Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, United States.
Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, United States.
Methods Cell Biol. 2020;159:201-227. doi: 10.1016/bs.mcb.2020.03.001. Epub 2020 Apr 8.
Advances in human pluripotent stem cell (hPSC) biology now allow the generation of organoids that resemble different regions of the gastrointestinal tract. Generation of region-specific organoids has been facilitated by developmental biology studies carried out in model organisms such as mouse, frog and chick. By mimicking embryonic development, hPSC-derived human colonic organoids (HCOs) can be generated through a stepwise differentiation, first into definitive endoderm (DE), then into mid/hindgut spheroids which are then patterned into posterior gut tissue which gives rise to HCOs following prolonged in vitro culture. HCOs undergo transitions similar to those observed in the developing colon of model organisms and human embryos. HCOs develop into tissue that resembles fetal colon on the basis of morphology, gene expression and presence of differentiated cell types. Generation of HCOs without the proper training or expertise can be a daunting task. Here, we describe a detailed protocol for differentiating hPSCs into HCOs, we include suggestions for troubleshooting these differentiations, and we discuss experimental design considerations. We have also highlighted the key advantages and limitations of the system.
人类多能干细胞（hPSC）生物学的进展现在允许生成类似于胃肠道不同区域的类器官。通过在模型生物（如鼠、蛙和鸡）中进行发育生物学研究，促进了区域特异性类器官的生成。通过模拟胚胎发育，可以通过逐步分化将 hPSC 衍生的人类结肠类器官（HCO）生成，首先分化为确定内胚层（DE），然后分化为中/后肠球体，然后将其图案化为后肠组织，经过长时间的体外培养后产生 HCO。HCO 经历与模型生物和人类胚胎发育过程中观察到的相似的转变。HCO 根据形态、基因表达和分化细胞类型的存在发育成类似于胎儿结肠的组织。没有适当的培训或专业知识，生成 HCO 可能是一项艰巨的任务。在这里，我们描述了将 hPSC 分化为 HCO 的详细方案，我们包括了对这些分化进行故障排除的建议，并讨论了实验设计的考虑因素。我们还强调了该系统的关键优点和局限性。
