Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages.
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.
Higuchi
Yuichiro
Y
Department of Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
Shiraki
Nobuaki
N
Yamane
Keitaro
K
Qin
Zeng
Z
Mochitate
Katsumi
K
Araki
Kimi
K
Senokuchi
Takafumi
T
Yamagata
Kazuya
K
Hara
Manami
M
Kume
Kazuhiko
K
Kume
Shoen
S
D000818:Animals; D001485:Basement Membrane / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004707:Endoderm / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D010179:Pancreas / Q000166:cytology* / Q000378:metabolism
D007797:Laminin; C097043:laminin alpha5