Using growth factor arrays and micropatterned co-cultures to induce hepatic differentiation of embryonic stem cells.
Using growth factor arrays and micropatterned co-cultures to induce hepatic differentiation of embryonic stem cells.
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DOI：
10.1016/j.biomaterials.2010.08.050
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发表时间：
2010-12
期刊：
影响因子：
14
通讯作者：
Revzin, Alexander
中科院分区：
文献类型：
--
作者：
Tuleuova, Nazgul;Lee, Ji Youn;Lee, Jennifer;Ramanculov, Erlan;Zern, Mark A.;Revzin, Alexander
将胚胎干细胞向肝谱系驱动的成功已经被采用大量昂贵的生长因子（GF）的分化方案的复杂性和成本所混淆。我们研究了在打印的GF阵列上培养和分化小鼠胚胎干细胞（mESC），而不是用可溶性GF补充培养基。将肝细胞生长因子（HGF）、碱性成纤维细胞生长因子（bFGF）和骨形态发生蛋白（BMP 4）与纤维连接蛋白（FN）和胶原（I）混合，然后打印在硅烷修饰的载玻片上，形成直径为500 μm的蛋白质斑点。将小鼠ESC在GF点上培养长达12天，并在不同时间点通过RT-PCR和免疫染色进行分析。驻留在含HGF的GF组合上的干细胞表现出肝分化的必要特征，包括多能性（Oct 4）的显著丧失、内胚层（Sox 17）的瞬时（向上和向下）表达以及早期肝标志物-白蛋白和甲胎蛋白的上调。通过将肝星状细胞添加到已经在GF斑点上含有mESC的表面，进一步增强肝分化。发现与非实质肝细胞共培养和最佳GF刺激的组合比单独使用的GF阵列或微图案化共培养物更早地诱导内胚层和肝表型，并且诱导程度更大。虽然这篇论文研究了小鼠胚胎干细胞的肝分化，但我们的研究结果和干细胞培养方法可能与人类胚胎干细胞培养有关。总体而言，结合印刷的GF阵列和异型共培养物的平台将广泛适用于鉴定ESC分化成各种组织类型的微环境生态位的组成。
The success in driving embryonic stem cells towards hepatic lineage has been confounded by the complexity and cost of differentiation protocols that employ large quantities of expensive growth factors (GFs). Instead of supplementing culture media with soluble GFs, we investigated cultivation and differentiation of mouse embryonic stem cells (mESCs) on printed arrays of GFs. Hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP4) were mixed in solution with fibronectin and collagen (I) and then printed onto silane-modified glass slides to form 500 μm diameter protein spots. Mouse ESCs were cultured on top of GF spots for up to 12 days and analyzed by RT-PCR and immunostaining at different time points. The stem cells residing on HGF-containing combinations of GFs exhibited requisite features of hepatic differentiation including pronounced loss in pluripotency (Oct4), transient (up and down) expression of endoderm (Sox17) and upregulation of early hepatic markers –albumin and alpha-fetoprotein. The hepatic differentiation was enhanced further by adding hepatic stellate cells to surfaces that already contained mESCs on GF spots. A combination of co-culture with non-parenchymal liver cells and the optimal GF stimulation were found to induce endoderm and hepatic phenotype earlier and to a much greater extent than the GF arrays or micropatterned co-cultures used individually. While this paper investigated hepatic differentiation of mouse ESCs, our findings and stem cell culture approaches are likely to be relevant for human ESC cultivation. Overall, the platform combining printed GF arrays and heterotypic co-cultures will be broadly applicable for identifying the composition of the microenvironment niche for ESC differentiation into various tissue types.
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