这项工作得到了 UCSF 突破性生物医学研究和桑德勒基金会的资助，March of Dimes 资助号 1-FY18-394 和 1DP2NS116769-01，NIH 主任新创新者奖 （DP2NS116769） 给 F.F. 和国家糖尿病、消化和肾脏疾病研究所 （R01DK121169） 给 F.F.，HM 得到了 Larry L. Hillblom 基金会博士后奖学金的支持， NIH T32-DK007418 奖学金和 UCSF 突破性生物医学研究计划独立博士后奖学金。

1. 培养基制备
注意：整个方案中提到的浓度是培养基成分的最终浓度。在无菌条件下在层流罩中准备所有培养基，并在 4 °C 下避光储存。2 周内用完。
<ol><li>hPSC 维持培养基：将无饲养层的 hPSC 维持培养基补充剂 （20 μL/mL） 与其基础培养基混合。</li>
	<li>培养基 A：将骨形态发生蛋白 4 （BMP4;1 ng/mL）、SB431542 （10 μM） 和 CHIR99021 （600 nM） 添加到分化基础培养基中。</li>
	<li>培养基 B：向分化基础培养基中加入 SB431542 （10 μM） 和 CHIR99021 （1.5 μM）。</li>
	<li>培养基 C：向分化基础培养基中加入 SB431542 （10 μM）、CHIR99021 （1.5 μM） 和 RA （1 μM）。</li>
	<li>神经嵴完全 （NCC） 培养基：向神经基础培养基中加入 FGF2 （10 ng/mL）、CHIR99021 （3 μM）、N2 添加剂 （10 μL/mL）、B27 添加剂 （20 μL/mL）、L-谷氨酰胺添加剂 （10 μL/mL） 和 MEM NEAA （10 μL/mL）。</li>
	<li>ENC 培养基：向神经基础培养基中加入 GDNF （10 ng/mL）、抗坏血酸 （100 μM）、N2 添加剂 （10 μL/mL）、B27 添加剂 （20 μL/mL）、L-谷氨酰胺添加剂 （10 μL/mL） 和 MEM NEAA （10 μL/mL）。</li>
	<li>乙二胺四乙酸 （EDTA） 1x：在 PBS 中稀释 500 mM EDTA （1000x） 至终浓度为 500 μM。 注：除非另有说明，否则在整个方案中使用不含 Ca2+ 和 Mg2+ 的 PBS。</li>
</ol>2. 培养板的包被
<ol><li>基底膜基质包被板：快速解冻，并使用 50 mL 血清移液管在 50 mL 冷冻 DMEM：F12 中稀释 500 μL 冷冻等分试样的基底膜基质。用稀释的基底膜基质溶液（每 cm2 孔表面积 100 μL）包被孔，并在 37 °C 下孵育过夜。 使用前吸出涂层介质。为防止基底膜基质糊化，请尽快执行此步骤并使用冷 DMEM：F12 溶解冷冻等分试样。 注意：在解冻（冰上）和分装（冰上的试管）期间，基底膜基质温度应保持低温，以防止凝结。</li>
	<li>PO/FN/层粘连蛋白包被板：通过稀释 1000x 储备液，在 PBS 中制备 15 μg/mL 多鸟氨酸 （PO） 溶液。每 cm2 孔表面积用 100 μL PO/PBS 溶液包被孔，并将板在 37 °C 下孵育过夜。 第二天，吸出 PO/PBS，并以每 cm2 孔表面积 100 μL 的浓度包被孔，加入新鲜制备的含有 2 μg/mL 纤连蛋白 （FN） 和 2 μg/mL 层粘连蛋白的 FN/层粘连蛋白/PBS 溶液。在 37 °C 下孵育至少 2 小时后，可以使用板。 使用前吸出 FN/层粘连蛋白/PBS。</li>
</ol>3. 维持 hPSC 培养
注：所有细胞孵育步骤均在 5% CO2 和 37 °C 的加湿培养箱中进行。
<ol><li>从 hPSC 培养物中吸出 hPSC 培养基，并每隔一天加入新鲜培养基（每 cm2 孔表面积 200 μL），直到菌落达到 ~80% 汇合。</li>
	<li>传代时，吸出 hPSC 培养基，每 cm2 孔表面积用 100 μL PBS 洗涤细胞。 注：使用不含 Ca2+ 和 Mg2+ 的 PBS 洗涤细胞非常重要。</li>
	<li>添加 500 μM EDTA（每 cm2 孔表面积 100 μL）。装回板盖。通过倒置显微镜观察并监测细胞是否分离菌落边缘（2-4 分钟）。</li>
	<li>使用 5 或 10 mL 血清移液器将相同体积的 hPSC 培养基转移到每个孔中，并通过上下吹打几次机械收获细胞。 注：最佳 EDTA 孵育时间取决于 iPSC 细胞系。避免对分离的集落进行剧烈移液，因为这会导致过度的集落解离和细胞死亡。当传代以开始分化板时，将细胞与 EDTA 孵育 1-2 分钟以上。</li>
	<li>将细胞悬液转移到 15 mL 锥形管中。旋转细胞（2 分钟，290 x g ，20-25 °C）并小心弃去上清液。</li>
	<li>将细胞重悬于适当体积的 hPSC 培养基中，并转移到新的基底膜基质包被板的孔中。 注：对于常规的 hPSC 维持，请使用 1：18 - 1：24 的传代比例（1：18 意味着将从 6 孔板的单个孔中解离的细胞的三分之一转移到全新的板中）。要开始分化板，请遵循 5：6 的传代比例。</li>
	<li>在 5% CO2 和 37 °C 下孵育。</li>
</ol>4. 神经嵴诱导
注意：此步骤如图 1A 所示。当细胞汇合度约为 80% 时开始 NC 分化。当细胞以 5：6 的比例传代时，这将在 1-2 天内实现（见上文）。从第 0 天开始用多能和完全未分化的 hPSC 细胞进行 NC 分化。为了提高效率，集落边界应具有最少的分化细胞。当集落较大时，传代和接种 hPSC 以进行分化，或者使用倒置显微镜监测时集落中心开始增厚/变暗。关注汇合度和形态往往比铺板细胞的数量更可靠，因为这可能根据细胞系的不同而变化，并且可能在每厘米 50,000-200,000 个2 之间变化。
<ol><li>在第 0 天，用含有 1 ng/mL BMP4、10 μM SB431542、600 nM CHIR99021（每 cm2 孔表面积 200 μL 培养基）的培养基 A 替换 hPSC 维持培养基。</li>
	<li>第 2 天，细胞应 100% 汇合。轻轻吸出用过的培养基 A，并用含有 10 μM SB431542、1.5 μM CHIR99021（每 cm2 孔表面积 200 μL 培养基）的培养基 B 喂养细胞。 注：随着 NC 诱导期间培养物的生长，细胞可能会从下面的单层分离。通过轻轻去除旧培养基并将新鲜培养基添加到孔的侧面，避免过多的细胞损失。</li>
	<li>第 4 天，再次用培养基 B（每 cm2 孔表面积 200 μL 培养基）喂养细胞。</li>
	<li>第 6 天，用含有 10 μM SB431542、1.5 μM CHIR99021 和 1 μM 视黄酸（每 cm2 孔表面积 400 μL 培养基）的培养基 C 替换培养基 B。</li>
	<li>在第 8 天和第 10 天，按第 6 天饲喂。</li>
</ol>5. ENC 球体形成
注意：此步骤如图 1B 所示。
<ol><li>第 12 天，轻轻吸出培养基 C，并用每 cm2 孔表面积 100 μL PBS 洗涤细胞。加入相同体积的细胞分离溶液，并在 5% CO2 和 37 °C 下孵育 30 分钟。</li>
	<li>通过添加相同体积的 NCC 培养基来稀释细胞分离溶液，该培养基含有 10 ng/mL FGF2、3 μM CHIR99021、10 μL/mL N2 添加剂、20 μL/mL B27 添加剂、10 μL/mL L-谷氨酰胺添加剂和 10 μL/mL MEM NEAA。使用血清移液管收获单细胞悬液，并将其添加到 15 mL 锥形管中。</li>
	<li>旋转细胞（2 分钟，290 x g ，20-25 °C）并小心弃去上清液。</li>
	<li>使用 10 mL 血清移液管，加入 12 mL（用于完整的 6 孔板）NCC 培养基，并通过机械上下吹打 1-2 次来完全重悬细胞。将细胞悬液转移到超低贴壁板上。 注：将大约 10 cm2 的 ENC 单层细胞重悬于 2 mL NCC 培养基中，并转移至超低吸附板的一个孔中。这相当于将完整的 6 孔 NC 诱导板转移到完整的 6 孔超低连接板中。</li>
	<li>第 14 天，轻轻旋转板，直到小球体聚集在每个孔的中心。使用 P1000 微量移液器以慢速圆周运动，在每个孔的圆周上吸出用过的 NCC 培养基。尽量避免删除自由浮动的椭球体。</li>
	<li>用原始体积的 NCC 培养基喂养细胞。</li>
</ol>6. EN 感应
注意：此步骤如图 1B 所示。
<ol><li>在第 15 天，应用与第 14 天相同的技术，轻轻去除培养基并用 PBS 洗涤球体。尽可能多地去除 PBS，避免球状体。</li>
	<li>加入细胞分离溶液（每 cm2 孔表面积 100 μL），并在 5% CO2 和 37 °C 下孵育 30 分钟，将球体解离成单细胞。</li>
	<li>使用 10 mL 血清移液器，向每个孔中加入等体积的 ENC 培养基（含有 10 ng/mL GDNF、100 μM 抗坏血酸、10 μL/mL N2 补充剂、20 μL/mL B27 补充剂、10 μL/mL L-谷氨酰胺补充剂和 10 μL/mL MEM NEAA）到每个孔中，并通过 2-3 轮机械移液打破剩余的球体。将细胞转移至 50 mL 锥形管中。 注：避免因强制移液或使用吸头开口较小的移液器而可能导致的过度剪切应力和细胞死亡。</li>
	<li>旋转细胞（2 分钟，290 x g ，20-25 °C）并小心弃去上清液。使用 10 mL 血清移液管，加入 ~ 5-10 mL ENC 培养基，并通过上下吹打 1-2 次完全重悬细胞。</li>
	<li>使用台盼蓝和细胞计数方法（如血细胞计数器）计数活细胞的浓度。 注意：台盼蓝是一种可疑的致癌物。小心处理，妥善处理。</li>
	<li>添加足够体积的 ENC 培养基，以每 cm2 表面积接种约 300,000-400,000 个活细胞，密度约为 ~ 1,000,000 个细胞/mL。从板的孔中吸出 FN/层粘连蛋白/PBS 溶液。 注意：根据计划用于 EN 培养的测定选择板格式，因为此阶段标志着方案的最后重新接种步骤。肠道神经元祖细胞可以在第 15 天冷冻。用细胞分离溶液解离球体后，在 10% DMSO 或 Stem-cellbanker 等冷冻培养基中以大约 5 x 106 个细胞/mL 的比例重悬祖细胞。需要时，在含有 5 μM Y-27632 ROCK 抑制剂的温热 ENC 培养基中解冻。以所需的板格式对细胞进行板板处理。几个小时后，用新的 ENC 更换介质。</li>
	<li>轻轻地将细胞加入 PO/FN/层粘连蛋白包被的孔中。避免气泡滞留在细胞悬液下，防止细胞附着在 PO/FN/层粘连蛋白包被上，尤其是在使用较小孔尺寸的板（如 384 孔板）时。这可能导致细胞死亡。</li>
	<li>每隔一天用 ENC 培养基（每厘米 2 孔表面积 200 μL）喂养细胞，直到第 30-40 天，之后减少喂食频率（至每周一次或两次）但将喂食量加倍（至每 cm2 孔表面积 400 μL 培养基）。 注意：这很重要，因为过量的去除和添加培养基会促进神经元培养物从孔表面分离。为了前瞻性地防止脱离，特别是对于长期培养，每周一次用 FN （2 μg/mL） 和 laminin （2 μg/mL） 补充 ENC。</li>
</ol>

从人多能干细胞衍生肠道神经元

<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>

作者没有什么可披露的。

此处描述的分化方案提供了一种强大的体外方法，可在 30-40 天内从 hPSC 获得肠道神经元（图 1E）和在较老的培养物中表达神经胶质纤维酸性蛋白、GFAP 和 SOX10 的肠胶质细胞（>第 55 天）13、14、19、22。这些神经元和神经胶质细胞是通过 hPSC 逐步分化为迷走神经和肠神经嵴?...

肠神经系统 （ENS） 是周围神经系统中最大的组成部分。ENS 包含超过 4 亿个神经元，这些神经元位于胃肠道内，几乎控制着肠道的所有功能1。对肠道神经病中 ENS 发育和功能及其缺陷的分子理解需要获得可靠和真实的肠道神经元来源。对人体原代组织的访问是有限的，动物模型无法概括许多肠道神经病中的关键疾病表型。事实证明，人类多能干细胞 （hPSC） 技术在提供所需细胞类型的无限来源方面非常有益，尤其是那些难以从主要来源中分离的细胞类型 2,3,4,5,6,7。在这里，我们提供了从 hPSC 获得 ENS 培养物的逐步和稳健的体外方法的详细信息。这些可扩展的 hPSC 衍生培养物为发育研究、疾病建模和高通量药物筛选开辟了途径，并且可以为再生医学提供可移植细胞。
肠道神经元谱系来源于胚胎发生过程中遵循 ENS 发育路径的神经嵴 （NC） 细胞。在胚胎中，NC 细胞沿着折叠神经板的边缘出现。它们增殖、迁移并产生许多不同的细胞类型，包括感觉神经元、雪旺细胞、黑色素细胞、颅面骨骼和肠道神经元以及神经胶质细胞 8,9,10。细胞命运的决定取决于 NC 细胞出现的前后轴的不同区域，即颅 NC、迷走神经 NC、躯干 NC 和骶部 NC。ENS 由迷走神经和骶部 NC 细胞发展而来，前者由于沿肠道长度的广泛迁移并定植于肠道，因此在肠道神经元群中占主导地位11。
从 PSC 衍生的 NC 细胞通常涉及双重 SMAD 抑制和 WNT 通路激活的组合12,13。直到最近，所有 NC 诱导方案都涉及培养条件下的血清和其他动物产品添加剂。化学上未定义的培养基不仅降低了 NC 诱导的可重复性，而且还挑战了机制发育研究。为了克服这些挑战，Barber 等人14 使用化学定义的培养条件开发了一种 NC 诱导方案，因此优于依赖血清替代因子（例如 KSR）的替代方法13,14。这是通过基础培养基更换和优化最初由 Fattahi 等人提出的从 hPSC 衍生肠道 NC 细胞的方案获得的。这种改进的系统是此处介绍的 hPSC 分化方案的基础。它首先通过在视黄酸 （RA） 之外精确调节 BMP、FGF、WNT 和 TGFβ 信号转导，在 15 天内诱导肠神经嵴 （ENC） 细胞。然后，我们通过用神经胶质细胞系衍生的神经营养因子 （GDNF） 处理细胞来获得 ENS 谱系。所有培养基成分均经过化学成分确定，可在 30-40 天内产生稳健的肠道神经元培养物。
除了此处描述的单层细胞培养系统外，还开发了使用自由浮动胚状体的替代 NC 诱导方法15,16。这些培养物中的迁移细胞已被证明表达 NC 标志物，其中亚群代表迷走神经 NC。与原代肠道组织共培养物已用于富集这些培养物中的肠道 NC 前体。这些研究中的培养基成分包含不同因素的组合，例如神经生长因子、NT3 和脑源性神经营养因子。在这个阶段，尚不完全清楚这些因素如何影响肠道神经元前体定型。为了有效比较单层和基于胚状体的培养物中的肠道 NC 诱导，需要更多数据。鉴于这些策略中的不同培养布局，应考虑每种方法的使用，并根据具体应用进行优化。
此处介绍的方案是可重复的，并且已由我们和其他人使用不同的 hPSC（诱导和胚胎）细胞系成功测试8,14,16。

<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

人类肠道神经系统 （ENS） 是一个由神经胶质和神经元细胞类型组成的大型网络，具有显著的神经递质多样性。ENS 控制肠道蠕动、酶分泌和营养吸收，并与免疫系统和肠道微生物组相互作用。因此，ENS 的发育和获得性缺陷是导致许多人类疾病的原因，并可能导致帕金森病的症状。动物模型系统和对原代组织的访问的限制对人类 ENS 的研究构成了重大的实验挑战。在这里，提出了一个详细的方案，用于使用确定的培养条件从人多能干细胞 hPSC 中有效 体外衍生 ENS 谱系。我们的方案从 hPSC 在 15 天内定向分化为肠神经嵴细胞开始，并在 30 天内产生多种功能性肠道神经元亚型。该平台为发育研究、疾病建模、药物发现和再生应用提供了可扩展的资源。

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.

作者聚焦：来自人多能干细胞的人肠道神经元的稳健培养

肠神经系统谱系与人多能干细胞的分化

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.

该方案提供了一种使用化学定义的培养条件从 hPSC 获得肠神经嵴和肠神经元的方法（图 1A-B）。产生高质量的神经元取决于有效的肠道神经嵴诱导步骤。这可以通过检查自由浮动球体的形态来目视评估，这些球体应该看起来是圆形的，表面光滑，尺寸约为 0.1 - 0.4 毫米，如图 1C 所示。这些球体还应表达神经嵴标志物 SRY-Box 转录因子 10 （SOX10...

从人类多能干细胞 （hPSC） 中提取肠道神经系统 （ENS） 谱系为研究 ENS 发展和疾病以及用于再生医学提供了可扩展的细胞来源。在这里，提出了使用化学定义的培养条件从 hPSC 获得肠道神经元的详细 体外 方案。

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

在这里，我们提供了一种在完全定义的条件下从人类多能干细胞产生人类肠道神经元的方法，我们使用这些细胞来了解人类肠道神经系统的发育和功能。所以我认为人类肠道神经系统的发育、生理学和病理生理学一直是一个巨大的挑战，这是因为肠道神经系统细胞的稀有性和神经嵴祖细胞的短暂性，以及难以进入组织和分离组织。这是一个从 HPSC 生成真实且功能性人类肠道神经元的强大平台，通过克服组织通路方面长期存在的技术挑战，它为疾病建模、药物发现以及研究 ENS 的发展和功能提供了肠道神经系统成分的可扩展培养物。

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

Research

JoVE Journal

Neuroscience

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

845 次观看.  University of California, San Francisco. 在这里，我们提供了一种在完全定义的条件下从人类多能干细胞产生人类肠道神经元的方法，我们使用这些细胞来了解人类肠道神经系统的发育和功能。所以我认为人类肠道神经系统的发育、生理学和病理生理学一直是一个巨大的挑战，这是因为肠道神经系统细胞的稀有性和神经嵴祖细胞的短暂性，以及难以进入组织和分离组织。这是一个从 HPSC 生成真实且功能性人类肠道?...

视频: 作者聚焦：来自人多能干细胞的人肠道神经元的稳健培养

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.

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.

科研

神经科学

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.

从人类多能干细胞形成神经嵴用于肠道神经系统研究

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.