Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
Acta Pharmacol Sin. 2010 Nov;31(11):1478-86. doi: 10.1038/aps.2010.100. Epub 2010 Oct 18.
To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX.
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.
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.
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.
建立一种有效的培养体系，以支持胚胎干细胞（ES 细胞）分化为共表达 F-VIII 和 F-IX 的肝细胞。
将 E14 期小鼠 ES 细胞在含有丁酸钠（SB）、碱性成纤维细胞生长因子（bFGF）和/或骨形态发生蛋白 4（BMP4）的分化培养基中培养，以诱导内胚层细胞和肝祖细胞的分化。然后使用肝细胞生长因子、抑瘤素 M 和地塞米松诱导 ES 细胞来源的肝细胞成熟。在分化的各个阶段，通过 RT-PCR 和实时 PCR 检测内胚层特异性基因和肝细胞特异性基因（包括 F-VIII 和 F-IX 的水平）的 mRNA 表达水平。通过免疫荧光和 Western blot 检测蛋白表达。在分化的最后阶段，通过流式细胞术确定共表达 F-VIII 和 F-IX 的细胞百分比，通过 ELISA 检测分泌到培养基中的 F-VIII 和 F-IX 蛋白水平。
在 SB、bFGF 和 BMP4 的组合作用下，内胚层特异性和肝细胞特异性标志物的表达上调至最高水平。在肝祖细胞分化过程中使用这三种诱导剂可显著增强 ES 细胞来源的肝细胞中 F-VIII 和 F-IX 的 mRNA 和蛋白水平。更重要的是，在分化的最后阶段，F-VIII 和 F-IX 高效共表达，并分泌到培养基中。
我们建立了一种新型的 ES 细胞来源的肝细胞体外分化方案，可共表达 F-VIII 和 F-IX，这可能为血友病的干细胞替代治疗提供基础。
