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We have evaluated the effect of heart extracellular matrix (ECM) on the cardiomyocyte differentiation of mouse embryonic stem cells (ES cells) using de-cellularized heart tissue. Several lines of evidence indicate that ECM plays significant roles in cell proliferation, cell death and differentiation, but role of ECM possessing a 3D structure in differentiation has not been studied in detail. We found that there are substantial differences in the quantitative protein profiles of ECM in SDS-treated heart tissue compared to that of liver tissue, as assessed by iTRAQ™ quantitative proteomics analysis. When mouse ES cells were cultured on thin (60 μm) sections of de-cellularized tissue, the expression of cardiac myosin heavy chain (cMHC) and cardiac troponin I (cTnI) was high in ES cells cultured on heart ECM compared with those cultured on liver ECM. In addition, the protein expression of cMHC and cTnI was detected in cells on heart ECM after 2 weeks, which was not detectable in cells on liver ECM. These results indicate that heart ECM plays a critical role in the cardiomyocyte differentiation of ES cells. We propose that tissue-specific ECM induced cell lineage specification through mechano-transduction mediated by the structure, elasticity and components of ECM.

Background: The survival of stem cells upon transplantation into ischemic myocardium is a major concern in cell-based therapy. In this study, we tested the hypothesis that activation of opioid receptors would enhance the survival of mesenchymal stem cells (MSCs) upon exposure to an injury stimulus. Methods and Results: MSCs were obtained from rat bone marrow and cultured in basal DMEM cell culture medium. Delta-opioid receptor (DOR) was present in MSCs as examined by reverse transcription-polymerase chain reaction and immunochemistry. Activation of DOR with 5μmol/L SNC80 (DOR agonist) for 24h significantly enhanced MSC viability upon exposure to 5μg/ml actinomycin D as determined by TUNEL and MTT assays. The cytoprotection was abolished with 20μmol/L naltrindole hydrochloride (a DOR antagonist). Treatment of the cells with 1.5μmol/L chelerythrine (protein kinase C inhibitor) and 1.25μmol/L WP1066 (signal transducer and activator of transcription 3 (STAT3) inhibitor) blocked SNC80-induced cytoprotection. Furthermore, treatment of the cells with chelerythrine also blocked STAT3-phosphorylation and Mcl-1 gene expression. Conclusions: Taken together, the results indicate that DOR plays a critical role in MSC survival upon exposure to actinomycin D through activation of protein kinase C and its downstream signaling molecules STAT3 and Mcl-1. DOR may be a novel therapeutic target for stem cell survival during cell-based therapy. (Circ J 2012; 76: 204-212)

Stem cells are regulated by their surrounding microenvironments, called niche, such as cell–cell interaction and extracellular matrix. Classically, feeder cells as a niche have been used in the culture of iPS cells from both the mouse and the human. However, the regulation mechanism of stem cells by feeder cells as a niche still have been partially unclear. In this study, we used three murine iPS cell lines, iPS-MEF-Ng-20D-17, iPS-MEF-Ng-178B-5 and iPS-MEF-Fb/Ng-440A-3, which were generated by different reprogramming methods. In general, these cell lines commonly need the feeder cells as a niche to culture. Recently, the effect of substrate stiffness is known in stem cell study. First, we focused on the mechanical properties of feeder cells, and then we speculated that feeder-less culture might be made possible by using molecules in place of the mechanical properties of the niche. Finally, we found that the combination of disintegrin (echistatin) and 2i (GSK3 inhibitor and MEK inhibitor) is a sufficient condition for three murine iPS culture. This novel method of mimicking the murine iPS cell niche may be useful to understand signaling pathways to maintain the pluripotency of stem cells.

Substrate elasticity is a potent regulator of the cell state. Soft substrates have been shown to promote the homogeneous self-renewal of mouse embryonic stem cells through the down-regulation of cell-matrix tractions. We therefore investigated whether soft substrates promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. After retroviral infection with five factors, Oct3/4, Klf4, Sox2, Lin28 and Nanog, mouse embryonic fibroblasts (MEFs) were cultured on several artificial substrates of varying elasticity and examined for the expression of pluripotency genes. When MEFs were cultured on soft (0.1 kPa) polyacrylamide gels coated with gelatin, the expressions of Nanog and Oct3/4 genes were higher than in cells cultured on rigid plastic dishes (∼10(6) kPa). The same result was obtained at higher elasticity (0.5 kPa) for adult human dermal fibroblasts (HDFa). We also examined whether reprogramming could be enhanced on soft substrates without exogenous gene introduction, finding that cells cultured on soft substrates in the presence of chemicals known to promote cell reprogramming exhibited up-regulated stem cell markers. These results suggest that controlling the substrate stiffness can enhance the initiation of cell reprogramming, which may lead to effective and reproducible iPS cell production.