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SummaryWe 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 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 b-cells in vitro. Knockdown of ES cells with integrin b1 (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.

Key words: basement membrane/alveolar epithelial cells/pulmonary fibroblasts ABSTRA CT. To investigate basement membrane formation by cooperation between pneumocytes and pulmonary fibroblasts, we cultured type II alveolar epithelial cells obtained from rats transfected with SV40-large T antigen gene (SV40-T2 cells) on type I collagen matrices. On fibroblasts-embedded gel (T2-Fgel), SV40-T2 cells ultrastructurallly formed a continuous and thin layer of lamina densa, while on collagen gel without fibroblasts (T2-gel) SV40-T2 cells produced only discontinuous and diffuse deposits. Stripping SV40-T2 cells off the tissues by H2O2treatment revealed a continuous and plane surface of lamina densa assembled on the T2-Fgel tissue, whereas only amorphous deposits appeared on the T2-gel tissue. Immunolocalization of major basement membrane components showed that type IV collagen, laminin, perlecan and entactin (nidogen) were continuously integrated on the lamina densa in T2-Fgel. In T2-gel, all these components were discontinuously distributed beneath SV40-T2 cells. The contribution of pulmonary fibroblasts to the assembly of basement membrane through reorganization of collagen matrix and/or soluble factors was examined by the cultured of SV40-T2cells on the freeze-thawed fibroblast-tissue and/or with the fibroblast-conditioned medium. Both SV40-T2cells on the freeze-thawed fibroblast-tissue and SV40-T2 cells in T2-gel in the fibroblast-conditioned medium failed to produce a lamina densa. SV40-T2cells could assemble a lamina densa only on the freeze-thawed fibroblast-tissue in the fibroblast-conditioned medium.These results show that the basement membranecomponentsare assembled to a lamina densa by combination of the reorganization of collagen matrix and the supply of soluble factors by pulmonary fibroblasts.The alveolus is a unit of gas exchange in the lung and is composed of squamous type I and cuboidal type II epithelial cells, the endothelial cells of capillaries, and pulmonary fibroblasts in interstitium between epithelium and endothelium. Type I cells are involved in gas exchange and occupy approximately 97%of the entire alveolar surface, while type II cells are the progenitors of type I cells, secrete surfactants and cover the remaining area. A thin layer of basement membraneexists beneath the basolateral cell membraneof alveolar epithelial cells and separates the epithelial cell sheet from the interstitium and the endothelium (2). Although a major role of pulmonary fibroblasts is to construct the acellular interstitium that maintains the integrity of pulmonary structure during ventilation, another role is thought to be regulation of the growth and differentiation of epithelial cells during lung development (27) and after epithelial injury (12). Morphological studies of lung development have suggested that mesenchymalcells are involved not only in development and differentiation of epithelial tissues but in the assembly of basement membranes (8). Basement membranehas a highly integrated architecture composed of extra...

The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES) cells is partly mediated through the extracellular matrix, of which laminin α5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM) substratum, using an HEK293 cell line (rLN10-293 cell) stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin β1 (Itgb1) in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies.

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