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Aim:To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX.Methods:Mouse E14 ES cells were cultured in differentiation medium containing sodium butyrate (SB), basic fibroblast growth factor (bFGF), and/or bone morphogenetic protein 4 (BMP4) to induce the differentiation of endoderm cells and hepatic progenitor cells. Hepatocyte growth factor, oncostatin M, and dexamethasone were then used to induce the maturation of ES cell-derived hepatocytes. The mRNA expression levels of endoderm-specific genes and hepatocyte-specific genes, including the levels of F-VIII and F-IX, were detected by RT-PCR and real-time PCR during various stages of differentiation. Protein expression was examined by immunofluorescence and Western blot. At the final stage of differentiation, flow cytometry was performed to determine the percentage of cells coexpressing F-VIII and F-IX, and ELISA was used to detect the levels of F-VIII and F-IX protein secreted into the culture medium.Results:The expression of endoderm-specific and hepatocyte-specific markers was upregulated to highest level in response to the combination of SB, bFGF, and BMP4. Treatment with the three inducers during hepatic progenitor differentiation significantly enhanced the mRNA and protein levels of F-VIII and F-IX in ES cell-derived hepatocytes. More importantly, F-VIII and F-IX were coexpressed with high efficiency at the final stage of differentiation, and they were also secreted into the culture medium.Conclusion:We have established a novel in vitro differentiation protocol for ES-derived hepatocytes that coexpress F-VIII and F-IX that may provide a foundation for stem cell replacement therapy for hemophilia. [ABSTRACT FROM AUTHOR]

AbstractAim:The feasibility of inducing endocrine pancreatic differentiation of embryonic stem (ES) cells has been well documented. However, whether ES cells possess the potential for exocrine pancreatic differentiation requires further exploration. Here, we investigated whether sodium butyrate and glucocorticoids were conducive to the exocrine pancreatic differentiation of ES cells.Methods:E14 mouse ES cells were cultured in suspension to form embryoid bodies (EBs). These EBs were cultured in differentiating medium containing varying concentrations of sodium butyrate. The effects of activinA and dexamethasone (Dex) on exocrine differentiation were also explored. Finally, the combination of sodium butyrate, activinA, and Dex was used to promote the differentiation of exocrine pancreatic cells. Specific exocrine pancreatic gene expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and amylase expression was examined by immunofluorescence staining. Flow cytometry analysis was also performed to determine the percentage of amylase-positive cells after the treatment with activinA, sodium butyrate, and Dex.Results:Exposure of ES cells to 1 mmol/L sodium butyrate for 5 days promoted exocrine pancreatic gene expression. Further combination with Dex and other pancreatic-inducing factors, such as activinA, significantly enhanced the mRNA and protein levels of exocrine pancreatic markers. Additionally, flow cytometry revealed that approximately 17% of the final differentiated cells were amylase-positive.Conclusion:These data indicate that the exocrine pancreatic differentiation of ES cells can be induced by activinA, sodium butyrate, and Dex, providing a potential tool for studying pancreatic differentiation and pancreas-related diseases. [ABSTRACT FROM AUTHOR]

Aim: There is increasing evidence indicating that embryonic stem (ES) cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is necessary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte differentiation from ES cells by utilizing cholestatic serum. Methods: One week after the induction of E14 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen storage of the differentiated cells was detected by Periodic acid-Schiff (PAS) reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. Results: In the presence of conditional selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were α-fetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation. Conclusion: The conditional selective medium containing cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to improve the hepatic differentiation ratio of ES cells. [ABSTRACT FROM AUTHOR]