Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, Düsseldorf, Germany.
J Cell Physiol. 2010 Nov;225(2):545-54. doi: 10.1002/jcp.22237.
The hepatic-like phenotype resulting from in vitro differentiation of unrestricted somatic stem cells (USSC) derived from human umbilical cord blood (CB) was analyzed with regard to functional and metabolic aspects. USSC can be differentiated into cells of all three germ layers in vitro and in vivo and, although they share many features with mesenchymal stroma cells (MSC), can be distinguished from these by their expression of DLK1 as well as a restricted adipogenic differentiation potential. For the differentiation procedure described herein, a novel three-stage differentiation protocol resembling embryonic developmental processes of hepatic endoderm was applied. Hepatic pre-induction was performed by activinA and FGF4 resulting in enhanced SOX17 and FOXA2 expression. Further differentiation was achieved sequentially by retinoic acid, FGF4, HGF, EGF, and OSM resulting in a hepatic endodermal identity, characterized by the expression of AFP and HNF1alpha. Thereafter, expression of G6PC, ARG1, FBP1, and HNF4alpha was observed, thus indicating progressive differentiation. Functional studies concerning albumin secretion, urea formation, and cytochrome-p450-3A4 (CYP3A4) enzyme activity confirmed the hepatic-like phenotype. In order to characterize the differentiated cells at a metabolic level, USSC were incubated with [1-(13)C]glucose. By tracing the fate of the molecule's label via isotopomer analysis using (13)C NMR spectroscopy, formation of both glycogen and some gluconeogenetic activity could be observed providing evidence of a hepatocyte-like glucose metabolism in differentiated USSC. In conclusion, the results of the present study indicate that USSC represent a stem cell source with a substantial hepatic differentiation capacity which hold the potential for clinical applications.
体外分化无限制体干细胞（USSC）源自人脐血（CB）的肝样表型在功能和代谢方面进行了分析。USSC 可以在体外和体内分化为所有三个胚层的细胞，尽管它们与间充质基质细胞（MSC）有许多共同特征，但可以通过其表达 DLK1 以及有限的成脂分化潜力与这些细胞区分开来。对于本文描述的分化程序，应用了一种新的三阶段分化方案，类似于肝内胚层的胚胎发育过程。通过激活素 A 和 FGF4 进行肝前诱导，导致 SOX17 和 FOXA2 表达增强。通过视黄酸、FGF4、HGF、EGF 和 OSM 依次进行进一步分化，导致肝内胚层特性，特征为 AFP 和 HNF1alpha 的表达。此后，观察到 G6PC、ARG1、FBP1 和 HNF4alpha 的表达，表明进行了逐步分化。关于白蛋白分泌、尿素形成和细胞色素 P450-3A4（CYP3A4）酶活性的功能研究证实了肝样表型。为了在代谢水平上表征分化细胞，将 USSC 与 [1-(13)C]葡萄糖孵育。通过使用 (13)C NMR 光谱通过同位异构体分析追踪分子标记的命运，可以观察到糖原的形成和一些糖异生活性，这提供了在分化的 USSC 中存在类似肝细胞的葡萄糖代谢的证据。总之，本研究的结果表明，USSC 代表了具有实质性肝分化能力的干细胞来源，具有临床应用的潜力。
