Redefining definitive endoderm subtypes by robust induction of human induced pluripotent stem cells

- Definitive endoderm (DE) cells contain three subtypes.
- Anterior DE cells are divided into anterior and posterior domains.
- We induced hiPSCs into posterior DE through late anterior primitive streak.
- Three DE subtypes differentiate into anterior and posterior foreguts and midhindgut.

Many reports have described methods that induce definitive endoderm (DE) cells from human pluripotent stem cells (hPSCs). However, it is unclear whether the differentiation propensity of these DE cells is uniform. This uncertainty is due to the different developmental stages that give rise to anterior and posterior DE from anterior primitive streak (APS). Therefore, these DE cell populations might be generated from the different stages of APS cells, which affect the DE cell differentiation potential. Here, we succeeded in selectively differentiating early and late APS cells from human induced pluripotent stem cells (hiPSCs) using different concentrations of CHIR99021, a small molecule Wnt/β-catenin pathway activator. We also established novel differentiation systems from hiPSCs into three types of DE cells: anterior and posterior domains of anterior DE cells through early APS cells and posterior DE cells through late APS cells. These different DE cell populations could differentiate into distinct endodermal lineages in vitro, such as lung, liver or small intestine progenitors. These results indicate that different APS cells can produce distinct types of DE cells that have proper developmental potency and suggest a method to evaluate the quality of endodermal cell induction from hPSCs.