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

Research

JoVE Journal

Neuroscience

695 ビュー.  University of California, San Francisco. ここでは、ヒト多能性幹細胞からヒト腸管ニューロンを全条件で作製する方法を提供し、これらの細胞を用いてヒト腸神経系の発達と機能を理解するために用いる。ですから、ヒトの腸管神経系の発達、生理学、病態生理学は大きな課題であり、それは腸神経系細胞の希少性や神経堤前駆細胞の一時的な性質、組織へのアクセスや組織の分離の難しさによるものだと思?...

ビデオ: 著者スポットライト:ヒト多能性幹細胞からのヒト腸内ニューロンの堅牢な培養

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...

https://cloudfront.jove.com/CDNSource/teasers/66133_b.jpg

the specification of telencephalic glutamatergic neurons from human pluripotent stem cells

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and...

replating differentiated neurons derived from human pluripotent stem cells

The video showcases a method to detach and replant human pluripotent stem cell-derived neurons using a mild proteolytic enzyme. After filtration and centrifugation, the neurons are replated on a coated multi-well plate for incubation, promoting attachment and...

Replating Differentiated Neurons Derived from Human Pluripotent Stem Cells

Apply at least 1 milliliter of proteolytic enzyme per 10-centimeter plate, and return the cells to the incubator for 40 to 45 minutes. During the incubation, check neurons on a phase-contrast microscope, and continue the protease treatment until the neural network completely detaches from the plate and starts to break apart. When the neurons have detached, stop digestion by adding 5 milliliters of fresh DMEM media for every 1 milliliter of...

generating postganglionic sympathetic neurons from human pluripotent stem cells

This video demonstrates an in vitro method to generate postganglionic sympathetic neurons from human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural crest cells (NCCs) on a basement membrane matrix support using a differentiation medium. The NCCs are then dissociated and formed into spheroids using another differentiation medium. Finally, the spheroids are transformed into postganglionic sympathetic neurons using a sympathetic neuron...

Generating Postganglionic Sympathetic Neurons from Human Pluripotent Stem Cells

After the second wash, treat the cells with seven milliliters of 0.5 millimolar EDTA for 15 minutes in the cell culture incubator, before aspirating the floating human pluripotent stem cells and transferring the harvested cells into a 50-milliliter...

formation of neural crests from human pluripotent stem cells for enteric nervous system research

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

co-culturing microglia and cortical neurons differentiated from human induced pluripotent stem cells

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and...

synaptic microcircuit modeling with 3d cocultures of astrocytes and neurons from human pluripotent stem cells

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array...

application of live mitochondria staining in cell-sorting to purify hepatocytes derived from human induced pluripotent stem cells

Author Spotlight: Advancing Hepatocyte Purification from Human Induced Pluripotent Stem Cells for Regenerative Medicine

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166)...

Purification of Human iPSC-Derived Hepatocytes by Mitochondrial-ALCAM Sorting

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three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem...

efficient derivation of human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small molecule induction

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural...

differentiation of enteric neural crest spheroid and neuronal induction from human pluripotent stem cells for enteric nervous system research

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

generation of spheroids from human pluripotent stem cells

This video demonstrates the transformation of human pluripotent stem cells (hPSCs) into spherical brain organoids. By using specific inhibitors, the hPSCs are guided to differentiate towards a neuronal lineage, forming neuroectodermal colonies. Special growth factors are then introduced, which encourage the self-organization and proliferation of the colonies, ultimately leading to the formation of spherical brain...

Generation of Spheroids from Human Pluripotent Stem Cells

generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s...

differentiating engineered human induced pluripotent stem cells into functional motor neurons

This video showcases the process of differentiating human iPSCs into motor neurons. The rationale involves utilizing iPSCs that have been genetically modified to express specific transcription factors. When cultured in a media containing doxycycline, this antibiotic triggers the expression of transcription factors, thereby promoting iPSC differentiation into motor neuron progenitors. Ultimately, the progenitors are cultured in a neuronal medium to yield mature and functional motor...

Differentiating Engineered Human Induced Pluripotent Stem Cells into Functional Motor Neurons

directed dopaminergic neuron differentiation from human pluripotent stem cells

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s...

rapid and efficient generation of neurons from human pluripotent stem cells in a multititre plate format

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled...

an automated culture system for maintaining and differentiating human-induced pluripotent stem cells

Author Spotlight: Automating iPSC Culture for Enhanced Reproducibility 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of...

Induced Pluripotent Stem Cell Differentiation Using an Automated Culture System

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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.

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.