Differentiation and isolation of hepatic-like cells from human embryonic stem cells
Differentiation and isolation of hepatic-like cells from human embryonic stem cells
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DOI：
10.1111/j.1432-0436.2004.07205002.x
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发表时间：
2004-06-01
期刊：
影响因子：
2.9
通讯作者：
Benvenisty, N
中科院分区：
文献类型：
--
作者：
Lavon, N;Yanuka, O;Benvenisty, N
人类胚胎干细胞是一种多能性细胞，可以作为移植医学的细胞来源，也可以作为研究人类胚胎发生的工具。我们在这里研究人类胚胎干细胞分化为肝细胞的潜力。我们的特点是肝脏富集基因的表达水平在未分化和分化的人胚胎干细胞的DNA微阵列。我们的分析揭示了胎儿肝脏富集基因的一个子集，在人胚胎干细胞分化成胚状体时表达。为了分离肝样细胞，我们将由肝细胞特异性启动子调控的报告基因导入人胚胎干细胞。我们分离了在体外分化时表达增强型绿色荧光蛋白的人胚胎干细胞克隆。通过免疫组化染色，我们发现这些细胞中的大多数表达白蛋白，而一些细胞仍然表达早期表达的蛋白甲胎蛋白。使用荧光激活细胞分选仪，我们能够分选出荧光分化的细胞，并将其扩增几周。这是第一份证明纯化人胚胎干细胞分化衍生物并进一步培养的可能性的报告。通过共聚焦显微镜，我们发现在20天的胚状体和畸胎瘤肝样细胞集群。正如在胚胎发育过程中观察到的那样，我们表明，在畸胎瘤中，肝样内胚细胞在心脏中胚细胞旁边发育。为了检查参与肝分化诱导的分泌因子，在各种生长因子的存在下培养人胚胎干细胞，证明酸性成纤维细胞生长因子在分化中的潜在参与。总之，在一定的生长条件和遗传操作下，我们现在可以从人类胚胎干细胞中分化和分离肝样细胞。
Human embryonic stem cells are pluripotent cells that can serve as a cell source for transplantation medicine, and as a tool to study human embryogenesis. We investigate here the potential of human embryonic stem cells to differentiate into hepatic cells. We have characterized the expression level of liver-enriched genes in undifferentiated and differentiated human embryonic stem cells by DNA microarrays. Our analysis revealed a subset of fetal hepatic enriched genes that are expressed in human embryonic stem cells upon differentiation into embryoid bodies. In order to isolate the hepatic-like cells, we introduced a reporter gene regulated by a hepatocyte-specific promoter into human embryonic stem cells. We isolated clones of human embryonic stem cells that express enhanced green fluorescent protein upon in vitro differentiation. Through immunostaining, we showed that most of these cells express albumin, while some cells still express the earlier expressed protein alpha-fetoprotein. Using fluorescence activated cell sorter, we were able to sort out the fluorescent differentiated cells and expand them for a few more weeks. This is the first report to demonstrate the possibility of purifying differentiated derivatives of human embryonic stem cells and culturing them further. Through confocal microscopy, we detected clusters of hepatic-like cells in 20-day-old embryoid bodies and in teratomas. As observed during embryonic development, we showed that in teratomas, the hepatic-like endodermal cells develop next to cardiac mesodermal cells. In order to examine the secreted factors involved in the induction of hepatic differentiation, human embryonic stem cells were grown in the presence of various growth factors, demonstrating the potential involvement of acidic fibroblast growth factor in the differentiation. In conclusion, given certain growth conditions and genetic manipulation, we can now differentiate and isolate hepatic-like cells from human embryonic stem cells.