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 (600 nM) to differentiation base medium.</li>
	<li>Medium B: Add SB431542 (10 &#956;M), and CHIR99021 (1.5 &#956;M) to differentiation base medium.</li>
	<li>Medium C: Add SB431542 (10 &#956;M), CHIR99021 (1.5 &#956;M) and RA (1 &#956;M) to differentiation base medium.</li>
	<li>Neural crest complete (NCC) Medium: Add FGF2 (10 ng/mL), CHIR99021 (3 &#956;M), N2 supplement (10 &#956;L/mL), B27 supplement (20 &#956;L/mL), L-glutamine supplement (10 &#956;L/mL), and MEM NEAAs (10 &#956;L/mL) to neurobasal medium.</li>
	<li>ENC Medium: Add GDNF (10 ng/mL), ascorbic acid (100 &#956;M), N2 supplement (10 &#956;L/mL), B27 supplement (20 &#956;L/mL), L-glutamine supplement (10 &#956;L/mL), and MEM NEAAs (10 &#956;L/mL) to neurobasal medium.</li>
	<li>Ethylenediaminetetraacetic acid (EDTA) 1x: Dilute 500 mM EDTA (1000x) to a final concentration of 500 &#956;M in PBS. 
	NOTE: Use Ca2+ and Mg2+ free PBS throughout this protocol unless otherwise stated.</li>
</ol>
2. Coating of culture plates
<ol>
	<li>Basement membrane matrix coated plates: Quickly thaw, and dilute a 500 &#956;L frozen aliquot of basement membrane matrix in 50 mL of chilled DMEM:F12 by vigorously pipetting using a 50 mL serological pipette. Coat the wells with the diluted basement membrane matrix solution (100 &#956;L per cm2 of well surface area) and incubate them overnight at 37 &#176;C. Aspirate the coating medium before use. To prevent gelatinization of basement membrane matrix, perform this step as quickly as possible and use cold DMEM:F12 for dissolving the frozen aliquot. 
	NOTE: Basement membrane matrix temperature should be kept cold during thawing (on ice) and aliquoting (tubes on ice) to prevent coagulation.</li>
	<li>PO/FN/laminin coated plates: Prepare a 15 &#956;g/mL polyornithine (PO) solution in PBS by diluting a 1000x stock. Coat wells with 100 &#956;L of PO/PBS solution per cm2 of well surface area and incubate the plates overnight at 37 &#176;C. The following day, aspirate PO/PBS and coat the wells at 100 &#956;L per cm2 of well surface area with freshly made FN/laminin/PBS solution containing 2 &#956;g/mL of fibronectin (FN) and 2 &#956;g/mL of laminin. Plates can be used after a minimum of 2 h incubation at 37 &#176;C. Aspirate FN/laminin/PBS before use.</li>
</ol>
3. Maintenance of hPSC culture
NOTE: All cell incubation steps are at 5% CO2 and at 37 &#176;C in a humidified incubator.
<ol>
	<li>Aspirate hPSC medium from the hPSC culture and add fresh medium (200 &#956;L per cm2 of well surface area) every other day until colonies are ~80% confluent.</li>
	<li>To passage, aspirate hPSC medium and wash cells with 100 &#956;L of PBS per cm2 of well surface area. 
	NOTE: It is important to use Ca2+ and Mg2+ free PBS to wash the cells.</li>
	<li>Add 500 &#956;M EDTA (100 &#956;L per cm2 of well surface area). Replace the lid of the plate. Watch through an inverted microscope and monitor the cells for detachment of colony edges (2-4 min).</li>
	<li>Use a 5 or 10 mL serological pipette to transfer the same volume of hPSC medium into each well and mechanically harvest the cells by pipetting up and down a few times. 
	NOTE: Optimal EDTA incubation time depends on the iPSC line. Avoid vigorous pipetting for detaching colonies as it can lead to excessive colony dissociation and cell death. When passaging to start a differentiation plate, incubate cells with EDTA for 1-2 min longer.</li>
	<li>Transfer the cell suspension into a 15 mL conical tube. Spin the cells (2 min, 290 x g, 20-25 &#176;C) and carefully discard the supernatant.</li>
	<li>Resuspend the cells in an appropriate volume of hPSC medium and transfer to wells of a new basement membrane matrix coated plate. 
	NOTE: For regular hPSC maintenance use a 1:18 - 1:24 passaging ratio (1:18 means transferring one third of the cells dissociated from a single well of a 6-well plate into a full new plate). To start a differentiation plate, follow a 5:6 passaging ratio.</li>
	<li>Incubate at 5% CO2 and 37 &#176;C.</li>
</ol>
4. Neural crest induction
NOTE: This step is schematically represented in Figure 1A. Begin NC differentiation when cells are about 80% confluent. This will be achieved within 1-2 days when cells are passaged at a 5:6 ratio (see above). Start NC differentiation on day 0 with pluripotent and fully undifferentiated hPSC cells. For a high efficiency, the colony borders should have minimal differentiating cells. Passage and plate hPSC for differentiation when colonies are large, or the center of the colony starts to thicken/darken when monitored using an inverted microscope. Paying attention to the confluency and morphology tends to be more reliable than the number of plated cells as this might vary depending on the cell line and can vary from 50,000-200,000 per cm2.
<ol>
	<li>On day 0, replace hPSC maintenance medium with Medium A containing 1 ng/mL BMP4, 10 &#956;M SB431542, 600 nM CHIR99021 (200 &#956;L of medium per cm2 of well surface area).</li>
	<li>On day 2, cells should be 100% confluent. Gently aspirate spent Medium A and feed cells with Medium B containing 10 &#956;M SB431542, 1.5 &#956;M CHIR99021 (200 &#956;L of medium per cm2 of well surface area). 
	NOTE: As cultures grow during the NC induction, cells may detach from the underlying monolayer. Avoid excess cell loss by gently removing old media and adding fresh media to the side of the well.</li>
	<li>On day 4, feed cells again with Medium B (200 &#956;L of medium per cm2 of well surface area).</li>
	<li>On day 6, replace medium B with Medium C containing 10 &#956;M SB431542, 1.5 &#956;M CHIR99021 and 1 &#956;M retinoic acid (400 &#956;L of medium per cm2 of well surface area).</li>
	<li>On days 8 and 10, feed as day 6.</li>
</ol>
5. ENC spheroid formation
NOTE: This step is schematically represented in Figure 1B.
<ol>
	<li>On day 12, gently aspirate Medium C and wash cells with 100 &#956;L of PBS per cm2 of well surface area. Add the same volume of cell detachment solution and incubate for 30 min at 5% CO2 and 37 &#176;C.</li>
	<li>Dilute cell detachment solution by adding the same volume of NCC medium containing 10 ng/mL FGF2, 3 &#956;M CHIR99021, 10 &#956;L/mL N2 supplement, 20 &#956;L/mL B27 supplement, 10 &#956;L/mL L-glutamine supplement, and 10 &#956;L/mL MEM NEAA. Using a serological pipette harvest the single cell suspension and add it to a 15 mL conical tube.</li>
	<li>Spin the cells (2 min, 290 x g, 20-25 &#176;C) and carefully discard the supernatant.</li>
	<li>Using a 10 mL serological pipette, add 12 mL (for a full 6-well plate) of NCC medium and resuspend the cells completely by mechanically pipetting up and down 1-2 times. Transfer the cell suspension to an ultra-low attachment plate. 
	NOTE: Approximately 10 cm2 of ENC monolayer cells are resuspended in 2 mL of NCC medium and transferred to one well of an ultra-low attachment plate. This equals to a full 6-well NC induction plate transferred into a full 6-well ultra-low attachment plate.</li>
	<li>On day 14, gently swirl the plates until small spheroids are gathered in the center of each well. Using a P1000 micropipette and in a slow circular motion, aspirate the spent NCC medium around the circumference of each well. Try to avoid removing the free-floating spheroids.</li>
	<li>Feed the cells with the original volume of NCC medium.</li>
</ol>
6. EN induction
NOTE: This step is schematically represented in Figure 1B.
<ol>
	<li>On day 15, apply the same technique used on day 14 to gently remove the medium and wash the spheroids with PBS. Remove as much PBS as possible avoiding the spheroids.</li>
	<li>Add cell detachment solution (100 &#956;L per cm2 of well surface area) and dissociate the spheroids into single cells by incubating for 30 min at 5% CO2 and 37 &#176;C.</li>
	<li>Using a 10 mL serological pipette, add an equal volume of ENC medium (containing 10 ng/mL GDNF, 100 &#956;M ascorbic acid, 10 &#956;L/mL N2 supplement, 20 &#956;L/mL B27 supplement, 10 &#956;L/mL L-glutamine supplement, and 10 &#956;L/mL MEM NEAA in neurobasal medium) to each well and break the remaining spheroids by 2-3 rounds of mechanical pipetting. Transfer the cells to a 50 mL conical tube. 
	NOTE: Avoid excessive sheer stress and cell death that can occur from forced pipetting or use of pipettes with smaller tip opening.</li>
	<li>Spin the cells (2 min, 290 x g, 20-25 &#176;C) and carefully discard the supernatant. Using a 10 mL serological pipette, add ~ 5-10 mL of ENC medium and resuspend the cells completely by pipetting up and down 1-2 times.</li>
	<li>Count the concentration of viable cells using trypan blue and a cell counting method such as a hemocytometer. 
	CAUTION: Trypan blue is a suspected carcinogen. Handle with care, dispose appropriately.</li>
	<li>Add enough volume of ENC medium aiming to plate approximately 300,000-400,000 viable cells per cm2 of surface area at a density of about ~ 1,000,000 cells per mL. Aspirate FN/laminin/PBS solution from wells of the plate. 
	NOTE: Choose the plate format according to the assays that are planned for the EN cultures as this stage marks the final re-plating step of the protocol. Enteric neuron progenitors can be frozen on day 15. After dissociating the spheres with cell detachment solution, resuspend the progenitors at approximately 5 x 106 cells/mL in freezing medium such as 10% DMSO or Stem-cellbanker. When needed, thaw in warm ENC media with 5 &#956;M Y-27632 ROCK inhibitor. Plate cells in the desired plate format. Replace the media with fresh ENC after a couple of hours.</li>
	<li>Gently add the cells into the PO/FN/laminin coated wells. Avoid bubbles getting trapped under the cell suspension preventing cells attachment to the PO/FN/laminin coating, especially when using plates with smaller well size such as 384-well plates. This could lead to cell death.</li>
	<li>Feed cells every other day with ENC medium (200 &#956;L per cm2 of well surface area) until day 30-40, after which reduce the feeding frequency (to once or twice a week) but double the feeding volume (to 400 &#956;L of medium per cm2 of well surface area). 
	NOTE: This is important as excessive removing and adding media could facilitate detachment of neuronal culture from the surface of the wells. To prospectively prevent detachment especially for longer-term cultures, supplement ENC with FN (2 &#181;g/mL) and laminin (2 &#181;g/mL) once a week.</li>
</ol>

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 authors have nothing to disclose.

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

Research

JoVE Journal

Neuroscience

Formation of Neural Crests from Human Pluripotent Stem Cells for Enteric Nervous System Research

To induce neural crest formation, replace the maintenance medium of the human pluripotent stem cell culture with 200 microliters of medium A per square centimeter of the well-surface. Incubate the culture under 5%carbon dioxide supplementation at 37 degrees Celsius for two days. Gently aspirate the medium from the well-containing 100%confluence cells then pipette about 200 microliters of medium B per square centimeter of the well-surface area.On day four, feed the cells with 200 microliters of medium B per square centimeter of the well-surface. Incubate the cells for two more days, then replace the medium B with 400 microliters per square centimeter of medium C.

Differentiation of Enteric Neural Crest Spheroid and Neuronal Induction from Human Pluripotent Stem Cells for Enteric Nervous System Research

To begin, take 12-day cultures of human pluripotent stem cells that have been subjected to neural crest induction. Gently aspirate medium C from the cells. Add 100 microliters of PBS per square centimeter of well surface area to wash the cells, and then remove the PBS.Add an equal volume of cell detachment solution to the cells and incubate for 30 minutes under 5%carbon dioxide supplementation at 37 degrees Celsius. Add an equal volume of NCC medium into the plate. Then, use a serological pipette to harvest the cell suspension and transfer it to a 15-milliliter conical tube.Spin the cells at 290G for two minutes between 20 to 25 degrees Celsius. Carefully discard the supernatant without disturbing the cell pellet. Use a 10-milliliter serological pipette to pipette 12 milliliters of NCC medium to the cell pellet.Mechanically pipette the suspension up and down to create a suspension. Then, transfer the cell suspension to an ultra low attachment plate. On day 14, gently swirl the cell culture plates to gather small spheroids at the center of each well.Use a P1000 micro pipette to slowly aspirate the spent medium from the circumference of each well. Refeed the cells with the original volume of NCC medium. On day 15, carefully remove the medium.Add PBS to the wells to wash the spheroids. Then, remove as much PBS as possible, ensuring spheroids are not disturbed. Next, add 100 microliters of cell detachment solution per square centimeter of well surface area.Incubate the cells in the solution for 30 minutes under 5%carbon dioxide at 37 degrees Celsius. With a 10-milliliter serological pipette, add an equal volume of ENC medium to each well. Mechanically pipette the solution to break any remaining spheroids.Then transfer the cells to a 50-milliliter conical tube. Discard the supernatant after centrifuging as before. Then, resuspend the cells in 5 to 10 milliliters of ENC medium.Count the concentration of viable cells using trypan blue and hemocytometer. Now, add enough volume of ENC medium to plate about 300, 000 to 400, 000 viable cells per square centimeter of surface area, and a density of about 1 million cells per milliliter. Then, aspirate the solutions from the wells.Next, add the cells to a plate with PO/FN laminin coated wells. Feed cells every other day with 200 microliters of ENC medium per square centimeter of well surface area until days 30 to 40. High quality free floating spheres with smooth surfaces were observed after neural crest induction.The spheroids expressed the neural crest marker SOX10. Neurites were observed between days 25 to 30 during the enteric neuron induction.

845 Views.  University of California, San Francisco.  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.

Derivation of Enteric Neurons from Human Pluripotent Stem Cells

Summary