Division of Human Stem Cell Technology, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
Biochem Biophys Res Commun. 2010 Jun 11;396(4):933-8. doi: 10.1016/j.bbrc.2010.05.026. Epub 2010 May 9.
The research of human pluripotent stem cells is important for providing the molecular basis for their future application to regenerative medicine. To date, they are usually cultured on feeder cells and passaged by partial dissociation with either enzymatic or mechanical methods, which are problematic for the research using them in the convenience and reproducibility. Here we established a new culture system that allows handling as easily as culturing feeder-free mouse ES cells. This newly developed culture system is based on the combinatorial use of ROCK inhibitor and soluble fibronectin, which enables us to expand human pluripotent stem cells from single cell dissociation on gelatin-coated surface without any feeder cells. In this new culture system, these human pluripotent stem cells can stably grow, even if in clonal density with keeping expression of stem cell markers. These cells also have abilities to differentiate into three germ layers in vivo and in vitro. Furthermore, no chromosomal abnormalities are found even after sequential passage. Therefore this system will dramatically simplify genetic engineering of these human pluripotent stem cells or defining process of their signal pathway.
人类多能干细胞的研究对于为它们未来在再生医学中的应用提供分子基础非常重要。迄今为止，它们通常在饲养细胞上培养，并通过酶或机械方法的部分解离进行传代，这对于使用它们在便利性和重现性方面的研究来说存在问题。在这里，我们建立了一种新的培养系统，其操作简便，类似于无饲养层的鼠 ES 细胞培养。这个新开发的培养系统基于 ROCK 抑制剂和可溶性纤连蛋白的组合使用，使我们能够在没有任何饲养细胞的情况下，从单个细胞解离在明胶包被的表面上扩大人类多能干细胞。在这个新的培养系统中，这些人类多能干细胞可以稳定生长，即使在克隆密度下，也能保持干细胞标志物的表达。这些细胞还具有在体内和体外分化为三个胚层的能力。此外，即使经过连续传代，也没有发现染色体异常。因此，该系统将极大地简化这些人类多能干细胞的基因工程或它们信号通路的定义过程。
