distributions. Overrepresentation analysis for genesets from the gene ontology database within the DEGs was performed using the web-based tool DAVID. To evaluate the DEG signature specificity in colonic cell types, scRNA-Seq of the C57Bl / 6 mouse colon was downloaded from the Broad Institute single cell portal / / singlecell.broadinstitute.org / single_cell / study / SCP1038 / . Counts data were randomly subsampled for 1000 cells per cell type using the original authors annotations for cell types (Drokhlyansky et al. (2020) Cell 182, 1606-1622.e1623). Data were then processed by Seurat by running the NormalizeData, ScaleData, FindVariableFeatures, RunPCA, FindNeighbors(dims = 1:20) followed by the RunUMAP(dims = 1:20) commands for dimensionality reduction and visualization. Scoring of the DEGs downregulated in DT-administered BAF53b::iDTR mice was performed using the AddModuleScore (ctrl = 100) function of Seurat which scores the expression of the input gene lists to each cell individually using methods derived from Tirosh et al. (2016; Science (New York, N.Y.) 352, 189-196). In vivo tracing of neuronal projections To label ENs and their projections an Adeno-Associated Virus Serotype 1 (AAV1) with a flip- excision switch (FLEX) vector for conditional Cre-On gene expression of ChR2 and mCherry (AAV1- FLEX-ChR2-mCherry, Addgene, 18916-AAV1) was delivered to the colons of BAF53b::iDTR mice. This approach allowed simultaneous tracing of nerve fibers, driven by BAF53b-Cre in the proximally injected colon, and neuronal ablation in the colon distally via DT. Mice were anesthetized under isoflurane, with a midline incision made to access the colon. AAV1- FLEX-ChR2-mCherry was administered to the muscularis propria of the midcolon towards the cecum at a volume of 2μL and a titer of 1.2×1013vg / ml using a NanoFilTM microliter syringe (33 G). DT was administered as described in the section ‘Neuron and ECG ablation’ 5mm distal to the AAV1- FLEX-ChR2-mCherry injection site in the opposing direction towards the rectum. Mice were sacrificed after 2 and 8 weeks post procedure and tissues were collected for immunohistochemistry and visualization of the distally projecting BAF53b::mCherry processes. Images were acquired using a Nikon AXR confocal microscope (Nikon Cambridge, MA). For quantification of mCherry density four images of a total field of view of 3mm2were assessed per mouse using ImageJ software. The Top Hat function was utilized for background equalization and the percentage area of PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 mCherry was calculated within the area occupied by EGCs by binarizing the S100B+immunohistochemically labelled area using the ‘Otsu’ threshold method to create a region of interest, before measuring the area occupied by mCherry binarized by the ‘Huang’ threshold method. Organ bath force contraction Organ bath experiments to measure the contractile force of the colonic smooth muscle in response to stimuli were performed as previous described (Robinson et al. (2017) American Journal of Physiology-Gastrointestinal and Liver Physiology 312, G85-G102; Stavely et al. (2022) Science Translational Medicine 14, eabl8753). Briefly, colons were excised and cut into 5mm rings. In experiments involving DT ablation, tissue staining with India ink was utilized to denote the ablation region and prepare colon rings for analysis. In experiments utilizing cell transplantation, the fluorescence of transplanted tdT+cells were visualized under a stereoscopic microscope to prepare colonic rings of the engraftment site. Colonic rings were mounted to force displacement transducers in a muscle strip myograph bath (Model 820 MS; Danish Myo Technology, Aarhus, Denmark) containing Krebbs solution (oxygenated with 95% O2 and 5% CO2) and were maintained at 37°C. Tissues were exposed to electric field stimulation (EFS) with pulse trains of 40 V for 15 sec, with pulse duration of 0.3 ms, at a frequency of 5 Hz using a CS4+ constant voltage stimulator with MyoPulse software (Danish Myo Technology, Aarhus, Denmark) to evaluate contractile responses to neuronal stimulation. For optogenetics experiments, blue light stimulation (BLS) was performed at the transplantation site at 10 ms pulse width, 10 Hz, with a 15 second train duration using a 200 µm diameter optic fiber and a diode-pumped solid-state laser system (470 nm, 200 mW, Model number: MDL-III-470; OptoEngine, LLC, Midvale, UT). In vivo recordings of smooth muscle activity To assess colonic smooth muscle activity in mice in vivo, electromyography (EMG) and intraluminal pressure were assessed simultaneously. Mice were subjected to anesthesia by isoflurane where an incision along the midline was made to access the colon. Intact colons were ligated and cannulated with the distal end connected to a pressure transducer (CWE Inc., Ardmore, PA). The colon was filled with physiological saline to an initial pressure of 10-15 mmHg in a closed system, with transient increases in pressure signifying colonic contractions. To perform EMG recordings, custom made three- lead needle electrodes (Motion Lab Systems, Inc. LA, CA) positioned in the muscularis propria of the colon and were connected to a four channel Bio-amplifier (CWE Inc., Ardmore, PA) through an ISO-Z Isolated Head Stage amplifier (CWE Inc., Ardmore, PA). Digitized recordings were made with the Power Lab 16 / 35 data acquisition system (ADInstruments, NSW, Australia) and data were analyzed with Lab Chart Pro Software v8.1.16 (ADInstruments). Quantification and Statistical Analysis Data analysis was performed using GraphPad Prism v7 (GraphPad Software Inc., San Diego, USA). For multiple comparisons, a one-way ANOVA was performed with Holm-Sidak test for post hoc analysis on parametric data and a Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test for nonparametric data. An unpaired t-test was conducted for pairwise comparisons. For all analyses P≤0.05 PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 was considered significant. All data were presented as mean ± standard error of the mean (SEM), unless otherwise stated. TABLE 1: PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Example 2: Highly neurogenic glia from human and mouse myenteric ganglia generate functional neurons following culture and transplantation into the gut Summary In this example, protocols to enrich for intraganglionic EGCs by isolating intact fragments of MyGa, generating cultures with higher neuronal purity than traditional methodologies isolating intramuscular single cells (IM-SCs) are described. MyGa-derived EGCs transdifferentiate into more PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 neurons than IM-SC-derived EGCs, confirming their neurogenic predisposition. Following transplantation to mouse intestine, MyGa-derived neurons generate calcium transients and activate smooth muscle in response to optogenetic stimulation. In human intestine, MyGa-derived cells are similarly highly neurogenic, are enriched for a distinct progenitor population identified by scRNA-seq, and exhibit neuromuscular connectivity following xenogeneic transplantation to mice. Highly neurogenic ENSCs are preferentially located within the MyGa and their selective isolation offers considerable potential for therapy. Introduction Cell therapies for ENS disorders would be vastly improved by increasing the number and proportion of ENSCs delivered to the recipient and by optimizing the capacity of the donor cells to generate neurons. Below the neurogenic potential of ENS cells residing within the myenteric ganglia (MyGa) to those within the intramuscular space (intramuscular single cells; IM-SCs) are compared. Described are protocols to selectively isolate MyGa vs IM-SCs to examine the neurogenic potential of each. Using lineage tracing models in mice, MyGa were shown to favor enteric neurogenesis when compared to IM-SCs, leading to the expansion of a cell population markedly enriched for enteric neurons and their progenitors. MyGa-derived cells recapitulate appropriate enteric neuronal subpopulations and demonstrate neuronal functionality, including the generation of calcium transients following electrical field stimulation and neuromuscular connectivity following transplantation to mouse intestine. Isolation procedures for the generation of ENSCs from human samples were developed and were found to yield a cell population with greater ENSC purity, high neurogenic potential, and neuronal functional properties following transplantation in vivo. These results are included in the results section below and include quantification assays including quantitative PCR, transgenic fluorescent reporters, immunohistochemistry and force contraction studies post-transplantation to the intestinal wall. These results confirm that highly neurogenic glia are spatially restricted to the myenteric ganglia and the ability to selectively isolate and expand this population represents an important advance for the development of ENS cell therapy. Results Intact myenteric ganglia can be isolated from the mouse intestine To visualize the myenteric plexus in mice, we utilized transgenic fluorescent reporter strains specifically marking enteric neurons and EGCs. For all experiments, mice were utilized after 3 months of age when they had reached adulthood to adequately reflect the postnatal environment (Flurkey et al. (2007) The-Mouse-in-Biomedical-Research, 2nd ed. Volume 3, Normative Biology, Husbandry, and Models). Enteric neurons were traced using BAF53b-Cre driver mice crossed with Rosa26-tdTomato (R26-tdT) reporter mice to generate BAF53b-Cre::R26-tdT mice (BAF53b::tdT). BAF53b-driven tdT fluorescence labels neuronal soma and their nerve fiber processes in the myenteric plexus (Zhan et al. Genesis 53, 440–448.). EGCs were visualized in mice with the glia-specific Plp1 promoter driving expression of EGFP. Double transgenics (BAF53b::tdT; Plp1-EGFP) allowed simultaneous visualization of PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 neurons and glia in the myenteric plexus (FIG.14A). EGCs were observed adjacent to enteric neurons within the myenteric ganglia (MyGa) and throughout the intramuscular (IM) region with bipolar processes projecting along enteric neuronal fibers (FIG.14B). The sizes of the MyGa were quantified in BAF53b::tdT; Plp1-EGFP mice in different regions of the gastrointestinal tract including the duodenum, jejunum, ileum, proximal colon, and distal colon (FIG.14C – FIG.14G). The minimum Feret’s diameter was calculated, which describes the smallest dimension across the MyGa (FIG.14C). Calculations for minimum Feret’s diameters were similar across the intestinal segments, with the exception of the proximal colon, where MyGa were larger (FIG.14H). Using these data, it was calculated that 85% of the MyGa had a minimum diameter of 20µm- 100µm and developed processes to isolate the MyGa involving enzymatic digestion and particle size- based filtration. First the muscularis propria harboring the myenteric plexus was removed by manual microdissection and digested in an enzymatic cocktail to generate single cell suspensions while retaining the enzymatically resistant MyGa as intact particles. Samples were filtered to collect the fraction <20µm in size, comprising IM-SCs, and the fraction between 20µm-100µm in size, containing enteric ganglia (FIG. 14I). Visualization of BAF53b::tdT; Plp1-EGFP fluorescence confirmed that the latter fraction was enriched for enteric neurons and EGCs as compared to the IM-SC fraction (FIG.14J). The fraction containing material >100µm was discarded. Validation by PCR showed that MyGa-enriched samples exhibited upregulation of neuronal (Tubb3, Phox2b, Chat, Nos1) and EGC / ENSC (Ngfr) markers, and downregulation of genes specific for mesenchymal cells (Pdgfra, Col1a1). These results confirm that the MyGa isolation strategy leads to a significant enrichment for ENS cells with reduced contamination by non-ENS cells as compared to the commonly utilized single cell suspension (FIG.14K). MyGa-derived neurospheres have a high proportion of enteric neurons, glia, and progenitors To compare the neurogenic potential and cellular composition of IM-SCs versus MyGa preparations, serially filtered samples from the small intestine of BAF53b::tdT; Plp1-EGFP mice were cultured in free-floating conditions to form neurospheres (FIG.15A). Both preparations generated neurospheres evidencing the presence of ENSCs. The expression of Plp1-EGFP and BAF53b::tdT was markedly elevated in MyGa-derived neurospheres (FIG.15B), although the number of spheres generated was lower (FIG.15C). As in FIG.14K, neuronal and EGC / ENSC markers were upregulated in the MyGa- derived neurospheres, while the mesenchymal marker, Pdgfra, was downregulated, indicating that MyGa- derived neurospheres yielded a purer neuroglial and progenitor population in vitro (FIG.15D). To confirm this, neurospheres generated from MyGa and IM-SC preparations from BAF53b::tdT; Plp1-EGFP mice were cultured on fibronectin-coated plates, which facilitated cell migration into monolayers in both groups (FIG.15E – FIG.15E’). Following trypsinization into single cell suspensions for flow cytometry (FIG.15F), MyGa-derived cultures were noted to be enriched for both Plp1-EGFP, representing ENSCs and EGCs, and BAF53b::tdT+ neurons (FIG.15G – FIG.15H). Conversely, IM-SC cultures had greater proportions of contaminating non-ENS cells, as shown by their high expression of double-negative cells expressing neither neuronal nor glial markers (FIG.15G – FIG.15H). To determine how the neurosphere source affected their differentiation potential, neurospheres derived from IM-SCs and MyGa from BAF53b::tdT; Plp1-EGFP mouse intestine were cultured on fibronectin and subpopulation markers were labeled by immunofluorescence (FIG.16A – FIG.16H’). The PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 proportion of tdT+ neurons and GFP+ EGCs was markedly higher in cultures derived from MyGa compared to IM-SCs (FIG.16I). Furthermore, the glia to neuron ratio was far greater in IM-SC samples, suggesting that MyGa give rise to a more neurogenic cell population (FIG.16J). Although the total number of enteric neurons was increased in MyGa-derived cultures, no differences were observed in the proportions of excitatory (calretinin) (FIG.16A – FIG.16B’, FIG.16K) versus inhibitory (nNOS) neuron (FIG.16C – FIG.16D, FIG.16L) subtypes between the two preparations when expressed as a percentage of BAF53b::tdT+enteric neurons. This indicated that neurospheres generated from the MyGa and IM-SCs form similar neuronal subtypes important for recapitulating the ENS. There was no significant difference in the proportion of Plp1-EGFP cells co-expressing the EGC marker GFAP (FIG.16E – FIG. 16F’; FIG.16M), while the proportion of Plp1-EGFP cells co-expressing the ENSC marker P75 was markedly elevated in the MyGa-derived fraction (FIG.16G – FIG.16H’, FIG.16N), suggesting the expansion of a pro-neurogenic ENSC population. MyGa-derived ENSCs exhibit higher rates of neurogenesis MyGa-derived neurospheres exhibited high levels of progenitor and neuronal markers suggesting that these cells may contain a highly neurogenic population consistent with our hypothesis that ENSCs are preferentially located within the myenteric ganglia (Guyer et al. (2023) Cell Reports 42, 112194). To experimentally validate the higher neurogenic potential of cells from the MyGa compared to IM-SCs, neurospheres were generated from BAF53b::tdT; Plp1-EGFP mice, cultured on fibronectin to form a monolayer, and trypsinized into single cell suspensions for evaluation by fluorescence activated cell sorting (FACS). In this experiment, cells were sorted for Plp1-EGFP, specifically selecting for a pure EGC / ENSC population and omitting differentiated enteric neurons (BAF53b::tdT). Cells expressing Plp1- EGFP were cultured in free floating conditions and the absence of BAF53b::tdT expression confirmed (FIG.16O). After 6 days in culture, both MyGa and IM-SCs formed neurospheres and contained neurons, as indicated by the presence of BAF53b::tdT fluorescence, confirming the neurogenic potential of Plp1- EGFP expressing ENSCs (FIG.16P). Neurospheres were then plated onto fibronectin-coated culture dishes to form monolayers and allow quantification of neuronal proportions (FIG.16Q). MyGa gave rise to populations with a lower glia to neuron ratio (FIG.16R) and a markedly greater proportion of BAF53b::tdT enteric neurons compared to IM-SCs (FIG.16S). The high rate of neurogenesis indicates that MyGa are indeed a reservoir for ENSCs with significant neurogenic potential. MyGa-derived neurospheres engraft, migrate, and differentiate into functional neurons following transplantation in vivo As the MyGa isolation process produced a cell population with increased neurogenesis and concomitant reduction in the contaminating mesenchymal population, MyGa offers an improved therapeutic source of cells for ENS regeneration. To determine whether MyGa-derived cells exhibit neuronal properties, including responses to electrical field stimulation (EFS) and receptivity to the excitatory neurotransmitter ACh, calcium imaging was performed on MyGa-derived cells cultured from BAF53b::tdT-GCaMP mice, in which neurons express a genetically encoded calcium indicator. Application of ACh to the culture medium elevated Ca2+levels in the MyGa-derived cells (FIG.17A – FIG.17B) and PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 EFS evoked Ca2+transients consistent with neuronal action potentials (FIG.17C – FIG.17D), confirming the functionality of MyGa-derived neurons. Next we evaluated whether MyGa-derived neurospheres transplanted to the mouse intestine could successfully integrate with the endogenous ENS. MyGa-derived neurospheres were generated from BAF53b::tdT; Plp1-EGFP mice as described above and 100 neurospheres transplanted into the colonic wall of wild-type recipient mice. Four weeks after transplantation, expression of Plp1-EGFP and BAF53b::tdT from transplanted cells was observed, with extensive cell migration and projection of BAF53b::tdT nerve fibers within the intestinal smooth muscle (FIG.17E – FIG.17E’’). In contrast, IM-SC- derived neurospheres did not engraft post transplantation at the same cell dose (MyGa 6 / 6 vs IM-SC 0 / 4). tdT+ enteric neurons derived from MyGa co-expressed the neuronal marker TUBB3 and formed connections with host-derived TUBB3 immunoreactive nerve fibers as well as the myenteric ganglia of recipient mice, demonstrating integration with the endogenous ENS (FIG.17F). As seen in FIG.16H and FIG.16N, we confirmed that transplanted MyGa-derived cells continued to express high levels of P754 weeks after transplantation (FIG.17G). To demonstrate that donor neurons derived from MyGa were functionally competent in the host intestine, we conducted a series of transplant experiments using optogenetic stimulation (Madisen et al. (2012) Nature Neuroscience 15, 793–802) of donor cells and recorded evoked contractile responses in recipient intestinal smooth muscle. MyGa were isolated from the small intestine of Chat-Cre::ROSA26- tdTomato-channelrhodopsin-2 (Chat::tdT-ChR2) mice, in which ChR2 is expressed by excitatory cholinergic neurons, which express choline acetyltransferase (ChAT). Neurospheres from MyGa were more likely to contain tdT expression compared to IM-SCs (FIG.17H, FIG.17J), and the neuronal specificity of the tdT expression was confirmed by co-labeling with the pan-neuronal antibody, Hu (FIG. 17I). Transplantation of Chat::tdT-ChR2 neurospheres from MyGa to Plp1-EGFP mouse recipients resulted in successful engraftment within the muscularis propria, with survival of transplanted cells for at least four weeks (FIG.17K). Recipient mice were anesthetized and intraluminal colonic pressure was recorded in response to blue light stimulation (BLS), which optogenetically activates the transplanted Chat::tdT-ChR2 donor cells (FIG.17L). This was done in the presence of the nitric oxide (NO) donor sodium nitroprusside to inhibit spontaneous muscle activity. Stimulation of transplanted MyGa-derived cells by BLS evoked transient increases in luminal pressure indicative of their ability to cause contraction of the colonic smooth muscle as demonstrated by in vivo electromyography (EMG) (FIG.17M). Recipient colons were then excised and dissected to obtain intestinal rings for organ bath experiments to measure the force of smooth muscle contractions (FIG.17N). In these preparations, BLS evoked robust smooth muscle contractions, confirming that the transplanted MyGa give rise to excitatory enteric neurons capable of making functional neuromuscular connections with the intestinal smooth muscle (FIG.17O). These experiments were replicated using MyGa derived from BAF53b-Cre::R26-tdT-ChR2 (BAF53b::tdT- ChR2) mice, which express ChR2 on all neurons (FIG.17P). BLS of BAF53b::tdT-ChR2 expressing MyGa-derived neurons in these preparations resulted in contraction of the smooth muscle, and this was inhibited by the addition of the sodium channel blocker tetrodotoxin (TTX), confirming that these contractions were mediated by transplant-derived neurons (FIG.17P). Together these experiments confirmed that MyGa-derived neurons possessed electrical activity and responsiveness to neurotransmitters, integrate with the ENS after transplantation, and regulate PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 smooth muscle contractility, thus demonstrating their capacity to differentiate into functional enteric neurons and their potential utility as a regenerative cell therapy. Myenteric ganglia can be isolated from human intestinal muscularis propria Next, it was investigated whether 1) MyGa could be isolated from human intestinal samples and 2) if, as in mice, these are similarly highly neurogenic and functionally effective following transplantation. First, the MyGa were visualized in human wholemount preparations of the muscularis propria by immunohistochemistry for pan-neuronal markers, PGP9.5 and Hu (FIG.18A). The minimum Feret’s diameters were much larger in human specimens from both normal and Hirschsprung disease (HSCR) tissue than in mice, with minimum diameters ranging between 0.2 to 0.6 mm (FIG.18B). From this data we predicted that the MyGa from human samples could be isolated using filter sets between 100µm- 1000µm. To isolate the MyGa, full-thickness samples of intestine were obtained from clinically indicated surgical resections (Table 2, below). The muscularis propria was dissected as an intact sheet (FIG.18C), then minced and digested in a collagenase XI and dispase solution at 37˚C for four hours with gentle rotation throughout the digestion procedure before being serially passed through a filter set of 100µm and then 1000µm. Samples were collected between 100µm-1000µm in size, with immunohistochemistry for the pan-neuronal marker TUBB3 revealing the successful isolation of intact human myenteric ganglia using these parameters (FIG. 18D). For further validation, gene expression was compared between IM-SCs <100µm and the 100µm- 1000µm MyGa fraction. As in mice, the MyGa-enriched fraction contained significantly higher expression of the neuronal markers, ELAVL4 and PHOX2B (FIG.18E). To confirm whether neurospheres could be generated from human gut-derived MyGa, using a dissecting microscope, myenteric ganglia were manually ‘picked’, cultured, and visualized over 8 days. Timelapse imaging of individual specimens indicated that the MyGa remodel over time to form neurospheres in vitro (FIG.18F). This confirms the neurogenic potential that these ganglia possess. Neurospheres generated from human MyGa are highly neurogenic To determine whether isolation of human MyGa utilizing the filtration method could be leveraged for ENS regeneration, properties of neurospheres generated from IM-SCs traditionally used to culture enteric neurospheres (<100µm) were compared to those derived from the MyGa-enriched fraction (100µm-1000µm). MyGa-derived neurospheres formed under appropriate culture conditions (FIG.19A – FIG.19A’) were cultured on fibronectin to form a monolayer before trypsinization to yield single cell suspensions for quantification of cell yield. No difference was observed in the proliferative capacity of cells isolated from the MyGa versus IM-SC fractions during the first or second passage (FIG.19B). Furthermore, unlike in mice, the MyGa enrichment process did not reduce the overall cell yield before the cells were passaged (P0) or the anticipated cell yields per gram of tissue determined by population doubling level assays at P1 and P2 (FIG.19C). To determine the effects of MyGa enrichment on the properties of neurospheres, expression of neuronal markers (PHOX2B, TUBB3) and EGC / ENSC markers (PLP1, NGFR), were determined across the three passages. The pro-neuronal markers PHOX2B and TUBB3 were elevated in MyGa-derived neurospheres at P0 and P1, but no statistical differences in PLP1 and NGFR were observed until P2 (FIG.19D). To validate that the enrichment of ENSCs in the MyGa PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 fraction was responsible for these results, additional experiments were performed comparing neurospheres derived from ‘hand-picked’ MyGa to those from the MyGa-enriched and IM-SC preparations at the first passage. Neurospheres isolated from hand-picked MyGa demonstrated the highest levels of PHOX2B, TUBB3, PLP1 and NGFR. As these samples contain the purest source of MyGa, this confirms that the MyGa are the major source of ENSCs in human intestine and likely explain the elevated expression of these genes in the MyGa-enriched samples (FIG.19E). To further resolve the cell populations, scRNA-seq was performed on cultures derived from the commonly utilized IM-SC suspensions, MyGa-enriched fractions, and hand-picked MyGa (FIG.19F). In the combined dataset, we observed distinct clusters of fibroblasts, EGC / ENSCs, and a small population of neurons (FIG.19G). As seen from the PCR results, cultures derived from hand-picked MyGa exhibited the highest purity of EGC / ENSCs, and those derived from the MyGa-enriched fraction improved ENSC purity 6.1-fold compared to IM-SC preparations (FIG.19H). These findings highlight the important observation that ENS progenitors are spatially restricted to the myenteric ganglia. Cell populations were validated by the expression of bona fide markers for EGCs (SOX10, S100B, PLP1), ENSCs (SOX2, ERBB3, FOXD3, GFRA1) (Elmentaite et al. (2021) Nature 597, 250–255), enteric neurons (ELAVL4, MAP2, PHOX2B, L1CAM) and intestinal fibroblasts (PDGFRA) (FIG.19I and FIG.21A). Other markers of human muscularis EGCs (Drokhlyansky et al. (2020) Cell 182, 1606-1622.e23.) were also expressed by EGC / ENSCs further validating their identity (FIG.21A). The top 10 markers by fold expression in these populations (>60% of cells) included markers of neurons (VIP, GAL) and intestinal fibroblasts (COL6A3), while the EGC / ENSC population exhibited high expression of PLP1, the marker utilized in this study to isolate EGC / ENSCs from mice (FIG.19J). The heterogeneity of EGC / ENSCs was further explored and 3 main subpopulations were identified, including EGC / ENSCs actively undergoing cell proliferation and two transcriptionally distinct EGC / ENSC subsets (FIG.19K – FIG.19L). The 3 subpopulations of EGC / ENSCs shared markers such as PTPRZ1 and ITGA6 which have been detected in mouse embryonic EGC / ENSCs (Lasrado et al. (2017) Science 356, 722–726) (FIG.19M). MKI67 was confirmed in the proliferating EGC / ENSC cluster, while the traditionally utilized ENSC marker NGFR was not a reliable pan-ENSC marker in humans, but rather was predominately expressed in the EGC / ENSC-1 subpopulation (FIG.19M). To evaluate the pro- neurogenic properties of EGC / ENSCs, differential expression of enteric neuronal markers was assessed between the two EGC / ENSC subpopulations. Only 23 / 581 neuronal markers were upregulated in EGC / ENSC-1 compared to 133 / 581 in EGC / ENSC-2, including established pro-neurogenic genes HAND2 and PHOX2B, and neuronal markers MAP2 and NCAM1 (FIG.19N). To validate EGC / ENSC markers identified by scRNA-seq in human samples, cells were labeled via immunocytochemistry. The EGC / ENSC 1 marker NGFR labelled a subset of the predicted pan- EGC / ENSC marker ITGA6 as anticipated (FIG.19O – FIG.19P). Quantification of ITGA6, NGFR and TUBB3 indicated that ITGA6 was the most abundant EGC / ENSC marker, and furthermore that MyGa enrichment improves the purity of cell cultures for EGC / ENSCs and enteric neurons (FIG.19Q – FIG. 19R). To determine whether these EGC / ENSCs subpopulations were specific to human gut, their possible equivalencies were explored in mouse neurospheres (FIG.19S). Scoring of human enteric neurons and EGC / ENSC 1 and 2 subpopulation markers for homologous genes in the mouse revealed PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 strong correlations between mouse and human enteric neurons and both EGC / ENSC subpopulations (FIG.19T). Notably, the predicted mouse EGC / ENSC 2 population scored higher for neuronal genes and was situated near the neuroblast / neuronal cluster, suggesting that both species contain a highly neurogenic EGC / ENSC population (FIG.19S – FIG.19T). Shared characteristics of EGC / ENSC 1 in mice and human include higher expression of NGFR and RUNX2 (nerve regeneration), whereas EGC / ENSC 2 share higher expression of SLC35F1 (neurodevelopment) and PTGDS (neuromodulator) (FIG.21B). Markers of human EGC / ENSC 1 and 2 populations resembled two polarized EGC populations in the Drokhylansky et al, 2020 (Cell 182, 1606-1622.e23) human intestinal atlas suggesting that traits of EGC populations could remain in vitro (FIG.21C – FIG.21E). Transcriptional markers for mouse intraganglionic EGCs were evaluated in the datasets of cultured EGC / ENSCs from mice and human samples (FIG.21F). The EGC / ENSC 2 population exhibited higher expression of intraganglionic EGCs in both species (mouse: 9 / 11 genes, human: 10 / 11 genes) lending support to the possibility that EGC / ENSC 2 is an equivalent population in mice and humans that shares characteristics with MyGa EGCs (FIG. 21F). Human MyGa ENSCs are primed for regenerative cell therapy To determine whether human ENSCs derived from the MyGa could be utilized as a cell therapy, the following were assessed 1) their ability to evoke calcium transients (effector function) and 2) their ability to induce colonic smooth muscle contractions (affector function). Calcium responses to ACh were determined using time-lapse imaging of monolayer cultures containing MyGa using the cell permeable calcium indicator Fluo-4 (FIG.20A – FIG.20C). To determine whether ACh-induced transients were neuronally mediated, cultures were preincubated with TTX to inhibit neuronal activity. Maximum intensity projections across time-lapse imaging show a higher number of cells responding to ACh compared to those in TTX conditions, suggesting calcium responses are consistent with neuronal activity (FIG.20A). Typically, cells remained inactive until the application of ACh, which induced a sharp rise in intracellular Ca2+within seconds followed by a steady decline in Ca2+levels over approximately 1 minute (FIG.20B – FIG.20B’). Evaluation of intracellular Ca2+responses to ACh (n = 226 cells) and to ACh in the presence of TTX (n=304 cells) indicated that 35.5% of MyGa-derived neurons responded to ACh as determined by a doubling in fluorescence levels and only 2.1% responders in the presence of TTX (FIG.20C). To evaluate the ability of human MyGa-derived neurons to regulate smooth muscle activity, ENSCs were transduced with an AAV vector expressing channelrhodopsin-2 (ChR2) and a GFP reporter (FIG.20D). These cells were cultured to form neurospheres (FIG.20E) and then transplanted into the colonic muscularis propria of NOD-scid IL2Rgammanull(NSG) immunocompromised mice. After 3 weeks, transplants were assessed for the presence of GFP expression, indicative of engraftment by successfully transduced human cells (FIG.20F – FIG.20G). Colonic rings were collected from the transplantation site for organ bath experiments to measure smooth muscle contractility by optogenetics using blue light stimulation (BLS). Representative traces show a consistent pattern of smooth muscle contractions in response to BLS of the transplanted region (FIG.20H). The peak force was quantified and shows that BLS of transplanted human MyGa-derived cells enhances smooth muscle contractions, indicating that they develop functional neuromuscular connectivity after transplantation (FIG.20I). Elevations in peak PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 force in response to BLS were negated in the presence of TTX confirming these effects were neurally mediated (FIG.20J). Discussion In this example, protocols have been developed that allow for EGCs residing within the myenteric ganglia in both mice and humans to be isolated and cultured independently. Using a lineage tracing mouse model, MyGa-EGC / ENSCs were demonstrated as more neurogenic than the EGC / ENSCs inhabiting the intramuscular space, leading to the expansion of a purer cell population that is markedly enriched for enteric neurons and their progenitors with reduced contamination by non-ENS cells. Importantly, MyGa-derived neurons recapitulate appropriate neurochemical subpopulations, including excitatory (calretinin) and inhibitory (nNOS) neurons, and demonstrate functionality by generating calcium transients following electrical field stimulation and by exhibiting direct control over intestinal muscle contractility following transplantation and optogenetic activation. Similar experiments were conducted on human samples, yielding a population with higher ENSC purity that favored neuronal differentiation and demonstrated functional neuronal properties following in vivo transplantation to mice. This example provides an extensive single cell transcriptome dataset of human-derived enteric neurospheres. Sequencing data not only validates the efficacy of our isolation process, confirming both the enrichment of ENS cells from MyGa as well as their associated enhanced neurogenic potential, but also offers insights into potential subpopulations of EGC / ENSCs and their transcriptional markers. Two subpopulations of EGC / ENSCs have been categorized that exhibit distinct profiles across samples and find the ENSC-2 population to be enriched in MyGa. In particular, the ENSC-2 subpopulation exhibited higher expression of pro-neurogenic and neuronal markers, including MAP2, consistent with our in vitro findings that MyGa-derived cells have increased neurogenic differentiation potential. Furthermore, using a viral vector expressing ChR2 and a GFP reporter, successful engraftment of human derived enteric neurons into an immunocompromised mouse host and subsequent effective smooth muscle contraction following blue light stimulation was demonstrated. These results provide evidence of functional human gut-derived cells post transplantation, which is useful for regenerative therapy. The specific isolation, propagation, and transplantation of MyGa-derived cells provides significant therapeutic benefits for the treatment of ENS disorders in the form of cell therapy. Regenerative therapies represent a promising strategy to replace the missing neurons and glia by restoring function in specific ENS disorders, including gastroparesis and Hirschsprung disease (as is described herein). ENSCs can differentiate into enteric neurons and EGCs following transplantation into both the embryonic and postnatal intestine, and are capable of specifically differentiating into neurons of the appropriate neurochemical phenotype. Using our proposed isolation process that enriches for MyGa, we simultaneously increase the proportion of ENSCs and decrease the proportion of non-progenitor cells that are delivered to the recipient, while also enhancing the neurogenic differentiation potential of the transplanted ENSCs. This cell isolation strategy provides significant value towards improving regenerative cell therapies for the treatment of ENS diseases. The above results were obtained using the following materials and methods. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 EXPERIMENTAL MODEL AND SUBJECT DETAILS Ethical Statement Animal experimentation was performed according to experimental protocols approved by the Institutional Animal Care and Use Committees IACUC (2009N000239) of Massachusetts General Hospital. This study was approved by the human research ethics committees of Massachusetts General Hospital (IRB protocol #2010P00669), The Royal Children’s Hospital, and Monash University (HREC 38262). Written consent was collected for all subjects prior to participation in this study. Mice Details for all mice utilized in this study are provided in Table 4. Mice were purchased from Jackson laboratory (Bar Harbor, ME) with the exception of Plp1-EGFP mice which were kindly donated by Wendy Macklin PhD, University of Colorado (Mallon et al. Journal of Neuroscience 22, 876–885). All mice were housed at the Center for Comparative Medicine animal facility at Massachusetts General Hospital under specific-pathogen-free conditions. All experiments were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee IACUC (2009N000239). Rodents were housed in Allentown Inc rectangular caging (160 cages per individually ventilated cage racks; which uses blower at 60 air changes per hour) under a 12h:12h light:dark cycle from 7 am – 7 pm. Bedding consisted of Hardwood Sanichip; with Carefresh nesting material and mice had access to Prolab Isopro RMH 3000 chow mix (ScottPharma) ad libitum. For all experiments, both male and female mice were utilized after 3 months of age when they had reached adulthood (Flurkey et al. (2007) The-Mouse-in- Biomedical-Research, 2nd ed. Volume 3, Normative Biology, Husbandry, and Models). Human specimens Specimens of ganglionated Hirschsprung disease (HSCR) colon were taken from patients (4-21 months of age) during pull-through surgery at the Royal Children’s Hospital, Melbourne, Australia. Control samples were obtained during closure of stoma surgery for anorectal malformation (ARM) from patients ranging 9-20 months of age. ARM tissue has normal intrinsic enteric innervation and hence is considered as a ‘healthy’ control in this study. Excess colonic or ileal intestinal samples resected as part of required patient care at Massachusetts General Hospital were collected from subjects between 2 months to 61 years old (Table 2). Table 2. Details of subjects utilized for cell isolation. See FIG.19. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 METHOD DETAILS Quantification of ganglia sizes in mouse samples To simultaneously visualize enteric neurons and EGCs in the intestine, dual reporter BAF53b- Cre::R26-tdT; Plp1-EGFP mice (BAF53b::tdT; Plp1-EGFP) were bred as we previously reported (Guyer et al. (2023) Cell Reports 42, 112194; Stavely et al. (2022) Science Translational Medicine 14). Mice were euthanized by CO2 asphyxiation and the small and large bowel were collected to prepare wholemount preparations by opening the intestinal lumen by cutting along the mesenteric border, pinning the tissue onto silicone-lined petri-dishes and removing the mucosa and submucosa under a dissection microscope prior to fixation in 4% PFA for 4 hours. The remaining muscularis propria containing the myenteric plexus were then imaged using a Keyence BZX-700 All-In-One Microscopy system (Keyence America Itasca). Morphological characterization of the ganglia was performed in 10 random ganglia captured within a total field of view of 2 mm2per tissue segment and per mouse. Individual ganglia containing neuronal soma were manually traced using Fiji open-source software (ImageJ v1.54f (Schindelin et al. (2012) Nat Methods 9, 676–682) and Feret’s diameters for each ganglia were calculated using the in-built measurements function in Fiji. Quantification of ganglia sizes in human tissue samples Tissue specimens from HSCR and ARM subjects were collected following surgical removal and transported in cold phosphate buffered saline (PBS, 0.1M, pH7.4) for processing. Mesentery and associated fat were removed, then tissues were placed in PBS containing nicardipine (10 µM) to limit contractions. Tissue was stretched to form a flat sheet and pinned mucosa downwards on a Sylgard-lined 10 cm dish then fixed (4% PFA, overnight at 4°C). Tissues were washed (3x1 h with PBS) and stored in PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 PBS with 0.1% sodium azide at 4°C. Longitudinal muscle-myenteric wholemounts were prepared by separating the external muscle layer from the submucosa. Circular muscle strips were then removed to expose the underlying myenteric plexus. Myenteric wholemounts were blocked and permeabilized using blocking buffer (5% normal horse serum, 0.5% Triton X-100 in PBS with 0.1% sodium azide) overnight at 4°C. Tissues were incubated for one week at 4°C with primary antibodies against PGP9.5 (rabbit, 1:2000, RRID: AB_10891773; Abcam) and HuC / D (mouse, 1:500, RRID: AB_221448; clone 16A11, Molecular Probes) diluted in blocking buffer. Tissues were then washed with PBS (3x30 min), then incubated with secondary antibodies conjugated to Alexa Fluor 488, 568, 594, or 647 (1:500 dilution in PBS, overnight, 4°C; ThermoFisher). Tissues were stained with DAPI (1:1000, 2 h at RT; Sigma Aldrich), washed (3x30 min with PBS), then mounted onto slides using buffered glycerol. Coverslips were sealed with clear nail varnish. Images of myenteric ganglia were acquired using a Leica TCS-SP8 laser scanning confocal system (HC PL APO CS220x / 0.75 NA immersion objective) and Leica Application Suite X software (v3.5.5.19976, Leica Microsystems). Images capturing 3-6 different ganglia or regions were acquired per wholemount preparation for each patient. Images were excluded for analysis if ganglia appeared damaged due to dissection. As in mice, individual ganglia containing neuronal soma were manually traced using Fiji open-source software (ImageJ v1.54f (Schindelin et al. (2012) Nat Methods 9, 676–682)) and Feret’s diameters for each ganglia were calculated using the in-built measurements function in Fiji. Myenteric ganglia enrichment and isolation Mice were euthanized and the intestines collected as above. The muscularis propria containing the myenteric plexus was removed by mechanical dissection from colons on silicone-lined petri dishes, or the small bowel (Stavely et al. (2021) Stem Cells 39, 1236–1252). Tissues were subsequently minced and digested in collagenase type XI (1 mg mL−1; Sigma Aldrich, St. Louis, Missouri, CAT# C7657) and dispase (0.4U mL−1; STEMCELL Technologies, Vancouver, Canada) solution at 37˚C for one hour using a VWR Mini Incubated Shaker (Avantor CAT# 76407-108). Samples were triturated until all the sample could pass through a 1000 μL pipette tip. The digested suspension was then serially passed through a set of sterile stackable cell strainers (pluriStrainer®, pluriSelect, El Cajon, CA, USA) with a 20 µm pore size strainer on the bottom and 100 µm pore size strainer placed on top. The contents passing through both the 100 µm and 20 µm cell strainers were considered a single cell suspension (<20 µm), while the contents in the mid-section of the filters (20-100 µm) was collected to obtain tissue fragments enriched for MyGa. All contents >100 µm were discarded. Successful enrichment for MyGa was confirmed by transgenic tdTomato fluorescence in BAF53b-Cre neuronal reporter mice. For human studies specimens were stored overnight at 4°C. Under a stereoscopic microscope, the muscularis propria was removed from the mucosa, submucosal, and serosal layers by mechanical dissection with fine forceps and blunt dissection with microdissection scissors. Prewarmed enzymatic solution of collagenase type XI (1 mg mL−1) and dispase (0.6U mL−1) in DMEM / F12 was added to tissues at 1 mL of enzymatic solution added per 200mg of tissue. Tissues were then minced into approximately 5mm pieces with sterile microdissection scissors and incubated in a VWR® Mini Incubated Shaker at 37˚C for 4 hours. Samples were triturated using glass serological pipettes with progressively smaller bore diameters (3 mm, 2 mm, and 1 mm) until the sample became liquefied. Large undigested fragments of tissue were filtered out of the solution using a 1000 µm cell strainer (pluriStrainer®, pluriSelect, El Cajon). PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Samples were then passed through a 100 µm cell strainer to collect the single cell suspension (<100 µm), and the remaining contents that failed to pass through (100-1000 µm) were collected to isolate the human MyGa enriched fraction. In some experiments MyGa were manually picked from this fraction under a stereoscopic microscope using fine forceps. Red blood cell removal was performed by incubating samples in ACK Lysing Buffer (Gibco, ThermoFisher Scientific) for 7 minutes. To validate MyGa enrichment, samples were collected in RLT buffer for gene expression analysis by PCR or 4% PFA for immunohistochemistry with anti-Tubulin β3 (TUBB3, 1:200, TUJ1 clone conjugated to Alexa Fluor 488). Neurosphere generation and cell culture Culture of neurospheres from mouse intestinal tissues was conducted in defined enteric neurosphere culture media (Mueller et al. (2024) Stem Cells Translational Medicine, szae013). Mice were euthanized and the small intestine was processed as described above in quantification of ganglia sizes in mouse samples. Single cell suspensions (<20 µm) and enriched MyGa fragments (20-100 µm) were cultured in enteric neurosphere media for 10 days in free-floating conditions (Corning Costar® 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates Cat# 3473) to generate neurospheres utilized for spheroid quantification, gene expression analysis, and cell transplantation experiments. For monolayer cultures, neurospheres generated after 14 days in free-floating conditions were transferred to tissue-culture treated plates (CytoOne 24 well Plate, TC Treated) coated with fibronectin (1:100 for 2h at 37°C, Millipore Sigma Fibronectin Bovine Plasma Cat# F1141-5MG) in the same media for an additional 14 days to facilitate cell migration. Monolayer cultures were utilized for immunohistochemistry experiments to visualize and characterize EGC and enteric neuronal subpopulations and to promote the trypsinization of cultures into single cell suspensions for fluorescence-activated cell sorting (FACS) experiments. For the culture of human cells, single cell suspensions (<100 µm), enriched MyGa fragments (100-1000 µm) and hand-picked MyGa were cultured in free-floating conditions (Corning Costar® 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, Cat# 3471) in neurosphere generation media43with the addition of Antibiotic-Antimycotic (Gibco, ThermoFisher Scientific) in a standard cell culture incubator (37 degrees, 5% CO2, and atmospheric O2). Plating density was determined according to the initial tissue weight with samples plated in 1 mL of media per 250 mg of initial tissue weight. Samples were cultured for 10-14 days with the media volume doubled midway through culture to generate neurospheres (passage 0). Neurospheres were then transferred to tissue-culture treated plates (CytoOne 6 well Plate, TC Treated Cat# CC7682-7506) coated with fibronectin (1:100 for 2h at 37°C) for the formation of monolayers over 10-14 days of culture with the media completely replaced midway. This monolayer culture step facilitated the removal of non-living debris that failed to adhere to the fibronectin- coated wells and allowed samples to be trypsinized to single cell suspensions (end of passage 0). Cells were counted and reseeded at a density of 5000 cells / cm2in free-floating conditions with the cell culture steps repeated as above for the first and second passages. Neurospheres were collected in RLT buffer and frozen for gene expression studies across passage 0 to 3. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Quantitative PCR Quantitative PCR was performed to assess the gene expression of neural stem cell, glial and neuronal markers (Ott et al. (2023) International Journal of Molecular Sciences 24, 5211). Briefly, samples were transferred to a microcentrifuge tube and centrifuged at 500g for 5 minutes to remove the supernatant. Samples were resuspended in RLT buffer (Qiagen) and stored at -80˚C until the time of RNA extraction using an RNeasy Mini kit (Qiagen). The concentration of extracted RNA was quantified using the Qubit RNA HS Assay Kit (Invitrogen, Thermo Fisher Scientific) on a Qubit 4 Fluorometer (Invitrogen, Thermo Fisher Scientific). Total RNA was reverse transcribed and amplified via RT-qPCR with the iTaq Universal SYBR Green One-Step Kit (Bio-Rad) using a Bio-Rad CFX96 real-time thermal cycler with the reaction setup performed as per the manufacturer’s instructions. Primer sequences for gene amplification in mouse samples are provided in Table 3. Table 3. Primer sequences for PCR. See FIG.14, FIG.15, and FIG.19. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Ms = mouse; Hu = human The thermal cycling protocol was per the kit manufacturer’s instructions. Results were processed with the Bio-Rad CFX Manager software (version 3.1), with a standard threshold to quantity cross point (Ct) values. Ct values were normalized to Gapdh expression in each sample as an internal control and the Log2 of the fold change (FC) was calculated between corresponding sample conditions from the same mouse or subject. Reactions were performed in duplicate. Immunohistochemistry in vitro For mouse studies, cells in monolayer culture were fixed for 30 min with 4% PFA (Electron Microscopy Science, #15710), permeabilized with 0.1% triton in PBS for 20 minutes and blocked in solution containing 10% donkey serum (Sigma, #D9663) for 1 hour at room temperature. Primary antibodies were incubated in PBS containing 10% donkey serum at 4˚C overnight. Cells were incubated with secondary antibodies below for 1 hour in the same blocking solution at room temperature and stained with DAPI (Invitrogen, #D1306) for 10 minutes. Primary antibodies utilized in this study included: rabbit anti-Calretinin (1:200, Invitrogen, Thermo Fisher Scientific, 18-0211), rabbit nNOS (1:400, Invitrogen, Thermo Fisher Scientific, 61-7000), goat anti-GFAP (1:500, abcam, ab53554), rabbit anti-P75 (1:500, Millipore Sigma, AB1554) and human anti-Hu (Anna-1) positive serum (1:16,000, gifted from Dr. Vanda Lennon; Mayo Clinic). Secondary antibodies were utilized at 1:200 for donkey anti-rabbit 647, anti- goat 647 and donkey anti-human Alexa Fluor 488 (Invitrogen). The samples were washed between each step in PBS for 3×5min. For human studies, samples (passage 1) were cultured on fibronectin-coated tissue-culture plates (CytoOne 24 well Plate, TC Treated) for 10-14 days and were processed as above. Primary antibodies included: rat anti-CD49f (1:100, APC conjugated, BioLegend, Cat# 313616), mouse anti-CD271 / NGFR (1:100, FITC conjugated, BioLegend, Cat# 345104) and mouse anti-Tubulin β 3 (1:300, Alexa Fluor® 647 conjugated, BioLegend, Cat# 801210). Cell population image analysis To visualize and quantify EGC and enteric neuronal populations Plp1-EGFP;Baf53b::tdT dual =reporter mice were used. The immunohistochemically labelled cells and transgenic fluorescence were imaged on a Keyence BZX-700 All-In-One Microscopy system (Keyence America Itasca). Images were analyzed using Fiji open-source software (ImageJ v1.54f (Schindelin et al. (2012) Nat Methods 9, 676– 682)) using a custom Fiji macro to quantify the number nuclei within the area occupied by transgenic and immunohistochemical fluorescence within a 10 mm2field of view per sample. Briefly, images of Baf53b::tdT and Plp1-EGFP fluorescence were binarized using the ‘Intermodes’ and ‘Max Entropy’ methods, respectively. Binary images were despeckled (removal of black dots < 2µm2) and the region occupied by Baf53b::tdT and Plp1-EGFP expressing cells was added to the region of interest manager using the ‘create selection’ function. Channels containing DAPI were then analyzed to count the number of nuclei within the Baf53b::tdT and Plp1-EGFP occupied region of interest to quantify PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 the number of cells. This was automated by generating images in the DAPI channel containing only the regions of interest of Baf53b::tdT or Plp1-EGFP and binarizing images using the ‘Yen’ method. Images were further processed with the ‘Fill Holes’ and ‘Watershed’ functions, and the number of nuclei were quantified using the ‘Analyze Particles’ (>4µm2) function. Proportions of Baf53b::tdT or Plp1-EGFP expressing cells were compared to the number of nuclei in the whole field of the images as a reference to determine cell proportions. Subpopulations of enteric neurons (Baf53b::tdT) and EGC / progenitors (Plp1-EGFP) were determined as similar to described above. Channels of immunohistochemical staining were processed to include only the Baf53b::tdT or Plp1-EGFP defined region of interest and were then binarizing using the ‘Max Entropy’ method. The subsequent region of interest of overlapping expression was used to define the area for nuclei quantification yielding the number of double positive cells. This analysis was conducted in a total 10mm2area. Semi-automated quantification of antibody immunoreactivity in human cells was performed by generating regions of interest in the DAPI channel using the ‘Analyze Particles’ workflow as described above. The mean fluorescence intensity (mean gray values) for immunofluorescence for each antibody labeling was recorded for each cell and corrected for background fluorescence levels. Thresholds for positivity by corrected mean fluorescence intensity were manually determined by training on subsets of positive cells for each image with a minimum of 1400 cells included in the final analysis per sample (average: 8137 ± 2104 cells per sample). Flow cytometry analysis and neurogenesis assay Flow cytometric analysis was conducted using data generated on a BD FACSAria cell sorter (BD Biosciences, Franklin Lakes, New Jersey) using FlowJo software (FlowJo, LLC, OR). Single cell suspensions were produced by the trypsinization of monolayer cultures as described in Neurosphere generation and cell culture. BAF53b::tdT; Plp1-EGFP dual reporter mice were utilized to quantify the proportions of enteric neurons, EGC / progenitors, and double negative cells with DAPI serving as a dead cell marker. Samples were acquired on an Aurora Cytek Spectral analyzer and data analyzed using FlowJo Treestar software. To determine the ability of EGCs / progenitors to differentiate into enteric neurons BAF53b::tdT; Plp1-EGFP dual reporter mice were used. Monolayer cultures were produced as above and FACS was performed to purify the Plp1-EGFP expressing population. Plp1-EGFP cells were cultured in free-floating conditions to form neurospheres in defined enteric neurosphere culture media42for 10 days before being seeded on fibronectin-coated plates and cultured for a further 4 days in the same media to facilitate cell migration and the visualization of individual cells. Cultures were imaged on a Keyence BZX-700 All-In- One Microscopy system and the number of Plp1-EGFP expressing cells and the emerging population of BAF53b::tdT expressing cells were manually quantified using the cell counter plugin of Fiji within a 10 mm2field of view per sample. Calcium imaging For calcium imaging studies, ENSCs were generated from Baf53b-Cre mice crossed with GCaMP5-tdT mice (Baf53b-GCaMP5) mice with constitutive expression of the tdTomato reporter and the PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 genetically encoded calcium indicator, GCaMP5, expressed by enteric neurons. ENSCs were cultured on fibronectin-coated culture plates for 10 days. Media was replaced with BrainPhys media (STEMCELL Technologies) and intracellular calcium ([Ca2+]i) transients in enteric neurons cells were recorded using the Keyence BZX-700 All-In-One Microscopy system. Analysis of intracellular calcium level was performed using ImageJ. Briefly, regions of interest (ROI) were produced using images of tdTomato fluorescence and the mean fluorescence intensity (mean grey value) of GCaMP5 fluorescence, representative of intracellular calcium [Ca2+]i, was measured in each frame using ImageJ by calculating the change of fluorescence intensity expressed as the relative fluorescence (ΔF / F0)45. Acetylcholine was spritzed at a final concentration of 100µM, and electric field stimulation (EFS) was delivered by two parallel silver / platinum electrodes using a CS4 constant voltage stimulator with MyoPulse software (Danish Myo Technology) at a single pulse of 40-80 V and 0.2 ms pulse duration. Calcium imaging in human cell monolayer cultures (passage 2) was performed using the Fluo-4 Calcium Imaging Kit (Invitrogen™, Thermo Fisher Scientific, MA, #F10489) according to the manufacturers protocol using a BZX-700 All-In-One Microscope. Acetylcholine was spritzed at a final concentration of 100µM with or without a 10 min preincubation with the voltage-gated sodium channel blocker tetrodotoxin (TTX, 1μM). Recordings were processed in ImageJ and R studio. First, regions of interest were set for each cell in the field of view by creating maximum intensity projections from all video frames. These images containing elevated fluorescence in all cells at baseline or after stimulation were binarized, had the watershed function applied, and a particle analysis was performed using Fiji. These regions of interest were used to measure the mean fluorescence intensity of Fluo-4 across the video recordings for each individual cell. Data were normalized to ΔF / F0 as described in mice and matrices of ΔF / F0 values for cells over time were processed using the ComplexHeatmap package (Gu (2022) iMeta 1, e43) in R studio with modifications by the circlize and Pals packages. Cell transplantation To examine the engraftment, spread, and differentiation of mouse MyGa-derived neurospheres, neurospheres were generated from BAF53b::tdT; Plp1-EGFP dual reporter mice and transplanted to 3 month old colorless wildtype mice. Recipient mice were anesthetized by isoflurane inhalation with a midline abdominal incision made to expose the mid-colon. Microinjections of neurosphere suspensions in sterile PBS were administered in a volume of 4 μL to the muscularis propria of the colon with 100 neurospheres delivered to each recipient. Optogenetics studies were performed to assess neuronal function from transplanted MyGa-derived neurospheres by delivering cells isolated from ChAT-Cre or Baf53b-Cre mice crossed with ROSA26-tdTomato- channelrhodopsin-2 (R26-tdT-ChR2) mice. In these experiments neurospheres were produced and delivered in the same manner as above to Plp1-EGFP recipients to confirm integration of transplants with the recipient ENS. For human ENSC transplantation studies, cells cultured in monolayer during the first passage were transduced with an Adeno-Associated Virus Type 6 carrying channelrhodopsin-2 (ChR2) and a GFP reporter driven off a CAG promoter (AAV6-CAG-ChR2-GFP; provided by Dr. Edward S Boyden, Massachusetts Institute of Technology, and produced by the UNC Vector Core, University of North Carolina) (Boyden et al. (2005) Nature Neuroscience, 1263–1268). Cells were transduced at a multiplicity of infection (MOI) of 10,000 for 72h before the media was replaced and fluorescence of the GFP reporter PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 was confirmed after one week. Monolayers were then trypsinized and taken to the second passage in free-floating conditions as described in Neurosphere generation and cell culture. After 10 days, human- derived neurospheres were administered to the midcolon of immunodeficient NOD-scid IL2Rgammanull(NSG) mice as described above. For immunohistochemical and functional optogenetic studies mice were sacrificed 3 weeks after transplantation. Immunohistochemistry of transplanted cells in vivo Tissues were blocked and permeabilized in solution containing 10% donkey serum, 10% BSA and 1% triton in PBS for 1 hour at room temperature on a rocking platform. Primary antibodies anti- Tubulin β3 (TUBB3, 1:200, TUJ1 clone conjugated to Alexa Fluor 647) or rabbit anti-P75 (1:500, Millipore Sigma AB1554) were diluted in the same buffer and incubated overnight at 4˚C before washing tissues in PBS. For P75 labelling, the secondary antibody donkey anti-rabbit Alexa Fluor 647 (1:500, Thermo Fisher Scientific, MA) was applied for 3 hours in blocking buffer (PBS with 10% donkey serum and 10% BSA) before nuclei staining with DAPI, washing in PBS, and mounting onto glass slides with AquaPolymount (Polysciences). Imaging was conducted on a Keyence BZX-700 All-In-One Microscopy system (Keyence America Itasca) and a Nikon AXR confocal microscope (Nikon Cambridge, MA). Smooth muscle activity recordings To assess colonic smooth muscle activity in mice in vivo, electromyography (EMG) and intraluminal pressure were assessed simultaneously (Rahman et al. (2024) Cellular and Molecular Gastroenterology and Hepatology 17, 907–921). Briefly, mice were subjected to anesthesia by isoflurane with a midline incision made to access the colon for ligation and cannulation with a pressure transducer (CWE Inc., Ardmore, PA). The colon was then filled with physiological saline to 10-15 mmHg in a closed system with increases in luminal pressure signifying colonic contractions. For EMG recordings, custom- made three-lead needle electrodes (Motion Lab Systems, Inc. LA, CA) were positioned in the muscularis propria of the colon and connected to a four channel Bio-amplifier (CWE Inc., Ardmore, PA) through an ISO-Z Isolated Head Stage amplifier (CWE Inc., Ardmore, PA). Digitized recordings were made with the Power Lab 16 / 35 data acquisition system (ADInstruments, NSW, Australia) and data were analyzed with Lab Chart Pro Software v8.1.16 (ADInstruments). Blue light stimulation of transplanted ChR2 expressing neurons was applied at 25mW, 10 ms pulse width, 10 Hz, with a 20 second train duration using a 200 µm diameter optic fiber and a diode-pumped solid-state laser system (470 nm, 200 mW, Model number: MDL-III-470; OptoEngine, LLC, Midvale, UT). For organ bath experiments, the contractile force of the colonic smooth muscle was recorded in response to blue light stimulation (Stavely et al. (2022) Science Translational Medicine 14; Rahman et al. (2024) Cellular and Molecular Gastroenterology and Hepatology 17, 907–921). Colons were excised and cut into 5mm rings under a fluorescent stereoscopic microscope with the presence of transplanted cells verified by tdT fluorescence for mouse-derived neurospheres, or GFP fluorescence for human-derived neurospheres. Colonic rings were mounted to force displacement transducers in a muscle strip myograph bath (Model 820 MS; Danish Myo Technology, Aarhus, Denmark) containing Krebbs solution (oxygenated with 95% O2 and 5% CO2) and were maintained at 37°C. Contractile responses were recorded with Lab Chart Pro Software and blue light stimulation was performed as above at 25mW, 10 ms pulse width, 10 Hz, with a 15 second train duration. To confirm blue PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 light stimulation evoked contractions were neurally-mediated, tissue preparations were incubated with 1μM tetrodotoxin (TTX) for 5 minutes to block voltage-gated sodium channels. Single cell RNA-seq Human cells in monolayer culture at the end of the second passage were trypsinized and cell counts performed. cDNA libraries were prepared using the Chromium Single Cell 3' Reagent Kit v3.1 (10X Genomics, CA) using the standard 10X Genomics workflow according to manufacturer’s instructions and sequenced on an Illumina NextSeq platform (Illumina, Inc., CA) at the MGH NextGen Sequencing Core Facility. Output data were processed using the 10X genomics Cell Ranger Count v7.1.0 pipeline using the human reference genome (GRCh38) to generate barcode and feature count matrices for downstream analysis (Zheng et al. (2017) Nature Communications, 14049). Data were analyzed using the Seurat package in the RStudio integrated development environment (Stuart et al. (2019) Cell 177, 1888–1902). Data were preprocessed by corrections for ambient RNA using soupX (Young et al. (2020) GigaScience 9, giaa151), removing ribosomal, heat shock protein, immediate early, and sex specific genes. High quality cells were included with between 600-5000 unique features, greater than 1000 UMI counts, and less than 15% of mitochondrial RNA. Doublet detection was performed using the R package DoubletFinder (McGinnis et al. (2019) Cell Systems 8, 329-337.e4). Data was processed using the standard Seurat v5 workflow with the functions SCTransform(), FindVariableFeatures() and RunPCA(). Cell cycles were evaluated in each cell using the CellCycleScoring function of Seurat and were treated as variables to regress. RPCAIntegration was performed across samples using the IntegrateLayers() and JoinLayers() functions. Nearest neighbors were calculated and UMAPs were generated by the FindNeighbors() and RunUMAP() commands with reduction method set to "RPCA" with 1:30 dimensions. Clusters were determined using FindClusters() and the resolution was optimized for each in silico experiment based off the maximum number of unique clusters with supporting literature, bona fide markers, and further experimental validation. Visualization of cell annotations or gene expression in UMAP space and Dotplots were performed using Seurat with modifications by the packages ggplot2, viridis, and pals. Differentially expressed genes (DEGs) between clusters or treatments analysis was performed using the FindAllMarkers function with a minimum log2FC value of 0.5 and adjusted p value < 0.05. Mouse enteric neurosphere data were obtained from Guyer et al (GSE184981) (Guyer et al. (2023) Cell Reports 42, 112194) and processed using Seurat as above. Human EGC data was obtained from the Broad Institute Single Cell Portal (study number SCP1038) and processed as above using original authors’ annotations for non-subject specific glial populations (Drokhlyansky et al (2020) Cell 182, 1606- 1622.e23). Human neurosphere cell population markers were assessed in data from mouse neurospheres (Guyer et al. (2023) Cell Reports 42, 112194) and human EGCs (Drokhlyansky et al (2020) Cell 182, 1606-1622.e23) using the AddModuleScore function to identify corresponding cell populations. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analysis All details of statistical analysis can be found in the figure legends and data set. Data analysis was performed using GraphPad Prism v7 (GraphPad Software Inc., San Diego, USA). For all analyses PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 P≤0.05 was considered significant. All data were presented as mean ± standard error of the mean (SEM), unless otherwise stated. TABLE 4: PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Example 3: Generating and characterizing neurosphere formation from a ganglionated region of resected bowel tissue obtained from Hirschsprung disease (HSCR) subjects Neurosphere formation from the ganglionated region of resected bowel tissue obtained from Hirschsprung disease (HSCR) subjects was evaluated as follows. Samples were processed using the MyGa enrichment procedure (as described Example 1 except modified as discussed below regarding cell strainer size) in combination with an optimized culture medium containing fibroblast growth factor (FGF; 20 ng / mL), glial-derived neurotrophic factor (GDNF; 50 ng / mL), and retinoic acid (RA; 75 ng / mL). The process is summarized below. Tissue Collection and Preparation Full-thickness resection specimens were acquired and stored in phosphate-buffered saline (PBS) supplemented with antibiotics: Primocin (100 μg / mL), Metronidazole (25 μg / mL), and Amphotericin B (2.5 μg / mL). Tissues were placed in a Sylgard-coated petri dish, longitudinally opened along the mesenteric border (if identifiable), and pinned flat using 0.2 mm insect pins. Samples were washed repeatedly in PBS until visible fecal matter and blood were removed. The mucosa was separated from the muscularis propria via blunt dissection under a stereoscopic microscope, taking care to preserve the structural integrity of the muscular layer. Residual submucosa, serosa, and blood vessels were removed as needed (FIG.22A). The dissected muscularis was transferred to a clean petri dish for inspection and further dissection. The sample was then placed in 50 mL tubes containing ice-cold PBS (without Ca²⁺ / Mg²⁺) and shaken vigorously for 30 seconds. This wash step was repeated 10 times using fresh tubes. Tissue was processed immediately or stored overnight at 4°C in PBS supplemented with the antibiotic cocktail for subsequent enzymatic dissociation. Enzymatic Digestion and Filtration The enzymatic digestion solution was prewarmed to 37°C. The tissue was weighed, minced into 3– 5^mm fragments (FIG.22B), and incubated in enzymatic solution (1^mL per 200^mg of tissue) at 37°C with agitation (260^rpm) for 3^hours in a VWR® Mini Incubated Shaker. Mechanical dissociation was performed sequentially using: PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 • A 25^mL glass pipette (20 triturations), • A 10^mL glass pipette (20 triturations), • Followed by incubation for an additional 2.5^hours at 37°C, • Then triturated with a 1000^μL pipette tip (30 times) until a uniform suspension was obtained (FIG.22C). The resulting suspension was filtered sequentially through: 1. A 1000^μm pluriStrainer® filter to remove large tissue fragments (rinsed with 15^mL sterile DMEM) (FIG.22D). 2. A 40^μm cell strainer (1 filter per 0.5^g of starting tissue), triturated and rinsed with DMEM / F12 (4 × 5^mL rinses). 3. The 40^μm strainer was inverted, and retained material was eluted using 10^mL DMEM / F12. 4. Residual fragments were collected by washing the filter at a 45° angle, as described, until no material was visible by microscopy (typically 10 rinses). The suspension was centrifuged at 500^g for 5^minutes. The pellet was resuspended in sterile DMEM (1^mL per gram of starting tissue), and the filtration steps were repeated (FIG.22E). Final fragments were suspended in growth media and seeded into ultra-low attachment 6-well plates at a density of one well per 0.25^g of starting tissue (4^mL / well). Growth media consisted of the following: DMEM, high glucose, pyruvate (ThermoFisherScientific CAT# 11995065), Gibco N-2 Supplement 100X, Gibco B-27 Supplement, minus Vitamin A (Cat# 12587001), Human Recombinant GDNF (Stemcell Technologies Cat# 78058) (50ng / mL final concentration in media), Retinoic acid (Sigma-Aldrich cat# R2625) (75ng / mL final concentration in media) and Human Recombinant bFGF (Stemcell Technologies Cat# 78003) (20ng / mL final concentration in media). Also referred to as GRF media. The presence of ganglionic fragments (MyGa) can be verified microscopically in the solution to confirm the process was successful (FIG.22F). Neurosphere Culture and Passage From Days 1 to 3, neurospheres began to form. On Day 5, 2^mL of fresh growth media was added. On Day 10, neurospheres (FIG.22G) were transferred to fibronectin-coated (1:100, 2^h at 37°C) tissue- culture-treated wells (1:1 media transfer ratio). By Day 15, neurospheres demonstrated firm attachment and initial monolayer formation. Media was replaced with 4^mL of fresh growth media. Cell Harvest and Expansion On Day 20 cells were processed to obtain single cell suspensions, the steps included: • Media was removed and wells were washed with 5^mL PBS. • 4^mL trypsin was added for 7^minutes at 37°C (5% CO₂). PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 • Trypsin was quenched with 8 mL DMEM, and cells were pelleted (500 g, 5 min). • The pellet was resuspended in 1 mL growth media for counting using a hemocytometer. Cells were either cryopreserved, seeded for expansion (Culture passage 1), taken for gene expression, immunocytochemical characterization or cell transplantation assays. Notably, when compared to culture of single cell suspensions < 40 μm, the above methodology prevented contamination causing failure of the culture (n = 8) which is common in primary intestinal cultures due to the high bacterial burden. These data indicate that counter filtration methodology (FIG.22E) is an important step for the decontamination of cultured samples. Example 4: Verifying the presence of enteric neural stem cells (ENSCs) in samples from Hirschsprung disease patients Neurospheres were isolated from Hirschsprung disease (HSCR) patient samples and from a non- HSCR control (a 67-year-old female). At the end of the first passage seeding was done at a density of 5000 cells / cm² (~50,000 cells / well) in 6-well low-attachment plates containing 4 mL growth media. On Day 5 of Culture passage 1 (P1), an additional 2 mL of media was added. By Day 10 of P1, neurospheres were resuspended, and aliquots were taken for gene expression profiling. RNA was isolated to evaluate gene expression levels (FIG.23A). Data were compared to two controls: the SH-SY5Y neuroblastoma cell line (positive control) and a cell line not known to undergo neural differentiation (negative control) [TERT-immortalized human breast cancer-associated fibroblast; Kojima, Y., et al., Proc Natl Acad Sci U S A.107, 20009-20014, 2011]. Expression of PLP1 and PHOX2B, key markers of enteric neural stem cells (ENSCs), was assessed by qPCR. ΔCT values were normalized to GAPDH, and data were plotted as linear scale normalized expression (0 = myofibroblast control, 1 = SH-SY5Y). Across all HSCR samples, positive expression of both PLP1 and PHOX2B was observed, confirming the presence of ENSCs within the cultures (FIG.23A). In a separate immunocytochemical assay, neurospheres obtained during Culture passage 1 were plated on fibronectin coated tissue culture dishes to form monolayer cultures. After 10 days, the presence of ENSCs was further confirmed through the expression of ITGA6 and NGFR (FIG.23 B – FIG.23C), two surface markers we validated in our scRNA-seq studies of enteric neuroglial populations (Example 2 and FIG.19M, FIG.19O, FIG.19P, and FIG.19R). We also identify that L1CAM, a neural cell adhesion molecule is a canonical marker of developing enteric neurons in scRNA-seq Data (Example 2 and FIG. 21A), which is also expressed in these cultures, supporting the presence of committed neuronal progenitors with neurite projections (FIG.23 B – FIG.23C). Example 5: Transplantation and function of ENSCs from Hirschsprung disease subjects to aganglionic Hirschsprung disease tissues. In this example, the functional capacity of enteric neural stem cells (ENSCs) derived from Hirschsprung disease (HSCR) patients was evaluated following their transplantation into aganglionic colon tissues derived from other HSCR patients maintained in ex vivo culture. To prepare cells for PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 transplantation, neurospheres initially generated from HSCR patient-derived ENSCs were dissociated and plated on fibronectin-coated tissue culture dishes to promote monolayer formation. These monolayers were then transduced with an adeno-associated viral vector (AAV6) carrying the gene for channelrhodopsin-2 (ChR2) and a green fluorescent protein (GFP) reporter (AAV6-ChR2-GFP). The incorporation of ChR2 enabled subsequent optogenetic activation of the cells, while the GFP allowed for visualization and tracking of transplanted cells in recipient tissues. Following transduction, the monolayer cultures were enzymatically dissociated with trypsin and reseeded at a density of 5000 cells / cm² in ultra-low attachment culture dishes for 10 days to regenerate neurospheres, representing passage 1 cultures. These neurospheres were then transplanted into freshly dissected aganglionic colonic segments from two different HSCR patients.6 neurospheres per 0.5 cm2of human tissue were transplanted ex vivo and cultured. Tissues were maintained in ex vivo organ culture for a duration of two weeks following transplantation. Transplanted cells were identified in recipient tissues by their expression of GFP. Immunohistochemical analysis demonstrated that these cells expressed neuronal markers including TUBB3 (βIII-tubulin) and PGP9.5, and that they formed neuronal projections, thereby confirming their successful differentiation into enteric neurons within the previously aganglionic host tissues (FIG.24A). To assess the functionality of the transplanted neurons, colonic tissues containing ChR2- expressing ENSCs were subjected to optogenetic stimulation during organ bath experiments. Tissue segments were mounted in muscle strip myograph chambers (Model 820 MS, Danish Myo Technology) containing oxygenated Krebs solution (95% O₂ / 5% CO₂) maintained at 37°C. Contractile force measurements of the smooth muscle were recorded using force-displacement transducers and Lab Chart Pro software. Optogenetic stimulation was applied using blue light at an intensity of 25 mW, with 10 ms pulse width and a frequency of 10 Hz for a train duration of 30 seconds. Recorded contractile traces were subsequently analyzed using custom-written scripts in RStudio. A latency detection function was used to assess response timing and magnitude in relation to the stimulation window. Specifically, the function evaluated deviations from baseline activity using the parameters: a stimulation window of 30 seconds, a post-stimulation window of 30 seconds, a baseline window of 10 seconds, a threshold of two standard deviations above or below the baseline mean, and a requirement that the signal deviation be maintained for a minimum duration of 1500 milliseconds to be considered a true response (FIG.24B). To determine whether observed responses were attributable to stimulation rather than spontaneous smooth muscle activity, each recording was compared to 100 randomly selected control timepoints (random seeds) from within the same trace. This comparison was used to model pseudo- stimulation events and establish a null distribution for spontaneous contractile behavior (FIG.24C). Following blue light stimulation, tissue responses were significantly more frequent than those observed at random timepoints within the same recording (FIG.24D). Furthermore, the latency from the onset of stimulation to the onset of response was significantly shorter during the stimulation and post- stimulation periods compared to modelled pseudo-stimulation windows (FIG.24E). These findings confirmed that recipient tissues exhibited specific responses to optogenetic activation of the transplanted ENSCs, rather than random, spontaneous activity. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Quantitative analysis further demonstrated that blue light stimulation significantly increased the negative area under the force trace curve, (AUC) prolonged the duration of time the smooth muscle signal remained below baseline, and decreased the tone of the smooth muscle (FIG.24F). These effects are indicative of consistent inhibitory smooth muscle relaxation, which is a key functional property of certain enteric neurons. Importantly, this relaxation was observed in aganglionic HSCR segments that are otherwise known to exhibit tonic contraction due to the absence of inhibitory neuronal input. Together, these results support the conclusion that transplanted HSCR-patient derived ENSCs were capable of functional integration into aganglionic bowel tissue and mediated physiologically relevant smooth muscle responses following optogenetic stimulation. Example 6: Cryopreservation of ENSCs maintains their neurosphere forming ability and function. Human subject derived ENSCs were cultured as described in Example 3 until single cell suspensions were harvested. Cells were counted using a hemacytometer prior to being pelleted via centrifugation (500 g, 5 min). The pellet was resuspended in Bambanker serum-free cryopreservation media such that there were between 0.5-2x106cells per mL of solution. Aliquots of 1mL were transferred to cryopreservation tubes and placed into an isopropanol insulated cooling container which was then transferred to a freezer set at -80C° to achieve a cooling rate of approximately -1C°. Cryopreservation tubes were then transferred to a liquid nitrogen storage container for long term preservation. To thaw the cells, cryotubes were immersed in a water bath set at 37C° and were gently agitated to prevent cooling of the surrounding water. Upon thaw, the cells in cryopreservation solution were immediately triturated and diluted 1:10 with DMEM and were pelleted via centrifugation (500 g, 5 min). the supernatant was removed and replaced with 1mL of GRF media (growth media). Cell counts were conducted via hematocytometer and cells were cultured at a density of 5000 cells / cm² in ultra-low attachment culture dishes. After 48h neurospheres were observed to reform from the previously cryopreserved single cell suspensions (FIG.25A). Notably, cryopreservation of neurospheres directly resulted in failure of cultures to form after thawing, indicating that the single cell suspension process is an essential part of our method for efficient recovery. To establish whether the neurospheres after cryopreservation maintained their functional properties, they were transplanted to ex vivo preparations of mouse colon lacking enteric neurons. In this model the colon of BAF53b-iDTR transgenic mice is collected which harbors an inducible diptheria toxin receptor (iDTR) under the control of the BAF53b promoter which is specific to neurons. Diphtheria toxin (DT) was applied to these cultures at a concentration of 500ng per mL for 48h to selectively ablate enteric neurons prior to implantation of human neurospheres previously transduced with AAV6-ChR2-GFP as in Example 5. The effects of blue light stimulation of transplanted cells on contractile force measurements of the smooth muscle was assessed in the ex vivo preparations as of Example 5 (FIG.25B) and compared to 100 randomly selected control timepoints (random seeds) within the same trace (FIG.25C). The stimulation samples with blue light evoked more responses within the 30 second stimulus and 30 second post-stimulus (rebound contractions) time periods than observed at random timepoints indicating the responses were transplant derived (FIG.25D). Quantitative analysis indicated a higher positive force during the blue light stimulation period indicative of contractions evoked from transplanted cells (FIG. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 25E). Similarly mean force and the area under the curve (AUC) were elevated in the post-stimulus period of blue light stimulation compared to what would be expected at random for all samples, which is indicative of a rebound contraction in this experimental model (FIG.25E). Together these data show that ENSCs retain function after cryopreserved using this method. Example 7: Treatment of Gastroparesis in a Neuronal Ablation Mouse Model A neuronal ablation model of gastroparesis was generated using BAF53b-iDTR transgenic mice, in which all neurons express the diphtheria toxin receptor (DTR) under control of the BAF53b promoter, permitting targeted neuronal ablation upon administration of diphtheria toxin (DT). Mice were anesthetized and received targeted injections of DT into the corpus of the stomach to induce region- specific enteric neuronal ablation. Two weeks following ablation, gastric emptying of solids and liquids was assessed by intragastric gavage of radiopaque contrast agents. Specifically, mice were gavaged with 0.3 mL of barium sulfate suspension (E-Z Paque, E-Z-EM) and 10 steel beads (diameter 0.81–0.90 mm; Bal-tec). After 90 minutes, mice were imaged using a portable X-ray unit (50 kV, 1.2 mAs; ScanX14 Portable Digital Imaging System, ALLPRO Imaging) (FIG.26A). Radiographic images were analyzed in ImageJ. Solid gastric emptying was calculated as the percentage of steel beads located outside the stomach. Liquid gastric emptying was quantified by calculating the integrated density (total pixel intensity) of barium contrast outside the stomach, normalized to the integrated barium signal throughout the gastrointestinal tract. Neuronal ablation via DT resulted in impaired solid (FIG.26B) and liquid (FIG.26C) gastric emptying compared to untreated controls.To evaluate the therapeutic potential of ENSCs, BAF53b-iDTR mice with gastroparesis (induced as described above) were treated with ENSCs prepared from donor mice expressing a fluorescent neuronal reporter (BAF53b::tdTomato). The procedure for ENSC isolation included, dissection of bowel from BAF53b::tdTomato donor mice, isolation of the muscularis propria and enzymatic digestion to dissociate tissue, ganglia enrichment via counter-filtration as described, for instance, in Example 1 and culture in pro-neurogenic media containing GDNF, RA, and FGF as described in Example 8. Following ENSC transplantation into the stomach wall of gastroparetic mice, animals were monitored for gastric motility recovery. In these experiments, mice exhibited improved solid and liquid gastric emptying, with values outside the pathological range of untreated gastroparetic controls. Additionally, successful engraftment of fluorescently labeled enteric neurons (BAF53b::tdTomato) was confirmed by microscopy, with observed neurite extension and network formation within the gastric wall (FIG.26D). Example 8: Glial cell line-derived neurotrophic factor (GDNF) and retinoic acid (RA) Synergy Unlocks Neurogenesis in Adult Enteric Glia / Neural Progenitors This example demonstrates that declining rates of neurogenesis in adult enteric glial / neural progenitor cells can be restored using a combination of GDNF, retinoic acid, and basic fibroblast growth factor (bFGF). Further, in vitro programming strongly influences post-transplantation outcomes, highlighting the importance of culture conditions in developing enteric cell therapies. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 As demonstrated below GRF supplementation (with GDNF and RA) reactivates neurogenic potential in adult enteric glial / neural progenitor cells (EG / NPs), overcoming age-related decline. Importantly, ENS network formation post-transplant is heavily influenced by in vitro culture conditions, emphasizing the need to optimize pre-transplant programming for effective adult-derived enteric cell therapies. Results The neurogenic properties of ENS cells in culture decline with age. To determine whether the age of mice used as a source of gut tissue influences the properties of ENS cultures, cells were isolated from the muscularis propria of mice at 2 weeks, 2 months, 4 months, 8 months, and 1 year of age (FIG.27A). In these studies, the BAF53b::tdTomato(tdT); Plp1-EGFP dual- reporter mouse model was used as described herein which facilitates simultaneous and distinct transgenic labelling of EG / NPs (GFP) and ENs (tdT). Cells isolated from each age group were seeded at equal densities in free-floating conditions in media previously described to support the 3D culture generation of enteric neurospheres (Guyer et al. (2023) Cell Rep 2023;42(3):112194). Whole-well imaging of all cultures was conducted after 2 weeks (FIG.27A – FIG.27B). GFP corresponding to Plp1 expression was robustly expressed in neurospheres across all groups, however BAF53b-tdT expression markedly diminished with age as observed in neurospheres generated from 2 week and 1 year old mice. (FIG.27C). On the gene expression level, no differences in Sox10 expression were observed between neurospheres generated from 2 week and 1 year old mice; however, the neuronal maker Elavl4 was lower and the fibroblast marker Pdgfra was higher in neurospheres generated from 1 year old mice (FIG. 27D). To determine the neurogenic potential of neurospheres in a high-throughput manner, the mean fluorescence intensity (MFI) of tdT was measured in regions of interest (ROIs) generated using the GFP channel corresponding to each individual neurosphere (FIG.27E). A sharp decline in tdT MFI was observed in neurospheres generated from 2-month-old mice as compared to 2 week-old mice, with a further decline observed at 1 year of age (2 weeks: 32.4±1.9AU, 2 months: 9.9±0.5AU, 4 months: 8.5±0.3AU, 8 months: 11.7±0.5AU and 12 months: 5.1±0.2AU; 2 weeks vs 2-12 months: all p < 0.0001) (FIG.27F). These data suggest that postnatal progenitors of increasing age have decreased neuronal differentiation potential in culture. This highlights the importance of considering the age of mice in experimental models and potential implications for cell therapy applications using cells derived from adults. Cell adherence affects the number and proportions of enteric neurons and glia generated in culture To explore how culture conditions affect the proportions of ENs and EG / NPs that are generated, the properties of EG / NP cultures grown in free-floating conditions which yields enteric neurospheres (3D culture) and those grown as adherent monolayers were compared. Cells were cultured from adult mice at 3 months of age and grown for two weeks in low-attachment culture plates to generate neurospheres. The neurospheres were then either transferred to fibronectin-coated culture dishes for one week to form monolayers, or were maintained in the low-attachment plates to continue the free-floating cultures (FIG. 28A – FIG.28C). Neurospheres grown under free-floating conditions maintained tight clustering of cells PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 within their 3D structure (FIG.28B), whereas in monolayer cultures, cells migrated outward from the neurospheres and formed neural projections (FIG.28C). PCR analysis, to determine gene expression differences (FIG.28D) highlighted that despite the more differentiated appearance of neural networks in monolayer conditions, no significant differences were observed in gene expression between neurospheres and monolayer cultures for the EG / NP markers Plp1 (-0.1±0.19 LogFC, p = 0.8444) and Ngfr (-0.5±0.22 LogFC, p = 0.2705). However, a decline in the EGC marker Gfap (-1.2±0.14 LogFC, p < 0.01), as well as in neuronal markers Phox2b (-1.4±0.5 LogFC, p < 0.01) and Elavl4 (-1.0±0.4 LogFC, p < 0.05), was observed in monolayer cultures (FIG.28D). In additional experiments, flow cytometry was conducted on single-cell suspensions generated from free-floating neurosphere and monolayer cultures (FIG.28E). Notably, these data appeared to oppose the gene expression study and indicated that higher proportions of EG / NPs (46.3±2.9% vs 6.0±2.6%, p < 0.001) and ENs (2.0±0.3% vs 0.1±0.1%, p < 0.01) were present in the single-cell suspensions from monolayers compared to neurospheres. The double-negative population, comprising fibroblasts / EMCs, was higher in the single-cell suspensions from neurospheres compared to those derived from monolayers (94.0±2.5% vs 51.5±2.7% < 0.0001) (FIG.28F). Notably, even after filtering at 35 µm, fewer singlet cells were present in samples derived from free-floating neurospheres than monolayer cultures (26.7±1.7% vs 32.3±0.44%, p < 0.01, two tailed t-test), indicating that monolayers are more favorable for generating single cell suspensions. Therefore, the conflicting results in gene expression and flow cytometry assays highlight important technical considerations between the use of free-floating 3D culture and monolayer culture systems. The gene expression results reflect uniform lysis of cells in both monolayer and neurosphere cultures, unlike the flow experiments, where enteric neurospheres with dense cell-cell contacts are more resistant to digestion to single cell suspensions, as previously shown in 3D culture of other types of neural progenitors (Jager et al. (2016) Adv Med Sci;61(1):78-84.). As cell-cell contacts between EGCs and ENs are more difficult to digest than those of mesenchymal cells, this could lead to skewed experimental data (Mueller et al. (2024) Cell Reports;43(11); Grundmann et al. (2015) Scientific Reports 2015;5(1):9226). Therefore, monolayer cultures offer advantages for protocols requiring cell suspensions, whereas neurogenesis is favored in free-floating conditions that generate neurospheres (Table 5). Table 5. Comparison between monolayer and neurosphere culture systems PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 bFGF supports EGC / progenitor expansion while GDNF and RA promote neurogenesis As highlighted in FIG.27, the neurogenic capacity of ENS cultures declines with age. As many ENS pathologies occur during adulthood, increasing neuronal numbers in cultures from older intestine may be an essential step for generating future autologous cell therapy products to rescue / repair the ENS in adults. One potential way to achieve this goal could be via the optimization of pro-neuronal differentiation factors in culture media. To test this, ENS cell culture composition was examined in response to several growth factors: basic fibroblast growth factor (bFGF), commonly used in EG / NP culture medias (Kruger et al. (2002) Neuron;35(4):657-69), as well as retinoic acid (RA) and glial cell line- derived neurotrophic factor (GDNF), which are known to play important roles in the development of the ENS (Sato et al. (2008) Developmental biology;320(1):185-98; Moore et al. (1996) Nature ;382(6586):76- 9; Niederreither et al. (2003) Development;130(11):2525-34; Fu et al. (2010) Development;137(4):631-40; McKeown et al. (2017) Stem Cell Reports;8(2):476-88; Mwizerwa et al. (2011) Dev Dyn;240(6):1402-11; Pichel et al. (1996) Nature;382(6586):73-6; Sato et al. (2008) Dev Biol;320(1):185-98; Simkin et al. (2013) PLoS One;8(5):e64077). Using the BAF53b::tdT; Plp1-EGFP mouse model to quantify whole plate screening of neurosphere area and neurogenicity as described in FIG.27, the effects of bFGF were compared to negative control media (-CM, without bFGF) and an optimized positive control media (+CM, containing bFGF and RA) known to efficiently grow enteric neurospheres (Guyer et al. (2023 Cell Rep 2023;42(3):112194; Joseph et al. (2011) The Journal of clinical investigation;121(9):3398-411; Kruger et al. (2002) Neuron;35(4):657-69; Bixby et al. (2002) Neuron;35(4):643-56) (media combinations detailed in Table 6) (FIG.29A – FIG.29D). After two weeks of culture, neurosphere formation was low in the -CM compared to +CM optimized for neurosphere growth (421±89µm2vs 10991±623µm2, p <0.0001) (FIG. 29E). The addition of bFGF alone to -CM (bFGF media) significantly increased the area covered by neurospheres (7876±504µm2, p <0.0001) and was only 28% less than that in the optimized +CM media (p <0.01), highlighting the potent mitogenic potential of bFGF (FIG.29E). MFI analysis of BAF53b::tdT, indicating neuronal differentiation, was not conducted in -CM due to low neurosphere numbers but MFI was lower in bFGF media compared to +CM media (11.4±0.4AU vs 13.5±0.3AU, p < 0.0001) (FIG.29F). Cell composition was assessed by flow cytometry using the monolayer culture protocol for single-cell generation described in FIG.28. Results indicated that bFGF is a potent mitogenic factor for EG / NPs (46.3±2.9% vs 5.1±1.5%, p < 0.0001) as bFGF alone is equivalent to +CM media (47.4±5.6%, p = 0.8369) (FIG.29G). However, all three media conditions examined contained low numbers of ENs (-CM: 0.7±0.1%, +CM: 2.0±0.3%, bFGF: 1.4±0.4%), highlighting that bFGF is not neurogenic and that there is a need to increase generation of ENs from adult gut tissues (FIG.29H). Table 6. Mouse Study Media Definitions PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 +CM, positive control media; -CM, negative control media; bFGF, fibroblast growth factor; RA, retinoic acid; GDNF, glial cell line-derived neurotrophic factor; GR, GDNF+RA; GFR, GDNF+RA+FGF; 2-ME, 2- mercaptoethanol; IGF, insulin-like growth factor, bFGF; Basic fibroblast growth factor. As both RA and GDNF have been shown to promote ENS formation and to have synergistic effects during embryonic ENS development (Sato et al. (2008) Developmental biology;320(1):185-98; Moore et al. (1996) Nature;382(6586):76-9; Niederreither et al. (2003) Development;130(11):2525-34), the effects of these factors using the same control media conditions and experimental assays described above (FIG.29I – FIG.29L) were tested. Compared to -CM the area occupied by neurospheres was not improved by the addition of GDNF (505±72µm2, p = 0.8427) or RA (1427±111µm2, p = 0.0852) applied individually (FIG.29M). However, in combination, GDNF and RA increased the area occupied by neurospheres (2934±139µm2, p < 0.001), albeit significantly less than +CM (p <0.0001), suggesting that GDNF and RA exhibit mild mitogenic properties (FIG.29M). Despite producing fewer neurospheres, BAF53b::tdT fluorescence was significantly higher in GDNF and RA media compared to neurospheres generated in the +CM media (23.9±0.7AU vs 13.5±0.3AU, p <0.0001 (FIG.29N). Flow cytometry data supported these observations of a synergistic effect. GDNF and RA together supported expansion of EG / NPs (17.9±3.2% vs -CM, p < 0.01) (FIG.29O), and exhibited a dramatic enhancement of neuronal differentiation (12.3±2.9%), which outperformed -CM (p < 0.001), GDNF (1.2±0.4%, p < 0.001), RA (0.7±0.3%, p < 0.001) as well as +CM (p < 0.001) 6.2-fold (FIG.29P). GDNF, RA and bFGF combine to induce neurogenesis and expand the progenitor pool from adult mice. As demonstrated above, neurospheres cultured in media containing GDNF and RA exhibited potent neurogenic properties with modest EG / NP proliferation. As terminally differentiated ENs do not proliferate (Stavely et al. (2024) Neuron.112(18):3143-60.e6), higher rates of neurogenesis could negatively impact cell expansion. To determine if it is possible to simultaneously expand the adult EG / NP pool while maintaining high levels of neurogenesis, similar to ENCCs during normal ENS development, we utilized media combining the mitogenic effects of bFGF and pro-neurogenic properties of GDNF and RA (GRF media) and compared this to the +CM (FIG.30A – FIG.30C). After two weeks, no differences in the expression of EG / NP markers Plp1 (0.2±0.3 LogFC, p = 0.5556), Gfap (0.2±0.4 LogFC, p = 0.5976) or Ngfr (0.2±0.4 LogFC, p = 0.6638) were observed between neurospheres cultured in GRF media and in +CM (FIG.30D). However, the expression of the fibroblast marker Pdgfra was lower (-1.4±0.5 LogFC, p < 0.05) in GFR media, while expression of neuronal markers Phox2b (1.8±0.3 LogFC, p < 0.001) and Elavl4 (2.3±0.4 LogFC, p < 0.001) were increased. Neurospheres were then plated in monolayer conditions for one week as described above for the generation of single cell suspensions for quantification using flow PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 cytometry (FIG.30E – FIG.30F). Consistent with the gene expression data, GRF media increased the proportion of ENs by 3.6 fold (2.8±0.7% vs 0.8±0.1%, p < 0.01), and marginally increased the number of EG / NPs 1.2 fold (67.2±3.5% vs 55.8±2.8%, p < 0.05) (FIG.30G – FIG.30H). Although we observed increased numbers of ENs in GRF media, this could be attributed to either increased levels of neurogenesis or to increased survival of pre-existing ENs. To distinguish between these two scenarios, a neuronal survival assay was performed by isolating a pure population of BAF53b::tdT+ENs using fluorescence activated cell sorting (FACS) as previously described (Stavely et al. (2024) Neuron.112(18):3143-60.e6) from BAF53b::tdT; Plp1-EGFP mice. This pure population of mature ENs, that do not undergo proliferation, was maintained for 2 weeks in GRF media and compared to ENs cultured in -CM and +CM (FIG.31A). Neuronal numbers were low, as expected, in the -CM media (3.0±1.7 ENs / well), and were not statistically different between the +CM and GRF media conditions (45.7±4.8 vs 31.3±7.2 ENs / well, p = 0.0944), suggesting that the increase in number of ENs in GRF media is not due to improving neuronal survival over +CM (FIG.31B – FIG.31C). To confirm that neurogenesis was responsible for the increased numbers of ENs in heterogenous cultures grown in GRF media, Plp1-EGFP+EG / NPs were purified by FACS and cultured for two weeks in -CM and +CM, as well as in media containing bFGF only, GDNF+RA, or GRF media (FIG.31D). Evaluation of BAF53b::tdT fluorescence, indicating birth of new neurons in culture, showed that EGCs / progenitors cultured in bFGF exhibited minimal neurogenesis (12.1±2.0 AU) while those grown in GRF media (152.3±10.8 AU) or GDNF+RA (142.5±12.6 AU) had significantly higher levels of neurogenesis compared to bFGF (both p < 0.0001) and -CM (2.7±0.7 AU, (both p < 0.0001) (FIG.31E). Furthermore, GRF media produced neurospheres with higher expression of BAF53b::tdT than +CM (115.2±6.4 AU, p < 0.05) (FIG.31E), supporting the finding that elevated numbers of ENs in GRF compared to +CM are driven by neurogenesis. Immunolabeled cross sections of neurospheres from bFGF and GRF culture conditions were consistent with the above results as bFGF produced more gliogenic neurospheres (Plp1-EGFP, SOX10, GFAP and S100B), while those grown in GRF maintained these markers in addition to containing more ENs (BAF53b::tdT and HuC / D) (FIG.31F – FIG.31N). Together, these data indicate that GRF culture media improves neurogenesis whilst maintaining expansion of the EG / NP pool to produce more neurogenic ENS cultures. In vitro culture conditions influence the fate of neural cells post-transplant To address the effects of EN and EG / NP proportions on cell transplantation efficacy, neurospheres were generated as above using either bFGF media favoring EG / NP composition or pro- neurogenic GRF media. These were then implanted to the colons of C57BL / 6J mice via surgical laparotomy (FIG.32A – FIG.32B). Mice were sacrificed 12 weeks after transplant and the fate of BAF53b::tdT+ENs and Plp1-EGFP+EG / NPs evaluated (FIG.32C – FIG.32D). Quantification of BAF53b::tdT+and Plp1-EGFP+cells at the transplant site indicated a 10-fold increase in the EN:EGC ratios of recipients that received neurospheres cultured in GRF compared to bFGF alone (0.52±0.25 vs 0.05±0.01, p < 0.05) (FIG.32E – FIG.32F). Notably, very few ENs were observed in recipients of bFGF neurospheres, indicating that low rates of neurogenesis persisted in the in vivo post-transplant environment (FIG.32F). Evaluation of network formation indicated a greater number of neurite branches (3512.0±582.3 vs 993.3±246.1, p < 0.05) and junctions (1629.0±280.9 vs 477.8±124.3, p < 0.05) in PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 recipients receiving GRF neurospheres (FIG.32H – FIG.32I). The properties of individual neurite branches were similar in maximum (294.7±76.3µm vs 272.6±76.8µm, p = 0.6857) and mean length (24.0±1.3µm vs 25.6±2.2µm, p = 0.6857) between networks generated from GRF and bFGF neurospheres, but the sum of branch lengths was higher in the networks from GRF neurospheres (83710±14247µm vs 24986±6972µm, p < 0.05) (FIG.32J – FIG.32L). These data indicate that the proportions of ENs and EGCs post-transplant are influenced by initial in vitro culture conditions, and furthermore, that generating ENs in vitro prior to transplant is an effective way to improve EN:EGC ratios that better recapitulate those of the normal ENS. GRF media promotes neurogenic properties in human ENS cultures. Human ENS cultures were grown in GRF and +CM media and evaluated for expansion and cellular composition. Full-thickness intestinal tissues were obtained from patients undergoing surgical resections, ranging from 9 weeks to 49 years of age (Table 7). Tissues were dissected to obtain the muscularis propria (FIG.33A – FIG.33B) and enzymatically digested to form single cell suspensions that were plated in free-floating conditions in either +CM or human GRF media. After 2 weeks of culture, neurospheres (FIG.33C) were collected for gene expression studies. Neurospheres grown in GRF media had higher expression of the EG / NP markers, PLP1 (3.5 fold, p < 0.01) and NGFR (2.9 fold, p < 0.01), and the neurogenic marker PHOX2B (6 fold, p < 0.001) compared to +CM media (FIG.33D). Neurospheres were plated in monolayer conditions for 7 days (FIG.33E) as described in mouse samples to generate single cell suspensions. Cell counts indicated lower yields of total cell numbers normalized per mg of starting tissue weight when grown in GRF compared to +CM (521,134±99,761 cells / mg vs 133,336±33,248 cells / mg, p < 0.05) (FIG.33F). Cultures were passaged by repeating the steps above with cells replated in free-floating conditions for an additional 2 weeks followed by transferring samples to fibronectin-coated plates again for 7 days. Cell populations were then assessed by immunohistochemistry for the EG / NP markers ITGA6 and NGFR (Mueller et al. (2024) Cell Reports;43(11)) and the neuronal marker TUBB3 (FIG.33G – FIG.33I). Similar to the gene expression data, cultures grown in GRF media contained higher populations of ITGA6+(43.2±9.7% vs 14.6±6.7%, p < 0.05), NGFR+(35.0±14.9% vs 7.1±5.6%, p < 0.05) and TUBB3+(26.4±4.7% vs 8.0±4.1%, p < 0.05) cells compared to the +CM cultures. Similar to our observations with mouse cells, these data indicate that GRF media expands EG / NP and provides highly-neurogenic cell populations in vitro. Table 7: Subject details PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Discussion We observed a significant decline in the neurogenic capacity of EG / NPs derived from mice between two weeks and two months of age, a trend which continued in mice up to one year. Notably, similar patterns have been observed in vivo. Postnatally, we observed that a subset of their progeny, the EGCs, continue to proliferate rapidly in the early postnatal period at P14 which declines to modest levels by 12 weeks of age (Guyer et al. (2023) Cell Rep 2023;42(3):112194). This pattern mirrors their higher rates of proliferation and plasticity in vivo during early postnatal life, akin to a neural progenitor. In our neurosphere cultures derived from mice of various ages, we observed a marked reduction in BAF53b::tdT-expressing cells when isolated from mice after 2 months of age, suggesting an age- related decline in neurogenesis. These findings indicated that the heightened neurogenic potential of EGCs in the early postnatal period is, to some extent, preserved in vitro. Altogether, these data indicate that EG / NPs, both in vivo and in vitro, progressively lose their neurogenic capacity with age. In this example the neurogenesis stimulating effects of GDNF are 10-fold higher in mouse cells in the presence of RA and more than 3-fold higher in human cells cultured with GRF media. RA promoted the expression of the GDNF receptor RET in early neural crest cells and also promotes GDNF induced chemoattraction in ENCCs, providing a link between synergistic signaling of RA and GDNF. In this example the mitogenic effect of bFGF on progenitors and on neuronal differentiation cues provided by exposure to GDNF+RA can be leveraged to maintain proliferation whilst inducing EN differentiation to maximize the balance between neurons and EG / NPs, thus imitating normal physiological development in vivo. In this example, we observed higher expression of Gfap and neuronal markers in neurospheres compared to monolayers, supporting the idea that 3D culture also promotes differentiation in EG / NPs. Nevertheless, in our experimental context monolayers can also be an advantageous approach for immunohistochemical studies and cell dissociation for downstream applications including flow cytometry, consistent control of cell numbers during passage, single-cell RNA-seq, and other applications. As this example has highlighted, combining features of both culture techniques is useful to enhance differentiation in cells from adult mice while preserving versatility for downstream applications. In this example, cells were both isolated from and transplanted to adult mice at 3 months of age, once postnatal enteric neurogenesis has significantly declined and the gut may be less permissive to EN differentiation. Within this environment, cells previously cultured in bFGF yield predominantly EG / NPs and form few ENs in vivo, with an EN:EGC ratio of about 1:20, even after a 3-month post-transplant follow-up. In contrast, cells cultured in GRF media, designed to enhance neuronal differentiation, achieved a more balanced EN:EGC ratio of approximately 1:2, closely resembling the 1:1 ratio found in endogenous mouse myenteric ganglia. In summary, this example underscores the influence of culture conditions on the formation of EG / NPs and ENs from adult mice and human tissues. Tailoring culture conditions, such as substrate composition and growth factor supplementation, is an essential approach to fine tune in vitro assay development and optimize the therapeutic efficacy of autologous cell therapy. This work represents meaningful advance toward achieving more favorable neuron-to-glia ratios in adult-derived ENS cultures and promoting ENS network formation in the adult gut environment. Through modifications to the culture PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 matrix and media, it has been demonstrated that the potential to reduce reliance on embryonic or neonatal sources for cell expansion and EN differentiation for experimental and, potentially clinical, applications. The above results were obtained using the following materials and methods. Materials and Methods Animals Plp1-EGFP mice were gifted to the Goldstein laboratory by Wendy Macklin (Mallon et al. (2002) Journal of Neuroscience;22(3):876-85). BAF53b::Cre (stock number 027826) and (R)26-tdTomato (stock number 007914) mice were purchased from The Jackson Laboratory. BAF53b::tdT; Plp1-EGFP dual glia and neuron reporter mice were generated as previously described (Stavely et al. (2024) Neuron. 112(18):3143-60.e6; Mueller et al. (2024) Cell Reports;43(11); Stavely et al. (2022) Science Translational Medicine;14(646):eabl8753). All mice were housed and bred under specific-pathogen-free conditions at the Center for Comparative Medicine animal facility at Massachusetts General Hospital (MGH). Rodents were housed in Allentown Inc. rectangular caging (160 cages per individually ventilated cage racks; which uses blower at 60 air changes per hour) under a 12h:12h light:dark cycle from 7 am – 7 pm. Bedding consisted of Hardwood Sanichip; with Carefresh nesting material and mice had access to Prolab Isopro RMH 3000 chow mix (ScottPharma) ad libitum. Generation of Heterogenous Mouse Cultures and Study Designs Mice were euthanized and the small intestine was removed from duodenum to terminal ileum. The smooth muscle-myenteric plexus (SMMP) layer was dissected from underlying tissue in PBS and digested for 35 minutes at 37 °C in dispase (250 μg / mL; #07923 STEMCELL Technologies, Vancouver, BC) and collagenase XI (#C7657-25MG 1 mg / mL; Sigma–Aldrich, Darmstadt, Germany). Following digestion, the cells were filtered through a 40-µm filter and plated in their respective media formulation (Table 6). Positive control media (+CM) contained a 1:1 mixture of DMEM (+ Glucose + Glutamine, Thermo Fisher, Waltham, MA) and NeuroCult Basal Media (#05700, STEMCELL Technologies,) supplemented with 20 ng / mL bFGF (STEMCELL Technologies, #78003), 20 ng / mL IGF-I (#RP-10931 Thermo Fisher, Waltham, MA)1, 2% B-27 supplement (#, 12587001, Gibco Thermo Fisher), 1% N-2 supplement (#17502001, Gibco Thermo Fisher), 50 mM 2-mercaptoethanol(#21985023, Thermo Fisher), 75 ng / mL all-trans retinoic acid (#R2625-100MG, Sigma-Aldrich) and 1% Antibiotic-Antimycotic (#15240062, Gibco Thermo Fisher) as previously reported (Guyer et al. (2023) Cell Rep 2023;42(3):112194). Negative control media (-CM) contained only 2% B27 supplement, 1% N2 supplement and 1% Antibiotic-Antimycotic in DMEM (#11995073, Gibco Thermo Fisher) and was supplemented with different combinations of bFGF (20 ng / mL), all-trans retinoic acid (75 ng / mL) and / or GDNF (50ng / mL) (#10788-108, FUJIFILM Irvine Scientific, Santa Ana, CA). All experiments were conducted using a standard humidified cell culture incubator (37˚C, 5% CO2 and atmospheric O2) with media supplemented once weekly. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 High throughput image-based analysis of BAF53b::tdT; Plp1-EGFP neurospheres. For experiments examining the effects of age and media compositions on heterogenous cell cultures, BAF53b::tdT; Plp1-EGFP mice were sacrificed and single cell suspensions were generated as above. Cells were plated at a density of 50,000 cells per well in 24-well ultra-low attachment plates (Corning Costar® 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates Cat# 3473). After two weeks, whole-plate tile scans were acquired using a Keyence BZX-700 All-In-One Microscopy System (Keyence America, Itasca). Image processing was performed in ImageJ (NIH, Bethesda), where individual wells were isolated and analyzed as follows: The Plp1-EGFP channel was binarized using auto- thresholding with the MaxEntropy algorithm, followed by despeckling to remove outliers smaller than 2 µm. Neurospheres were segmented using the Watershed function, and the “Analyze Particles” command was used to quantify and measure the area occupied by Plp1-EGFP+ neurospheres, filtering for objects >20 µm. Regions of interest (ROIs) for each neurosphere generated from the particle analysis were utilized to measure the mean grey value (mean fluorescence intensity, MFI) of BAF53b::tdT expression within each neurosphere. RNA Isolation / qPCR RNA isolation was performed via a RNeasy Mini kit (#74106, Qiagen, Hilden, Germany) as of manufacturer’s instructions. RNA was quantified using a Qubit 2.0 Fluorometer (#Q32866, Thermo Fisher Life Technologies). qPCR was prepared utilizing iTaq Universal SYBR Green Supermix (#1725151, Bio- Rad, Hercules, CA) and was performed on either a Bio-Rad CFX Touch Real-Time PCR Detection System (Bio-Rad) or a LightCycler 96 Instrument (Roche). Primer sequences for gene amplification in mouse and human samples are provided in Table 8. The thermal cycling protocol followed the manufacturer’s instructions. Data were analyzed using Bio-Rad CFX Manager software (version 3.1) with a standard threshold to determine quantification cycle (Ct) values. Ct values were normalized to Gapdh / GAPDH expression within each sample as an internal control, and the Log2 fold change (FC) was calculated between corresponding sample conditions from the same mouse or subject. All reactions were performed in duplicate. Table 8: Primer sequences PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Ms = mouse; Hu = human Flow cytometry analysis Analysis by flow cytometry was conducted on samples generated from BAF53b::tdT; Plp1-EGFP mice. To generate single cell suspensions, free-floating neurospheres were dissociated for 45 minutes using Accutase (STEMCELL Technologies), or monolayer cultures on fibronectin-coated plates (sigma, #F1141, 1:100 for 1h) were dissociated for 10 minutes into a single-cell suspensions using .025% Trypsin- EDTA (#25200056, Gibco Thermo Fisher) at 37C°C. Samples were filtered through a 35-µm cell strainer (#6475025, Electron Microscopy Sciences, Hatfield, PA) and stained with DAPI(#D1306, Invitrogen Thermo Fisher) serving as a viability marker. Cell sorting was performed with BD FACSAria cell sorter (BD Biosciences, Franklin Lakes, New Jersey) instruments. Flow cytometric analysis was conducted using FlowJo software (FlowJo, LLC, OR). Neurogenesis and neuronal survival assays For neurogenesis experiments cells were isolated from the small intestine of BAF53b::tdT; Plp1- EGFP mice as described above. EG / NPs and ENs were isolated using fluorescence activated cell sorting (FACS) as previously described herein. Briefly, the heterogenous population of cells was plated at a density of 5×104cells / cm2on a fibronectin-coated (sigma, #F1141, 1:100 for 1h) 24-well cell culture plate cultured for 2 days in media comprised of DMEM / F12 media (ThermoFisher, Gibco) containing 10% FBS (#10438-018, ThermoFisher, Gibco) and 1% penicillin-streptomycin (ThermoFisher, Gibco, 15140122). Monolayers were trypinized as above and FACS was conducted using a BD FACSAria cell sorter (BD Biosciences) to collect GFP+ EG / NPs and tdT+ ENs. For the neurogenesis assay GFP+ cells were plated in their respective media (Table 6) at a density of 5,000 cells / well in a 96-well ultra-low attachment round- bottom microplate. After 3 weeks the MFI of BAF53b::tdT from newly formed ENs was quantified in the Plp1-EGFP neurospheres as described above. For neuronal survival assays BAF53b::tdT+ ENs were PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 cultured in their respective media on 96-well fibronectin-coated flat-bottom plate at a density of 500 neurons per well. After two weeks in culture images of the entire wells were taken on a Keyence BZX-700 All-In-One Microscopy System and the number of neurons were quantified per well manually with ImageJ. Media was supplemented once weekly. Neurosphere Sectioning Neurospheres were fixed in 4% paraformaldehyde (PFA) for 1 hour, followed by overnight incubation in 15% sucrose at 4°C for cryoprotection. They were then infiltrated with 7.5% gelatin / 15% sucrose in PBS at 37°C before embedding. Each neurosphere was individually placed in a 10×10×5 mm cryomold (# 4565, Andwin Scientific, Simi Valley, CA) and allowed to gel at room temperature. To track sphere positioning, a Sharpie mark was made on the mold before rapid freezing in −50°C methylbutane. The frozen neurospheres were sectioned into 10 μm cryosections, which were then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, 9036-19-5) in PBS for 20 minutes. Sections were blocked with 10% donkey serum (#D9663-10ML, Sigma-Aldrich) for 1 hour before immunostaining. The following primary antibodies (diluted in 10% donkey serum) were applied overnight at 4°C: the neuronal marker Anti-HuC / D (clone: 16A11, 1:50, Thermo Fisher Scientific, A-21271), EG / NP markers Rabbit anti-S100B (1:200, ab52642, Abcam, Cambridge, UK), Rabbit anti-Sox10 (1:500, ab155279, Abcam) and Goat anti-GFAP (1:500, ab53554, Abcam). After primary antibody incubation, Alexa Fluor® 647-conjugated secondary antibodies (diluted in 10% donkey serum) were applied: Donkey anti-rabbit IgG (1:200, A31573, Thermo Fisher Scientific), Donkey anti-goat IgG (1:200, A21447, Thermo Fisher Scientific) or Donkey anti-human IgG (H+L) (1:200, AB2340578, Thermo Fisher Scientific). DAPI was used to stain cell nuclei. Sections were mounted in aqueous Poly / Mount and imaged using the Keyence BZX-700 All-In-One Microscopy system. In vivo Cell Transplantation Experiments Neurosphere transplantation was conducted in the midcolon as previously described (Rahman et al. (2024) JCI Insight;9(17); Mueller et al. (2024) Cell Reports;43(11); Pan et al. (2024) Nat Commun;15(1):2479). Briefly, 1-2 months old C57BL / 6J mice utilized as recipients were anesthetized by isoflurane inhalation and the midcolon was accessed via midline laparotomy. Neurospheres generated from the neurogenesis assay described above were implanted into the muscularis propria. After 12 weeks, mice were sacrificed, and the colon was collected to assess transplanted GFP+ enteric glial cells (EGCs) and BAF53b::tdT+ EN populations, as well as BAF53b::tdT+ neural network formation. Colons were fixed in 4% PFA, peeled to obtain the SMMP, and processed as whole-mount preparations. Imaging was conducted using a Keyence BZX-700 All-In-One Microscopy System. For quantification of EGC and EN ratios, samples were stained with DAPI and the number of GFP+ or tdT+ cells were enumerated in a 0.396 mm² area per sample. For analysis of neural networks images of BAF53b::tdT fluorescence were taken in a 9.88 mm² area and were analyzed via a macro in ImageJ as follows: a max intensity projection was applied to combine image Z-stacks, enhanced contrast of neurites using Contrast Limited Adaptive Histogram Equalization (CLAHE), background subtraction, Gaussian smoothing with auto-local- thresholding (Phansalkar method) to binarize neural structures, Despeckling to remove noise and the dilate function to maintain continuity in thin neurites, followed by skeletonization to converts neural PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 structures into simplified line representations which were analyzed with the analyze skeleton plugin to measure branch lengths, and counts of branches and junction node counts to assess network complexity. Human Gut Tissues Colonic or ileal samples resected as part of required patient care at Massachusetts General Hospital were collected from subjects between 2 months and 49 years old (Table 7). Tissues were stored overnight at 4°C in sterile PBS. The muscularis propria was mechanically separated from the mucosal, submucosal, and serosal layers using fine forceps and microdissection scissors under a stereoscopic microscope. For enzymatic dissociation, tissues were minced into approximately 5 mm fragments using sterile microdissection scissors and digested in a prewarmed enzymatic solution consisting of collagenase type XI (1 mg / mL) and dispase (0.6 U / mL) in DMEM / F12. The digestion was carried out at 37°C for 4 h in a Mini Incubated Shaker (#76407-108, VWR International, Portland, OR) with intermittent trituration using glass serological pipettes of progressively smaller bore diameters (3 mm, 2 mm, and 1 mm) until the sample became liquefied. Large undigested tissue fragments were removed using a 1000 μm cell strainer (#4355100003 pluriStrainer, pluriSelect USA, El Cajon, USA), and single cells were obtained by passing the suspension through a 70 μm strainer (#22-363-548 Fisher Scientific, Pittsburgh, PA). Red blood cells were lysed by incubating the cell suspension in ACK Lysing Buffer (#A1049201, Gibco, ThermoFisher Scientific) for 7 min at room temperature. Following isolation, cells were divided into equal aliquots and plated in their respective culture media at a density of 5–20 × 10³ cells / cm² in ultra-low attachment plates for direct comparisons. As a positive control media (+CM) we utilized a defined formulation (Hotta et al. (2023) Cell Transplantation 2023;32:09636897231215233) which consisted of DMEM / F12(#11320033, Gibco Thermo Fisher), supplemented with 1% Glutamax(#35050061, Gibco Thermo Fisher), 2% B27, 1% N2, of 0.2% heparin (#07980 STEMCELL Technologies), 2-mercaptoethanol (50 mM), 1% Antibiotic-Antimycotic, Primocin (100 μg / mL)(#ant-pm-05 Invivogen, San Diego, CA), metronidazole (50 μg / mL) (M3761-5G, Sigma Aldrich) and the growth factors EGF (20 ng / mL) and human recombinant bFGF (20 ng / mL). Media supplemented with GDNF, RA and bFGF consisted of DMEM, supplemented with 2% B27, 1% N2, 2- mercaptoethanol (50 mM), 1% Antibiotic-Antimycotic, Primocin (100 μg / mL), metronidazole (50 μg / mL) and the growth factors human recombinant bFGF (20 ng / mL) (#78006, STEMCELL Technologies), all- trans retinoic acid (75 ng / mL) and human recombinant GDNF (50 ng / mL) (#78058, STEMCELL Technologies). Media was supplemented once weekly. Cell yield per mg of tissue was calculated by i) counting the number of cells after passage to calculate their proliferation rate from original seeding densities, ii) multiplying the original total cell count after digestion by the proliferation rate and iii) dividing this value by the original tissue weight. For human neurosphere immunocytochemistry, neurospheres attached to fibronectin-coated plates were fixed with 4% PFA for 30 minutes at room temp, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, 9036-19-5) in PBS for 20 minutes, blocked with 10% donkey serum for one hour, then stained using the following conjugated primary antibodies diluted in 10% donkey serum: mouse anti- tubulin β3 (801210, 1:400, conjugated to Alexa Fluor 647, BioLegend, San Diego, CA); mouse anti- human CD271 (NGFR) (345105, 1:200, conjugated to FITC, Biolegend); rat anti-human / mouse CD49f / ITGA6 (313616, 1:200; conjugated to APC, Biolegend). Primary antibodies were applied overnight PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 at 4 °C or for 1 hour at room temperature. Cell nuclei were stained with DAPI (Invitrogen). Immunofluorescence images were acquired from six randomly selected fields, covering a total area of 49.4 mm² per sample. The proportions of NGFR+, ITGA6+, and TUBB3+ cells were quantified using an automated Fiji (ImageJ) macro as follows: Rolling ball background subtraction was applied to reduce uneven illumination. Auto-local-thresholding (Phansalkar method) was applied to segment nuclei, followed by mask conversion and watershed transformation to separate closely associated nuclei in DAPI channel images. Cell counts were obtained using particle analysis. For the immunohistochemically labeled channels contrast was enhanced using Contrast Limited Adaptive Histogram Equalization (CLAHE) to improve visibility of structures and binary thresholding was performed using the MaxEntropy method to segment labeled structures and measurements were extracted via ROI analysis from the nuclei masks to count the number of overlapping labeled cells and nuclei and calculate the proportions of immunoreactive cells. Statistical Analysis All details of statistical analysis can be found in the description of the figures. Data analysis was performed using GraphPad Prism v7 (GraphPad Software Inc., San Diego, USA). For all analyses p < 0.05 was considered significant. All data were presented as mean ± standard error of the mean (SEM), unless otherwise stated. Example 9: Autologous Cell Therapy for Hirschsprung Disease This example describes a laboratory protocol defining the process for conducting tissue preparation and digestion, including cell passaging, for the generation of an autologous neural cell therapy. Once the cells are passaged, they are ready for quality assays including gene and protein expression. A summary of equipment and reagents are defined below. Standard laboratory reagents, materials, and equipment are not defined (e.g. pipettes and tips, conical tubes, etc.) unless specifically noted. Under no circumstances should expired reagents and materials be used. Moreover, equipment should be maintained and calibrated according to manufacturer specifications. Standard terms and conditions Standard terms and conditions used throughout this protocol include: PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Tissue preparation Equipment and reagents ^ VWR® Mini Incubated Shaker, Supplier: Avantor CAT# 76407-108 ^ Scales (tissue weight g) ^ Sterile forceps (tissue transfer) ^ Sterile stainless-steel scissors approximately 12cm in length (mincing) ^ Sylgard (Ellsworth DOW SYLGARD 184 SKU#4019862) ^ Petri-dish 10cm (Corning cat# 430167) ^ Minutien Pins, Tip: 0.02 mm, 1 cm, 500 / pk (FisherScientific cat# NC9681411) Solutions to prepare ^ PBS with Antibiotics ^ PBS [Magnesium and Calcium free] base ^ Primocin (100ug / mL) ^ Metronidazole (25ug / mL) ^ Amphrocetin B (2.5ug / mL) 1. Prepare materials and solutions for tissue preparation in BSC prior to arrival. Make Sylgard-lined 10 cm dish and sterilize under UV light overnight. Place insect pins in 70% EtOH and was in sterile PBS. 2. Acquire full thickness resection specimen in PBS with Antibiotics. 3. Place tissue in sylgard-lined petri dish. 4. Cut tubular tissue longitudinally (along the mesenteric border if visible). 5. Using 0.2mm insect taxonomy pins, fix the tissue in a flat sheet on the sylgard-lined petri dish. 6. Wash tissue in PBS until any fecal matter and blood is removed and solution becomes clear. 7. Using blunt dissection with microdissection scissors, remove the mucosa from the muscularis propria. Mucosa can be lifted and held by fine forceps, being careful not to hold the muscularis propria to prevent damage. Blood vessels connecting the layers of the intestine are thick and may need to be cut if the mucosa does not come off easily. A stereoscopic microscope can assist in visualizing the layers and the removal of finer structures such as the submucosa, serosa and residual vessels. 8. Transfer muscularis to a fresh petri-dish and inspect the sample for quality (cleanliness, lack of submucosal tissue). Further dissect as required. 9. Place tissue in 50mL tube in ice cold sterile PBS without Ca2+or Mg2+and shake vigorously for 30 seconds. 10. Repeat 10 times with fresh 50mL tubes. 11. Store tissue in PBS containing antibiotic cocktail overnight at 4 degrees for later processing. Tissue digestion Equipment and reagents ^ pluriStrainer® 1000 µm (Cell Strainer), sterile (PluriSelect El Cajon, CA, USA; SKU 43- 51000-03) PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 ^ Fisherbrand™ Sterile Cell Strainer 40 µm (FisherScientific, USA; 22-363-547) ^ (Corning Costar® 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, Cat# 3471) ^ Collagenase type XI from Clostridium histolyticum, 2-5 FALGPA units / mg solid, ≥800 CDU / mg solid (Sigma-Aldrich, CAT# C7657) ^ 1 U / mL dispase in DMEM / F-12 (Stem cell technologies, CAT# 07923) ^ Gibco DMEM / F:12 Cat# 11320033 ^ DMEM, high glucose, pyruvate (ThermoFisherScientific CAT# 11995065) ^ Gibco N-2 Supplement 100X Cat#17502001 ^ Gibco B-27 Supplement (50X), minus Vitamin A Cat# 12587001 ^ Human Recombinant bFGF (Stemcell Technologies Cat# 78003) (20ng / mL final concentration in media) ^ Human Recombinant GDNF (Stemcell Technologies Cat# 78058) (50ng / mL final concentration in media) ^ Retinoic acid (Sigma-Aldrich cat# R2625) (75ng / mL final concentration in media) ^ InvivoGen Primocin Cat# ant-pm-1 ^ Millipore Sigma Metronidazole Cat# M3761-5G ^ Gibco 2-Mercaptoethanol Cat# 21985023 Solutions to prepare Complete Growth Media ^ DMEM [4.5g / L D-Glucose, L-Glutamine, 110mg / L Sodium Pyruvate] basal media ^ 50x B27 Supplement ^ 100x N2 Supplement ^ 100x 2-mercaptoethanol ^ All-trans Retinoic Acid (75ng / mL) ^ Human Recombinant GDNF (50ng / mL) ^ Human Recombinant bFGF (20ng / mL) ^ Primocin (100ug / mL) ^ Metronidazole (25ug / mL) ^ Amphrocetin B (2.5ug / mL) Enzymatic Solution ^ DMEM / F12 base ^ collagenase type XI (1 mg mL−1) ^ Dispase (0.6U mL−1) 12. Prewarm enzymatic solution to 37˚C. 13. Transfer tissue to an empty 50mL tube and weigh the sample. 14. Mince until fragments are approximately 3x3mm-5x5mm. 15. Add 1 mL of enzymatic solution per 200mg of tissue weight recorded earlier. 16. Incubate in a VWR® Mini Incubated Shaker at 37˚C for 3 hours at 260rpm. 17. Triturate using a 25mL glass pipette 20 times. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 18. Triturate using a 10mL glass pipette 20 times. 19. Incubate in a VWR® Mini Incubated Shaker at 37˚C for 2.5 hours at 260rpm. 20. Triturate using a 1000µL pipette tip 30 times until a fluid suspension is formed. 21. Apply suspension to a pluriStrainer® 1000 µm (Cell Strainer) in a 50mL tube and rinse around the whole filter with 15mL of sterile DMEM to remove any large tissue fragments. 22. Resuspend the flow through and apply to a 40 µm cell strainer in a 50mL tube using one filter per 0.5g of starting tissue. The solution should be triturated. 23. Rinse filter with sterile DMEM / F124x5mL waiting for the DMEM / F12 to almost completely pass through each time. Solution may need to be triturated. 24. Invert the filter in a new 50mL tube. Using a P1000 pass approximately 10mL of sterile DMEM / F12 to collect fragments. 25. To ensure all fragments are collected: Bring the filter back to the upright position and hold at a 45- degree angle. Apply 1mL sterile DMEM / F12 to the top of the slope and wash fragments down to the bottom using a P1000. Resuspend the fragments at the bottom of the slope of the filter by adding 1mL of media, quickly resuspending, and drawing up the solution for ganglia collection. Repeat until visual inspection of the filter shows no material. Should take approximately 10 attempts. 26. Centrifuge samples at 500g for 5 minutes. 27. Remove the supernatant and resuspend the sample in 1mL of sterile DMEM per gram of starting tissue. Mix thoroughly. 28. Repeat steps 19 to 23 using one 40 µm cell strainer and 50mL tube per 1g of starting tissue. 29. Resuspend fragments in 2mL of growth media per tube. 30. Seed fragments into low-attachment 6-well plates using one well per 0.25g of starting material in 4mL of growth media. 31. Verify the presence of MyGa enrichment within the tissue fragments using a light microscope. Ganglia come in a variety of shapes and sizes with examples of hand-picked ganglia shown in FIG.22F. 32. Culture in a standard cell culture incubator. Culture Phase 1 (passage 0) Equipment and reagents ^ Corning Costar® 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, Cat# 3471 ^ CytoOne 6 well Plate, TC Treated Cat# CC7682-7506 ^ Millipore Sigma Fibronectin Cat# F1141-5MG ^ DMEM, high glucose, pyruvate (ThermoFisherScientific CAT# 11995065) ^ Gibco N-2 Supplement 100X Cat#17502001 ^ Gibco B-27 Supplement (50X), minus Vitamin A Cat# 12587001 ^ Human Recombinant bFGF (Stemcell Technologies Cat# 78003) (20ng / mL final concentration in media) ^ Human Recombinant GDNF (Stemcell Technologies Cat# 78058) (50ng / mL final concentration in media) ^ Retinoic acid (Sigma-Aldrich cat# R2625) (75ng / mL final concentration in media) PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 ^ InvivoGen Primocin Cat# ant-pm-1 ^ Millipore Sigma Metronidazole Cat# M3761-5G ^ Gibco 2-Mercaptoethanol Cat# 21985023 ^ Gibco Trypsin-EDTA (0.25%), phenol red Cat # 25200072 33. Days 1 – 3: The MyGa enriched fraction should begin to form spheroids with irregular spheroid morphology. 34. On Day 5, add 2mL of growth media per well 35. On Day 10, commence the spheroid attachment and formation of cellular monolayers. Pre-coat tissue-culture treated plates (CytoOne 6 well Plate, TC Treated) with fibronectin (1:100 for 2h at 37°C). Transfer spheroids in the same media volume 1:1 to fibronectin coated wells. 36. On Day 15, check spheroid attachment to the plate and the early stages of cell migration into a monolayer. The majority of spheroids should have a firm attachment when the plate is gently rocked. Remove all media and replace with 4mL of growth media. Cultures should look clear of large debris. 37. Day 20: Conduct harvest of cells on the monolayer as follows: a) Remove all media. b) Add 5mL of sterile PBS to all wells, then remove to wash. c) Add 4mL of trypsin solution and incubate for 7 minutes in a standard cell incubator (37 degrees, 5% CO2, and atmospheric O2. Resuspend and validate under a light microscope. d) Quench trypsin by adding 8mL of DMEM and transfer to 15mL tubes. e) Pellet cells by centrifugation at 500G for 5min. f) Resuspend pellet in 1mL of growth media to perform cell counting via a hemocytometer. g) Use cell counts to prepare for the first passage, cryopreservation and other quality control assays (defined later in this protocol). Culture Phase 2 (passage 1) Equipment and reagents ^ DMEM, high glucose, pyruvate (ThermoFisherScientific CAT# 11995065) ^ Gibco N-2 Supplement 100X Cat#17502001 ^ Gibco B-27 Supplement (50X), minus Vitamin A Cat# 12587001 ^ Human Recombinant bFGF (Stemcell Technologies Cat# 78003) (20ng / mL final concentration in media) ^ Human Recombinant GDNF (Stemcell Technologies Cat# 78058) (50ng / mL final concentration in media) ^ Retinoic acid (Sigma-Aldrich cat# R2625) (75ng / mL final concentration in media) 38. Calculate number of cells in the single cell suspension (fresh or thawed from cryopreservation) and re-seed into low-attachment 6-well plates at a density of 5000 cells / cm2or approximately 50,000 cells per well in 4mL of growth media. Incubate until Day 5 of Culture Phase 2 (“P2”). 39. On Day 5 of P2, add 2mL of growth media per well. 40. On Day 10 of P2, check spheroid formation and validate quality. Resuspend neurospheres within the well and subsample for quantification of spheroid number, size and gene expression assays. Gene Expression PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Equipment and reagents ^ Qiagen RNeasy mini kit Cat# 74106 ^ Internal positive control ^ Internal negative control Primers used for qPCR: ^ GAPDH (hu) F 5’ GGA GCG AGA TCC CTC CAA AAT 3’ (SEQ ID NO: 17) ^ GAPDH (hu) R 5’ GGC TGT TGT CAT ACT TCT CAT GG 3’ (SEQ ID NO: 18) ^ PLP1 (hu) F 5’ TGC TGA TGC CAG AAT GTA TGG 3’ (SEQ ID NO: 19) ^ PLP1 (hu) R 5’ GCA GAT GGA CAG AAG GTT GGA 3’ (SEQ ID NO: 20) ^ NGFR (hu) F 5’ CCG TTG GAT TAC ACG GTC CAC 3’ (SEQ ID NO: 21) ^ NGFR (hu) R 5’ TGA AGG CTA TGT AGG CCA CAA 3’ (SEQ ID NO: 22) ^ TUBB3 (hu) F 5’ GGC CAA GGG TCA CTA CAC G 3’ (SEQ ID NO: 23) ^ TUBB3 (hu) R 5’ GCA GTC GTT TTC ACA CTC 3’ (SEQ ID NO: 24) ^ PHOX2B (hu) F 5’ AAC CCG ATA AGG ACC ACT TTT G 3’ (SEQ ID NO: 25) ^ PHOX2B (hu) R 5’ AGA GTT TGT AAG GAA CTG CGG 3’ (SEQ ID NO: 26) Major equipment ^ Bio-Rad CFX96 real-time thermal cycler (or equivalent) 41. Quantitative PCR performed as of manufacturer instructions using the iTaq Universal One-Step RT- qPCR Kit or equivalent qPCR assay. Immunocytochemistry Equipment and reagents ^ Falcon® 24-well Multiwell Flat Bottom TC-treated Cell Culture Plate, with Lid Cat# 353226 ^ Corning Costar® 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates Cat# 3473 ^ 4% PFA (Paraformaldehyde, 4% in PBS ) ^ PBS with 1% Triton ^ 10% donkey serum in PBS ^ pre-diluted DAPI (4′,6-diamidino-2-phenylindole) Antibodies used for ICC or flow cytometry: ^ Biolegend Cd49f APC conjugated monoclonal rat antibody Cat# 313616 ^ Biolegend FITC anti-human CD271 NGFR Conjugated monoclonal mouse IgG antibody Cat# 345104 Antibodies used for ICC only: ^ Biolegend Alexa Fluor® 647 anti-Tubulin β 3 (TUBB3) Antibody mouse IgG Cat# 801210 Major equipment ^ Keyence BZX-700 All-In-One Microscopy system (Keyence America Itasca) – can supplement with any inverted fluorescent microscope. 42. On Day 1, Fix tissue in 4% PFA for 30 minutes at room temperature (RT) PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 43. Wash cells 3 times, 5 minutes each, with PBS 44. Dilute 1% Triton stock to a final concentration of 0.1% Triton in PBS, add to cells and incubate for 20 minutes at RT for permeabilization. 45. Wash cells 3 times, roughly 5 minutes each, with PBS 46. Block cells for 1 hour at RT in 10% donkey serum in PBS 47. Wash cells 3 times, roughly 5 minutes each, with PBS 48. Add primary antibody, diluted in 10% donkey serum in PBS, and let incubate overnight at 4°C (covered). 49. On Day 2, Wash cells 3 times (about 10 mins each) 50. Add secondary antibody, diluted in 10% donkey serum in PBS for 1 hour at RT (covered) 51. Wash cells 3 times. 52. Add pre-diluted DAPI and let incubate for 10 mins at RT. 53. Wash 3 times. 54. Perform imaging on a Keyence BZX-700 All-In-One Microscopy system or equivalent inverted microscope. 55. Perform blinded cell counts of DAPI positive and immunoreactive cells. Flow Cytometry Procedure Equipment and reagents ^ Enzyme for enzymatic digestion ^ 40um strainer ^ Flow cytometry staining buffer (sterile PBS with 2% FBS) ^ Microtiter plates ^ DAPI undiluted stock Antibodies used for IHC or flow cytometry: ^ Biolegend Cd49f APC conjugated monoclonal rat antibody Cat# 313616 ^ Biolegend FITC anti-human CD271 NGFR Conjugated monoclonal mouse IgG antibody Cat# 345104 56. Prepare single-cell suspension by enzymatic digestion using trypsin. Filter via 40um strainer. 57. Aliquot 50 µL of cell suspension (from 105to 108) to each tube or well. 58. Combine the recommended quantity of each primary antibody in an appropriate volume of Flow Cytometry Staining Buffer so that the final staining volume is 100 µL (i.e.50 µL of cell sample + 50 µL of antibody mix) and add to cells. Pulse vortex gently to mix. Add 1 antibody each to separate samples of cells for compensation samples. 59. Incubate for 30 minutes at 37°C. Ensure protection from light. 60. Wash the cells by adding Flow Cytometry Staining Buffer. Use 2 mL / tube or 200 µL / well for microtiter plates. Centrifuge at 400-600 x g for 5 minutes at room temperature. Discard supernatant. Repeat 2 times. 61. Stain samples with DAPI 1:100 and resuspend. Analyze samples by flow cytometry. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Numbered Embodiments Embodiment 1. A composition comprising a population of human enteric neural stem cells (ENSCs), wherein the population comprises at least one of a cell expressing nerve growth factor receptor (NGFR), a cell expressing integrin alpha 6 (ITGA6), and a cell expressing L1 cell adhesion molecule (L1CAM). Embodiment 2. The composition of embodiment 1, wherein NGFR, ITGA6, and / or L1CAM are expressed at the protein level. Embodiment 3. The composition of embodiment 2, wherein expression of NGFR, ITGA6, or L1CAM protein is detected using an immunohistochemistry assay, flow cytometry, or Western blotting. Embodiment 4. The composition of any one of embodiments 1-3, wherein the population of human ENSCs further comprises a cell that expresses proteolipid protein 1 (PLP1) and / or a cell that expresses paired-like homeobox 2B (PHOX2B). Embodiment 5. The composition of embodiment 4, wherein PLP1 and / or PHOX2B are expressed at the mRNA level. Embodiment 6. The composition of embodiment 5, wherein expression of PLP1 or PHOX2B mRNA is detected using a polymerase chain reaction (PCR) assay or a quantitative PCR assay, or by RNA sequencing. Embodiment 7. The composition of any one of embodiments 4-6, wherein expression of PLP1 and / or PHOX2B mRNA in the population of human ENSCs is at a higher level than in an equivalent population of human fibroblast or myofibroblast cells. Embodiment 8. The composition of any one of embodiments 1-7, wherein the ENSCs are cryopreserved. Embodiment 9. The composition of any one of embodiments 1-7, wherein a portion of the ENSCs are cryopreserved. Embodiment 10. A container comprising the composition of any one of embodiments 1-7. Embodiment 11. The container of embodiment 10, wherein the composition is cryopreserved. Embodiment 12. A method of producing a human ENSC from intestinal tissue, said method comprising the steps of: a) dissociating the intestinal tissue using an enzyme b) passing the dissociated tissue through a series of cell strainers having pore sizes between 1000 μm and 40 μm and collecting material that passes through the 1000 μm pore size strainer, but not the 40 μm pore size strainer to collect material enriched for myenteric ganglia; and c) culturing myenteric ganglia; thereby producing a human ENSC from intestinal tissue. Embodiment 13. The method of embodiment 12, wherein the intestinal tissue comprises muscularis propria tissue. Embodiment 14. The method of embodiment 12 or 13, wherein the enzyme is a collagenase and / or a dispase. Embodiment 15. The method of embodiment 12, wherein the intestinal tissue is from a pediatric patient. Embodiment 16. The method of embodiment 12, wherein the intestinal tissue is from an adult patient. Embodiment 17. The method of embodiment 16, wherein the method comprises collecting material that passes through the 1000 μm pore size strainer, but not a 100 μm pore size strainer. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Embodiment 18. The method of any one of embodiments 12-17, wherein the intestinal tissue is cryopreserved and thawed prior to step (a). Embodiment 19. The method of any one of embodiments 12-18, wherein culturing myenteric ganglia comprises culturing in a medium containing fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF) and / or retinoic acid. Embodiment 20. The method of embodiment 12 further comprising harvesting the human ENSC. Embodiment 21. The method of any one of embodiments 12-20, wherein the human ENSC expresses NGFR, ITGA6, L1CAM, PLP1, and / or PHOX2B. Embodiment 22. The method of embodiment 21, wherein NGFR, ITGA6, and / or L1CAM are expressed at the protein level. Embodiment 23. The method of embodiment 21, wherein PLP1 and / or PHOX2B are expressed at the mRNA level. Embodiment 24. The method of any one of embodiments 12-23, further comprising cryopreserving the human ENSC. Embodiment 25. A method of cryopreserving a population of human ENSCs produced from intestinal tissue, the method comprising cryopreserving the population of human ENSCs produced from intestinal tissue in cryopreservation medium, wherein the population of human ENSCs comprises at least one of a cell expressing NGFR, a cell expressing ITGA6, and a cell expressing L1CAM. Embodiment 26. The method of embodiment 25, wherein NGFR, ITGA6, and / or L1CAM are expressed at the protein level. Embodiment 27. The method of embodiment 25 or 26, wherein the population of human ENSCs further comprises a cell that expresses PLP1 and / or a cell that expresses PHOX2B Embodiment 28. The method of embodiment 27, wherein PLP1 and / or PHOX2B are expressed at the mRNA level. Embodiment 29. The method of any one of embodiments 25-28, wherein the cryopreservation medium is Bambanker serum-free cryopreservation media. Embodiment 30. A tissue transplant comprising a population of human ENSCs, wherein the population comprises at least one of a cell expressing NGFR, a cell expressing ITGA6, and a cell expressing L1CAM. Embodiment 31. The tissue transplant of embodiment 30, wherein NGFR, ITGA6, and / or L1CAM are expressed at the protein level. Embodiment 32. The tissue transplant of embodiment 30 or 31, wherein the population of human ENSCs further comprises a cell that expresses PLP1 and / or a cell that expresses PHOX2B. Embodiment 33. The tissue transplant of embodiment 32, wherein PLP1 and / or PHOX2B are expressed at the mRNA level. Embodiment 34. The tissue transplant of any one of embodiments 30-33, wherein the tissue transplant is a neurosphere. Embodiment 35. A method of enhancing a function of an enteric nervous system in a patient in need thereof, the method comprising administering the composition of any one of embodiments 1-9 to the patient. PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 Embodiment 36. The method of embodiment 35, wherein the composition comprises an ENSC that is autologous to the patient. Embodiment 37. A method of enhancing a function of an enteric nervous system in a patient in need thereof, the method comprising administering the tissue transplant of any one of embodiments 30-34 to the patient. Embodiment 38. The method of embodiment 37, wherein the tissue transplant comprises an ENSC that is autologous to the patient. Embodiment 39. A method of treating gastroparesis in a patient in need thereof, the method comprising administering the composition of any one of embodiments 1-9 to the patient. Embodiment 40. The method of embodiment 39, wherein the composition comprises an ENSC that is autologous to the patient. Embodiment 41. A method of treating gastroparesis in a patient in need thereof, the method comprising administering the tissue transplant of any one of embodiments 30-34 to the patient. Embodiment 42. The method of embodiment 41, wherein the tissue transplant comprises an ENSC that is autologous to the patient. Embodiment 43. A method of treating Hirschsprung disease (HSCR) in a patient in need thereof, the method comprising administering the composition of any one of embodiments 1-9 to the patient. Embodiment 44. The method of embodiment 43, wherein the composition comprises an ENSC that is autologous to the patient. Embodiment 45. A method of treating HSCR in a patient in need thereof, the method comprising administering the tissue transplant of any one of embodiments 30-34 to the patient. 46. The method of embodiment 45, wherein the tissue transplant comprises an ENSC that is autologous to the patient. Additional Embodiments All references cited in this specification, including, database-accessioned information (e.g., in GENBANK, UNIPROT, PUBMED), are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention. It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only.

Claims

PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 CLAIMS 1. A composition comprising a population of human enteric neural stem cells (ENSCs), wherein the population comprises at least one of a cell expressing nerve growth factor receptor (NGFR), a cell expressing integrin alpha 6 (ITGA6), and a cell expressing L1 cell adhesion molecule (L1CAM).

2. The composition of claim 1, wherein NGFR, ITGA6, and / or L1CAM are expressed at the protein level.

3. The composition of claim 2, wherein expression of NGFR, ITGA6, or L1CAM protein is detected using an immunohistochemistry assay, flow cytometry, or Western blotting.

4. The composition of any one of claims 1-3, wherein the population of human ENSCs further comprises a cell that expresses proteolipid protein 1 (PLP1) and / or a cell that expresses paired-like homeobox 2B (PHOX2B).

5. The composition of claim 4, wherein PLP1 and / or PHOX2B are expressed at the mRNA level.

6. The composition of claim 5, wherein expression of PLP1 or PHOX2B mRNA is detected using a polymerase chain reaction (PCR) assay or a quantitative PCR assay, or by RNA sequencing.

7. The composition of claim 4, wherein expression of PLP1 and / or PHOX2B mRNA in the population of human ENSCs is at a higher level than in an equivalent population of human fibroblast or myofibroblast cells.

8. The composition of claim 1, wherein the ENSCs are cryopreserved.

9. The composition of claim 1, wherein a portion of the ENSCs are cryopreserved.

10. A container comprising the composition of claim 1.

11. The container of claim 10, wherein the composition is cryopreserved.

12. A method of producing a human ENSC from intestinal tissue, said method comprising the steps of: a) dissociating the intestinal tissue using an enzyme b) passing the dissociated tissue through a series of cell strainers having pore sizes between 1000 μm and 40 μm and collecting material that passes through the 1000 μm pore size strainer, but not the 40 μm pore size strainer to collect material enriched for myenteric ganglia; and c) culturing myenteric ganglia; thereby producing a human ENSC from intestinal tissue.

13. The method of claim 12, wherein the intestinal tissue comprises muscularis propria tissue.

14. The method of claim 12 or 13, wherein the enzyme is a collagenase and / or a dispase.

15. The method of claim 12, wherein the intestinal tissue is from a pediatric patient.PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 16. The method of claim 12, wherein the intestinal tissue is from an adult patient.

17. The method of claim 16, wherein the method comprises collecting material that passes through the 1000 μm pore size strainer, but not a 100 μm pore size strainer.

18. The method of claim 12, wherein the intestinal tissue is cryopreserved and thawed prior to step (a).

19. The method of claim 12, wherein culturing myenteric ganglia comprises culturing in a medium containing fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF) and / or retinoic acid.

20. The method of claim 12 further comprising harvesting the human ENSC.

21. The method of claim 12, wherein the human ENSC expresses NGFR, ITGA6, L1CAM, PLP1, and / or PHOX2B.

22. The method of claim 21, wherein NGFR, ITGA6, and / or L1CAM are expressed at the protein level.

23. The method of claim 21, wherein PLP1 and / or PHOX2B are expressed at the mRNA level.

24. The method of claim 12, further comprising cryopreserving the human ENSC.

25. A method of cryopreserving a population of human ENSCs produced from intestinal tissue, the method comprising cryopreserving the population of human ENSCs produced from intestinal tissue in cryopreservation medium, wherein the population of human ENSCs comprises at least one of a cell expressing NGFR, a cell expressing ITGA6, and a cell expressing L1CAM.

26. The method of claim 25, wherein NGFR, ITGA6, and / or L1CAM are expressed at the protein level.

27. The method of claim 25 or 26, wherein the population of human ENSCs further comprises a cell that expresses PLP1 and / or a cell that expresses PHOX2B 28. The method of claim 27, wherein PLP1 and / or PHOX2B are expressed at the mRNA level.

29. The method of claim 25, wherein the cryopreservation medium is Bambanker serum-free cryopreservation media.

30. A tissue transplant comprising a population of human ENSCs, wherein the population comprises at least one of a cell expressing NGFR, a cell expressing ITGA6, and a cell expressing L1CAM.

31. The tissue transplant of claim 30, wherein NGFR, ITGA6, and / or L1CAM are expressed at the protein level.

32. The tissue transplant of claim 30 or 31, wherein the population of human ENSCs further comprises a cell that expresses PLP1 and / or a cell that expresses PHOX2B.

33. The tissue transplant of claim 32, wherein PLP1 and / or PHOX2B are expressed at the mRNA level.PATENT Attorney Docket No.: 51654-004WO2 MGB Docket No.: MGH2023-459 34. The tissue transplant of claim 30, wherein the tissue transplant is a neurosphere.

35. A method of enhancing a function of an enteric nervous system in a patient in need thereof, the method comprising administering the composition of claim 1 to the patient.

36. The method of claim 35, wherein the composition comprises an ENSC that is autologous to the patient.

37. A method of enhancing a function of an enteric nervous system in a patient in need thereof, the method comprising administering the tissue transplant of claim 30 to the patient.

38. The method of claim 37, wherein the tissue transplant comprises an ENSC that is autologous to the patient.

39. A method of treating gastroparesis in a patient in need thereof, the method comprising administering the composition of claim 1 to the patient.

40. The method of claim 39, wherein the composition comprises an ENSC that is autologous to the patient.

41. A method of treating gastroparesis in a patient in need thereof, the method comprising administering the tissue transplant of claim 30 to the patient.

42. The method of claim 41, wherein the tissue transplant comprises an ENSC that is autologous to the patient.

43. A method of treating Hirschsprung disease (HSCR) in a patient in need thereof, the method comprising administering the composition of claim 1 to the patient.

44. The method of claim 43, wherein the composition comprises an ENSC that is autologous to the patient.

45. A method of treating HSCR in a patient in need thereof, the method comprising administering the tissue transplant of claim 30 to the patient.

46. The method of claim 45, wherein the tissue transplant comprises an ENSC that is autologous to the patient.