Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum
Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum
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用含有胆汁淤积血清的培养系统选择性富集小鼠胚胎干细胞的肝细胞
DOI：
10.1111/j.1745-7254.2007.00715.x
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发表时间：
2007-12-01
影响因子：
8.2
通讯作者：
Chen, Ji-sheng
中科院分区：
文献类型：
--
作者：
Min, Jun;Shang, Chang-zhen;Chen, Ji-sheng
摘要目的：越来越多的证据表明胚胎干细胞（ES细胞）在体外具有向肝细胞样细胞分化的能力。但要进一步提高其分化效率，促进其临床应用，还需进一步研究。在这里，我们报告了一个有效的培养系统，以支持肝细胞分化的ES细胞，通过利用胆汁淤积serum.Methods：一周后，诱导E14小鼠ES细胞与丁酸钠肝细胞，胆汁淤积血清加入到培养系统中的不同浓度和肝细胞样细胞诱导增殖。肝细胞的形态学和表型标志物的特点，分别采用光学显微镜，免疫细胞化学和RT-PCR。结果：在含胆汁淤积血清的条件选择培养基中，可观察到大量类似于肝细胞的上皮细胞。RT-PCR分析表明，未分化的ES细胞不表达任何肝特异性标志物;然而，在丁酸钠和含胆汁淤积血清的条件选择性培养基的存在下，检测到肝分化标志物。免疫荧光染色显示，ES源性肝细胞呈甲胎蛋白、白蛋白和细胞角蛋白18阳性，具有储存糖原的能力。结论：含胆汁淤积血清的条件选择性培养基是从混合分化的ES细胞中选择性富集肝细胞样细胞的最佳培养基，为提高ES细胞的肝分化率提供了一种新的方法。
AbstractAim: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.