Toyoda Taro, Kimura Azuma, Tanaka Hiromi, Osafune Kenji
Center for iPS Cell Research and Application (CiRA), Kyoto University;
Center for iPS Cell Research and Application (CiRA), Kyoto University.
J Vis Exp. 2019 Mar 27(145). doi: 10.3791/57641.
Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1 (PDX1) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1 cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1 cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17 definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1β and HNF4α, and eventual differentiation into PDX1 cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1 cells.
人多能干细胞（hPSC）来源的胰腺细胞是再生医学中很有前景的细胞来源，也是研究人类发育过程的一个平台。重现发育过程的逐步定向分化是生成包括胰腺/十二指肠同源盒蛋白1（PDX1）胰腺祖细胞在内的胰腺细胞的主要方法之一。传统方案在传代后不久就用小集落开始分化。然而，在集落或聚集体状态下，细胞容易出现异质性，这可能会阻碍向PDX1细胞的分化。在这里，我们展示了一个将hPSC分化为PDX1细胞的详细方案。该方案包括四个步骤，通过接种解离的单细胞开始分化。在诱导SOX17定形内胚层细胞后，会表达两种原始肠管标记物HNF1β和HNF4α，并最终分化为PDX1细胞。本方案操作简便，可能会提高和稳定一些以前发现向内胚层谱系或PDX1细胞分化效率低下的hPSC系的分化效率。
