Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Biomaterials. 2011 Mar;32(8):2032-42. doi: 10.1016/j.biomaterials.2010.11.045. Epub 2010 Dec 15.
Generation of specific lineages of cells from embryonic stem (ES) cells is pre-requisite to use these cells in pre-clinical applications. Here, we developed a recombinant E-cadherin substratum for generation of hepatic progenitor populations at single cell level. This artificial acellular feeder layer supports the stepwise differentiation of ES cells to cells with characteristics of definitive endoderm, hepatic progenitor cells, and finally cells with phenotypic and functional characteristics of hepatocytes. The efficient differentiation of hepatic endoderm cells (approximately 55%) together with the absence of neuroectoderm and mesoderm markers suggests the selective induction of endoderm differentiation. The co-expression of E-cahderin and alpha-fetoprotein (approximately 98%) suggests the important role of E-cadherin as a surface marker for the enrichment of hepatic progenitor cells. With extensive expansion, approximately 92% albumin expressing cells can be achieved without any enzymatic stress and cell sorting. Furthermore, these mouse ES cell-derived hepatocyte-like cells showed higher morphological similarities to primary hepatocytes. In conclusion, we demonstrated that E-cadherin substratum can guide differentiation of ES cells into endoderm-derived hepatocyte-like cells. This recombinant extracellular matrix could be effectively used as an in vitro model for studying the mechanisms of early stages of liver development even at single cell level.
从胚胎干细胞 (ES) 细胞中生成特定细胞谱系是将这些细胞用于临床前应用的前提。在这里，我们开发了一种重组 E-钙黏蛋白基质，用于在单细胞水平上生成肝祖细胞群体。这种人工细胞外基质饲养层支持 ES 细胞逐步分化为具有明确内胚层、肝祖细胞特征的细胞，最终分化为具有肝细胞表型和功能特征的细胞。肝内胚层细胞的高效分化（约 55%），同时不存在神经外胚层和中胚层标志物，提示内胚层分化的选择性诱导。E-cahderin 和甲胎蛋白的共表达（约 98%）提示 E-钙黏蛋白作为肝祖细胞富集的表面标志物的重要作用。经过广泛扩增，约 92%的白蛋白表达细胞可以在没有任何酶处理和细胞分选的情况下实现。此外，这些源自小鼠 ES 细胞的肝样细胞在形态上与原代肝细胞具有更高的相似性。总之，我们证明了 E-钙黏蛋白基质可以指导 ES 细胞分化为内胚层来源的肝样细胞。这种重组细胞外基质可有效用作体外模型，用于研究甚至在单细胞水平上的肝早期发育的机制。
