Baharvand Hossein, Hashemi Seyed M, Kazemi Ashtiani Saeid, Farrokhi Ali
Department of Stem Cells, Royan Institute, Tehran, Iran.
Int J Dev Biol. 2006;50(7):645-52. doi: 10.1387/ijdb.052072hb.
Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.
人类胚胎干细胞（hESCs）作为细胞替代疗法的细胞来源以及早期人类发育的模型具有巨大潜力。在本研究中，我们在二维和三维（2D和3D）培养系统中研究了hESCs向肝细胞分化的潜力。将胚状体（EBs）插入胶原支架3D培养系统或在胶原包被的培养皿上培养，并用外源性生长因子刺激以诱导肝脏组织发生。免疫荧光分析显示白蛋白（ALB）和细胞角蛋白-18（CK-18）的表达。二维和三维培养系统中的分化细胞表现出肝细胞的几个特征，包括转甲状腺素蛋白、α-1-抗胰蛋白酶、细胞角蛋白8、18、19、色氨酸-2,3-双加氧酶、酪氨酸转氨酶、葡萄糖-6-磷酸酶（G6P）、细胞色素P450亚基7a1的表达以及甲胎蛋白（AFP）和ALB的分泌以及尿素的产生。与二维培养相比，在三维培养中，更早检测到ALB和G6P，并且产生更高水平的尿素和AFP。分化的hESCs的电子显微镜检查显示出类似肝细胞的超微结构，包括糖原颗粒、发达的高尔基体、粗面和滑面内质网以及细胞间小管。在含有外源性生长因子的三维胶原支架内将hESCs分化为类肝细胞，产生具有肝细胞特征性形态特征、基因表达模式和代谢活性的细胞，并可能为肝病治疗提供分化细胞来源。
