The work was supported by grants from UCSF Program for Breakthrough Biomedical Research and Sandler Foundation, March of Dimes grant no. 1-FY18-394 and 1DP2NS116769-01, the NIH Director&#39;s New Innovator Award (DP2NS116769) to F.F. and the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK121169) to F.F., H.M. is supported by Larry L. Hillblom Foundation postdoctoral fellowship, NIH T32-DK007418 fellowship and UCSF Program for Breakthrough Biomedical Research independent postdoctoral fellowship.

1. Media preparation
NOTE: Concentrations mentioned throughout the protocol are final concentrations of the media components. Prepare all media under sterile conditions in a laminar flow hood, and store at 4 &#176;C in the dark. Use within 2 weeks.
<ol>
	<li>hPSC maintenance medium: Mix feeder-free hPSC maintenance medium supplement (20 &#181;L/mL) with its base medium.</li>
	<li>Medium A: Add bone morphogenetic protein 4 (BMP4; 1 ng/mL), SB431542 (10 &#956;M), and CHIR99021...

Derivation of Enteric Neurons from Human Pluripotent Stem Cells

<ol>
	<li>Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enteric+nervous+system:+a+second+brain.">The enteric nervous system: a second brain.</a> Hosp Pract. 34 (7), 35-38 (1999).</li><li>Haggarty, S. J., Silva, M. C., Cross, A., Brandon, N. J., Perlis, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+drug+discovery+for+neuropsychiatric+disorders+using+patient-specific+stem+cell+models.">Advancing drug discovery for neuropsychiatric disorders using patient-specific stem cell models.</a> Mol Cell Neurosci. 73, 104-115 (2016).</li><li>Bose, A., Petsko, G. A., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+a+tool+for+modeling+Parkinson's+disease.">Induced pluripotent stem cells: a tool for modeling Parkinson's disease.</a> Trends Neurosci. 45 (8), 608-620 (2022).</li><li>Liu, C., Oikonomopoulos, A., Sayed, N., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+diseases+with+induced+pluripotent+stem+cells:+from+2D+to+3D+and+beyond.">Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.</a> Dev Camb Engl. 145 (5), (2018).</li><li>Marchetto, M. C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+neural+development+and+treatment+of+Rett+syndrome+using+human+induced+pluripotent+stem+cells.">A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells.</a> Cell. 143 (4), 527-539 (2010).</li><li>Park, I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134 (5), 877-886 (2008).</li><li>Lee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461 (7262), 402-406 (2009).</li><li>Shyamala, K., Yanduri, S., Girish, H. C., Murgod, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest:+The+fourth+germ+layer.">Neural crest: The fourth germ layer.</a> J Oral Maxillofac Pathol. 19 (2), 221-229 (2015).</li><li>Crane, J. F., Trainor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+stem+and+progenitor+cells.">Neural crest stem and progenitor cells.</a> Annu Rev Cell Dev Biol. 22, 267-286 (2006).</li><li>Thomas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+crest+cells+display+molecular+and+phenotypic+hallmarks+of+stem+cells.">Human neural crest cells display molecular and phenotypic hallmarks of stem cells.</a> Hum Mol Genet. 17 (21), 3411-3425 (2008).</li><li>Heanue, T. A., Pachnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+nervous+system+development+and+Hirschsprung's+disease:+advances+in+genetic+and+stem+cell+studies.">Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies.</a> Nat Rev Neurosci. 8, 466-479 (2007).</li><li>Mica, Y., Lee, G., Chambers, S. M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neural+crest+induction,+melanocyte+specification,+and+disease-related+pigmentation+defects+in+hESCs+and+patient-specific+iPSCs.">Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs.</a> Cell Rep. 3 (4), 1140-1152 (2013).</li><li>Fattahi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+human+ENS+lineages+for+cell+therapy+and+drug+discovery+in+Hirschsprung+disease.">Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease.</a> Nature. 531 (7592), 105-109 (2016).</li><li>Barber, K., Studer, L., Fattahi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+enteric+neuron+lineages+from+human+pluripotent+stem+cells.">Derivation of enteric neuron lineages from human pluripotent stem cells.</a> Nat Protoc. 14 (4), 1261-1279 (2019).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+transplantation+of+enteric+neural+crest+cells+from+human+induced+pluripotent+stem+cells.">Characterization and transplantation of enteric neural crest cells from human induced pluripotent stem cells.</a> Mol Psychiatry. 23 (3), 499-508 (2018).</li><li>Workman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+human+pluripotent-stem-cell-derived+intestinal+tissues+with+a+functional+enteric+nervous+system.">Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.</a> Nat Med. 23, 49-59 (2017).</li><li>Southard-Smith, E. M., Kos, L., Pavan, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox10+mutation+disrupts+neural+crest+development+in+Dom+Hirschsprung+mouse+model.">Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model.</a> Nat Genet. 18 (1), 60-64 (1998).</li><li>Lee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+directed+differentiation+of+neural+crest+stem+cells+derived+from+human+embryonic+stem+cells.">Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells.</a> Nat Biotechnol. 25 (12), 1468-1475 (2007).</li><li>Majd, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hPSC-derived+enteric+ganglioids+model+human+ENS+development+and+function.">hPSC-derived enteric ganglioids model human ENS development and function.</a> BioRxiv. , (2022).</li><li>Majd, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deriving+Schwann+cells+from+hPSCs+enables+disease+modeling+and+drug+discovery+for+diabetic+peripheral+neuropathy.">Deriving Schwann cells from hPSCs enables disease modeling and drug discovery for diabetic peripheral neuropathy.</a> Cell Stem Cell. 30 (5), 632-647 (2023).</li><li>Samuel, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Schwann+cell+derived+melanocytes+from+hPSCs+identifies+pro-metastatic+factors+in+melanoma.">Generation of Schwann cell derived melanocytes from hPSCs identifies pro-metastatic factors in melanoma.</a> BioRxiv. , (2023).</li><li>Richter, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+muscarinic+receptor+signaling+protects+human+enteric+inhibitory+neurons+against+platin+chemotherapy+toxicity.">Inhibition of muscarinic receptor signaling protects human enteric inhibitory neurons against platin chemotherapy toxicity.</a> BioRxiv. , (2023).</li></ol>

The differentiation protocol described here provides a robust in vitro method to obtain enteric neurons from hPSCs within 30-40 days (Figure 1E) and enteric glia expressing glial fibrillary acidic protein, GFAP, and SOX10 in older cultures (&#62; day 55)13,14,19,22. These neurons and glia are induced by stepwise differentiation of hPSCs into vagal and ente...

The enteric nervous system (ENS) is the largest component of the peripheral nervous system. The ENS contains more than 400 million neurons that are located within the GI tract and control nearly all functions of the gut1. Molecular understanding of the ENS development and function and its defects in enteric neuropathies requires access to a reliable and authentic source of enteric neurons. Access to human primary tissue is limited, and animal models fail to recapitulate key disease phenotypes in many enteric neuropathies. Human pluripotent stem cell (hPSC) technology has proven exceedingly beneficial in providing an unlimited source of desired cell types, especially those that are difficult to isolate from primary sources2,3,4,5,6,7. Here, we provide details of a stepwise and robust in vitro method to obtain ENS cultures from hPSCs. These scalable hPSC-derived cultures open avenues for developmental studies, disease modeling, and high-throughput drug screening and can provide transplantable cells for regenerative medicine.
Enteric neuron lineages are derived from neural crest (NC) cells following the ENS developmental path during embryogenesis. In embryos, NC cells emerge along the margins of the folding neural plate. They proliferate, migrate and give rise to many different cell types including sensory neurons, Schwann cells, melanocytes, craniofacial skeleton and enteric neurons and glia8,9,10. The cell fate decision depends on the distinct region along the anterior-posterior axis that the NC cells emerge from, i.e., cranial NC, vagal NC, trunk NC and sacral NC. The ENS develops from vagal and sacral NC cells with the former dominating the population of the enteric neurons owing to the extensive migration along the length of the bowel and colonizing the gut11.
Derivation of NC cells from PSCs commonly involves a combination of dual SMAD inhibition and WNT pathway activation12,13. Until recently, all NC induction protocols involved serum and other animal product additives in the culture conditions. Chemically undefined media not only lower the reproducibility of NC induction but also challenge mechanistic developmental studies. To overcome these challenges, Barber et al14 developed an NC induction protocol using chemically defined culture conditions and was hence advantageous over alternative methods that rely on serum-replacement factors (e.g., KSR)13,14. This was obtained by basal media replacements and optimizing a protocol originally presented by Fattahi et al to derive enteric NC cells from hPSCs. This improved system is the basis of the hPSC differentiation protocol that is presented here. It begins with induction of enteric neural crest (ENC) cells in a 15-day period by precise modulation of BMP, FGF, WNT and TGF&#946; signaling in addition to retinoic acid (RA). We then derive the ENS lineages by treating cells with glial cell line-derived neurotrophic factor (GDNF). All media compositions are chemically defined and yield robust enteric neuronal cultures within 30-40 days.
In addition to the monolayer cell culture system described here, alternative NC induction approaches have been developed which use free-floating embryoid-bodies15,16. Migratory cells in these cultures have been shown to express NC markers with a subset representing the vagal NC. Co-cultures with primary gut tissue have been used to enrich enteric NC precursors in these cultures. Media compositions in these studies contain a combination of different factors such as nerve growth factor, NT3 and brain-derived neurotrophic factor. At this stage, it is not fully clear how these factors might affect the enteric neuron precursor commitment identities. For an efficient comparison of the enteric NC induction in the monolayer and the embryoid-body-based cultures more data is required. Given the different culture layouts in these strategies, the use of each method should be considered and optimized according to specific application in mind.
The protocol presented here is reproducible and has been successfully tested by us and others using different hPSC (induced and embryonic) lines8,14,16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement (serum free, minus vitamin A)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix, Geltrex</td>
			<td>Gibco</td>
			<td>A14133-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D systems</td>
			<td>314-BP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td>Eppendorf, model no. 5810R</td>
			<td>02262501</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell detachment solution, Accutase</td>
			<td>Stemcell Technologies</td>
			<td>07920</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes</td>
			<td>USA scientific</td>
			<td>1475-0511, 1500-1211</td>
			<td></td>
		</tr>
		<tr>
			<td>Differentiation base medium, Essential 6</td>
			<td>Life Technologies</td>
			<td>A1516401</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 no glutamine</td>
			<td>Life Technologies</td>
			<td>21331020</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Corning</td>
			<td>MT-46034CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Feeder-free hPSC maintenance medium, Essential 8 Flex Medium Kit</td>
			<td>Life Technologies</td>
			<td>A2858501</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>R&#38;D systems</td>
			<td>233-FB/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>1475</td>
			<td></td>
		</tr>
		<tr>
			<td>Human pluripotent stem cells, H9 ESC</td>
			<td>WiCell</td>
			<td>RRID: CVCL_1240</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator with controlled humidity, temperature and CO2</td>
			<td>Thermo Fisher Scientific</td>
			<td>Herralcell 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>EVOS FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Thermo Fisher Scientific</td>
			<td>1300 series class II, type A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Cultrex</td>
			<td>3400-010</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine supplement, Glutagro</td>
			<td>Corning</td>
			<td>25-015-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAAs</td>
			<td>Corning</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell plates, Falcon</td>
			<td>BD</td>
			<td>353934, 353075</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement</td>
			<td>CTS</td>
			<td>A1370701</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Ca and Mg free)</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette filler</td>
			<td>Eppendorf</td>
			<td>Z768715-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>USA scientific</td>
			<td>1111-2830</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-678-11E, 13-678-11F</td>
			<td></td>
		</tr>
		<tr>
			<td>PO</td>
			<td>Sigma-Aldrich</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>polymer coverslip bottom imaging plates, ibidi</td>
			<td>ibidi</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr>
			<td>RA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>R&#38;D systems</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low attachment plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-601</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>R&#38;D systems</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation of enteric nervous system lineages from human pluripotent stem cells

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

Author Spotlight: Robust Culture of Human Enteric Neurons from Human Pluripotent Stem Cells

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

Here we provide a method for generation of human enteric neurons from human pluripotent stem cells under fully defined conditions, and we use these cells in order to understand human enteric nervous system development and function. So I think human enteric nervous system development, physiology and pathophysiology has been a great challenge, and it's because of the rarity of the enteric nervous system cells and the transient nature of the neural crest progenitors, and difficulty in access to tissue and isolation of the tissue. This is a robust platform for generating authentic and functional human enteric neurons from HPSCs, and by overcoming longstanding technical challenges in tissue access, it provides scalable cultures of enteric nervous system components for disease modeling, drug discovery, and studying ENS developments and function.

This protocol provides a method to derive enteric neural crest and enteric neurons from hPSCs using chemically defined culture conditions (Figure 1A-B). Generating high-quality neurons depends on an efficient enteric neural crest induction step. This can be visually assessed by checking the morphology of the free-floating spheres that should look round with smooth surfaces with a size of approximately 0.1 - 0.4 mm as seen in Figure 1C. These sph...

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is presented.

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls ...

differentiation-enteric-nervous-system-lineages-from-human