Abstract
Efficient definitive endoderm (DE) differentiation is essential for the generation of hepatocytes from human embryonic stem cells (hESCs), which are in demand for drug development and clinical application. However, this has been hindered as signaling pathways regulating the differentiation remain unclear. Therefore, understanding the signaling pathways controlling DE formation is important for differentiation of hESCs to hepatic lineages. Generation of DE through the initial formation of mesendoderm is activin-dependent differentiation. Further induction by higher concentration of Activin A gives rise to DE, whereas lower concentration favors mesoderm lineage. On the other hand, phosphatidylinositol-3-kinase (PI3K) signaling, which is important for ESCs self-renew were shown to be potent inhibitors of the differentiation. It has been shown that supression of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then DE in conditioned medium. However, the differentiation efficiency and molecular mechanism underlying the mesendoderm and DE differentiation cannot be clearly addressed in this culture condition. Therefore, in this study, we have used chemically defined medium to assess differentiation potential by supression of PI3K in combination with Activin A. In this study, we demonstrate that suppression of PI3K signal in H1 hESCs causes downregulation of pluripotency marker (Oct4 and Sox2) and induces mesendoderm/endoderm gene expression (Brachyury, Gata 6 and FoxA2). Treatment of hESCs with combined Activin A (100 ng/ml) and PI3K inhibitor, Ly 294002 (20 μM) increased expression of mesendoderm and endoderm markers (Brachury, GSC, FOxA2, Sox17 and Wnt3a) several fold higher than that of Activin A (100 ng/ml) alone. As a result of improved DE formation, combined treatments also improved hepatocytes production, exhibited higher level of hepatic gene expression and increased Cyp3A4 activity (four-fold higher) than those derived from the treatment with activin alone. These results suggest that repression of PI3K is necessary for efficient DE differentiation and subsequently for the hepatocyte generation.
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