Induced pluripotent stem cells differentiate into intestinal organoids in three-dimensional niche in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND: Induced pluripotent stem cells (iPSCs) are a special type of cells with self-renewal and multi-differentiation potential, which can differentiate into intestinal organoids under certain conditions. OBJECTIVE: To explore whether iPSCs can differentiate into intestinal organoids under specific conditions in vitro.METHODS: iPSCs from B6J mice were recovered and cultured for 3 days until clone units covered about 80％ of the culture dish, and then the cells were cultured in the medium containing Activin A for 3 days until the deterministic endoderm formed. Further, the culture medium was replaced by the medium with fibroblast growth factor 4 and Wnt3A for 4 days to differentiate into the spheroids with CDX2+. After that, spheroids were collected and mixed with Matrigel,and then the mixture was dropped into the 4-well plate and cultured with Rspondin1, Noggin, epidermal growth factor, B27 and other growth factors to differentiate into intestinal organoids. Cell morphology was observed, FoxA2 and Sox17 expresson in the deterministic endoderm was detected, and CDX2, Sox9, CGA, MMP7 were measured.RESULTS AND CONCLUSION: iPSCs were cultured with Activin A for 3 days with higher cell fusion, initial differentiation and FoxA2/Sox17 expression (P < 0.05) than those of non-induced iPSCs. Spheroids began to appear at the 3rd day after culture with fibroblast growth factor 4 and WNT3A, and formed a lot at the 4th day. And CDX2 expression in spheroids was significantly increased compared with that in the deterministic endoderm (P < 0.05). Organoids gradually formed after 3 days culture, which contained all cell types of intestinal organoids, and expressions of specific markers, Sox9, CGA, MMP7, were significantly higher than those in spheroids (P < 0.05). To conclude, iPSCs can be induced to differentiate into intestinal organoids in three-dimensional niche in vitro.