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Objective To optimize the method of generating hepatocyte-like cells (HLCs) from pluripotent stem cells (PSCs)in vitro. Methods Considering the development of hepatocyte in vivo, a more efficient method was developed by optimizing biochemical factors to the stepwise differentiate PSCs into HLCs. Then the differentiation experiments were performed with human embryonic stem cell (hESC)line H9. The efficiency of the generation of HLCs was evaluated by morphological observation at different stages of differentiation. RT-qPCR and flow cytometry assay were used to examine the marker genes expression. Results A method of high-efficient hESCs differentiation to definitive endoderm (DE) was developed by adding cytokines activin A, bone morphogenetic protein 4(BMP4)and fibroblast growth factor 2(FGF2). It highly improved mRNA expression of marker genes at different stages. Venus fluorescence tracer and the results of flow-cytometry also showed that the population proportion of HLCs was significantly increased. Conclusions The high-efficient differentiation to DE improves differentiation efficiency of PSCs into HLCs.

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BACKGROUND: Similar to the human embryonic stem cells, human induced pluripotent stem cells possess the ability to differentiate into three germ layers and self-renewal capacity. It is of great clinical significance to generate functional hepatocytes from human induced pluripotent stem cells. OBJECTIVE: To establish a protocol for the directed differentiation into hepatocytes from human induced pluripotent stem cells. METHODS: Human induced pluripotent stem cells were characterized by a microscope and immunofluorescence. Pluripotent stem cells were differentiated into definitive endoderm, hepatic endoderm, hepatoblasts, and finally functional hepatocytes via sequential modulation of the signaling pathways using recombinant transcription factors. That was, activin A, fibroblast growth factor 4/bone morphogenetic protein 4, hepatocyte growth factor, and tumor suppressor M were added sequentially on days 0, 5, 10, and 15. The morphological features and molecular markers at different stages were dynamically observed by microscopy, real-time quantitative PCR, and ELISA. RESULTS AND CONCLUSION: (1) The human induced pluripotent stem cells exhibited the typical morphology of pluripotent stem cells and expressed highlevels of the induced pluripotent stem cells-specific markers, SSEA4, SOX2, and OCT4. (2) The human induced pluripotent stem cells were successfully induced sequentially into definitive endoderm cells, hepatoblasts, and hepatocytes. (3) Real-time quantitative PCR showed that with the progression of hepatocyte differentiation, the expression of induced pluripotent stem cells marker (OCT4) was decreased. The expression levels of definitive endoderm (GCS and CXCR4) and hepatic endoderm (HNF4α and HNF1β) markers were increased and then decreased. The expression of hepatocyte markers (CYP34A and ALB) was gradually increased. (4) ELISA results further showed that the hepatocytes after 15 days of induction began to secrete liver-specific proteins (albumin and urea), and their secretory function further increased with the prolongation of time. (5) We conclude that a protocol for differentiation of hepatocytes from human induced pluripotent stem cells was successfully established, which provides an unlimited source of hepatocytes for liver regeneration. [ABSTRACT FROM AUTHOR]