Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages
Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages
复制标题
DOI：
10.1242/jcs.066886
复制
发表时间：
2010-08-15
影响因子：
4
通讯作者：
Kume, Shoen
中科院分区：
文献类型：
--
作者：
Higuchi, Yuichiro;Shiraki, Nobuaki;Kume, Shoen
我们以前报道过，胚胎干细胞（ES）培养在M15细胞，中胚层来源的支持细胞系，有效地向内胚层的命运分化，最终采用各种消化器官，如胰腺和肝脏的特定谱系。我们在这里表明，M15细胞的内胚层诱导活性部分是通过细胞外基质介导的，层粘连蛋白。阿尔法5是其中一个关键成分为了建立一种无饲养层的ES细胞胰腺分化方法，我们使用了一种合成的基底膜（sBM）基质，该基质使用了稳定表达层粘连蛋白-511的HEK 293细胞系。在sBM上，小鼠ES或诱导多能干（iPS）细胞依次分化为定形内胚层、胰腺祖细胞，然后在体外分化为表达胰岛素的胰腺β细胞。用整合素β 1（Itgb 1）敲低ES细胞减少了向胰腺细胞的分化。硫酸乙酰肝素蛋白聚糖2（HSPG 2）敲除和肝素酶处理协同减少了表达Pdx 1的细胞数量。这些发现表明，基底膜的成分在定形内胚层谱系的分化中具有重要作用。这种新的方法将有助于研究ES或iPS细胞的胰腺分化，并为再生医学提供潜在的替代细胞来源。
We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin. alpha 5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511. On the sBM, mouse ES or induced pluripotent stem (iPS) cells sequentially differentiated into the definitive endoderm, pancreatic progenitor cells, and then insulin-expressing pancreatic beta-cells in vitro. Knockdown of ES cells with integrin beta 1 (Itgb1) reduces differentiation towards pancreatic cells. Heparan sulfate proteoglycan 2 (HSPG2) knockdown and heparitinase treatment synergistically decreased the number of Pdx1-expressing cells. These findings indicate that components of the basement membrane have an important role in the differentiation of definitive endoderm lineages. This novel procedure will be useful for the study of pancreatic differentiation of ES or iPS cells and the generation of potential sources of surrogate cells for regenerative medicine.