PDF(Pubmed)
Abstract：
Intestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture.
We established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells\' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies.
The proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture.
The high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.
We established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells\' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies.
The proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture.
The high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.
摘要：
背景:来源于人多能干细胞(hPSC)的肠上皮细胞通常作为类器官在体外维持和培养,因为它们在培养时不表现出粘附。然而,类器官的三维结构使得它们在再生医学和药物发现中的使用变得困难。间充质基质细胞在体内肠干细胞附近发现，并提供营养因子来调节干细胞的维持和增殖。如BMP抑制剂，WNT,还有R-spondin.在这项研究中,我们的目的是使用从hPSC衍生的肠道类器官中分离的间充质基质细胞来建立体外培养系统，该系统能够在粘附培养中稳定增殖和维持hPSC衍生的肠上皮细胞。
方法：我们建立了来自hPSC衍生的肠道类器官的肠上皮细胞和间充质基质细胞的分离方案以及这些细胞的共培养系统。然后我们评估肠上皮细胞和间充质基质细胞的形态，增殖能力，染色体稳定性,致瘤性，和基因表达谱。我们还评估了细胞在药代动力学和毒性研究中的有用性。
结果：增殖的肠上皮细胞呈柱状，微绒毛和糖萼的形成，细胞极性，以及药物代谢酶和转运蛋白的表达。肠上皮细胞也表现出屏障功能，转运活动，和药物代谢能力。值得注意的是,在没有间充质基质细胞的情况下，小肠上皮干细胞不能贴壁培养，也不能被其他饲养细胞替代。类器官来源的间充质基质细胞类似于维持小肠上皮干细胞所必需的滋养细胞，并在贴壁培养中起关键作用。
结论：高增殖扩张，生产力,hPSC来源的肠上皮细胞的功能可能在药代动力学和毒性研究以及再生医学中具有潜在的应用。
方法：我们建立了来自hPSC衍生的肠道类器官的肠上皮细胞和间充质基质细胞的分离方案以及这些细胞的共培养系统。然后我们评估肠上皮细胞和间充质基质细胞的形态，增殖能力，染色体稳定性,致瘤性，和基因表达谱。我们还评估了细胞在药代动力学和毒性研究中的有用性。
结果：增殖的肠上皮细胞呈柱状，微绒毛和糖萼的形成，细胞极性，以及药物代谢酶和转运蛋白的表达。肠上皮细胞也表现出屏障功能，转运活动，和药物代谢能力。值得注意的是,在没有间充质基质细胞的情况下，小肠上皮干细胞不能贴壁培养，也不能被其他饲养细胞替代。类器官来源的间充质基质细胞类似于维持小肠上皮干细胞所必需的滋养细胞，并在贴壁培养中起关键作用。
结论：高增殖扩张，生产力,hPSC来源的肠上皮细胞的功能可能在药代动力学和毒性研究以及再生医学中具有潜在的应用。
