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  • 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.

Journal of cell science (2010-07-22)
Yuichiro Higuchi, Nobuaki Shiraki, Keitaro Yamane, Zeng Qin, Katsumi Mochitate, Kimi Araki, Takafumi Senokuchi, Kazuya Yamagata, Manami Hara, Kazuhiko Kume, Shoen Kume
RESUMO

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 alpha5 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 beta1 (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.

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Anti-Laminin Antibody, Chemicon®, from rabbit