Abstract-Stem cell-based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into cells from all 3 germ layers. In the present study, we used the hanging drop method to differentiate maGSCs into cardiomyocytes and analyzed their functional properties. Differentiation efficiency of beating cardiomyocytes from maGSCs was similar to that from ESCs. The maGSC-derived cardiomyocytes expressed cardiac-specific L-type Ca 2ϩ channels and responded to Ca 2ϩ channel-modulating drugs. Cx43 was expressed at cell-to-cell contacts in cardiac clusters, and fluorescence recovery after photobleaching assay showed the presence of functional gap junctions among cardiomyocytes. Action potential analyses demonstrated the presence of pacemaker-, ventricle-, atrial-, and Purkinje-like cardiomyocytes. Stimulation with isoproterenol resulted in a significant increase in beating frequency, whereas the addition of cadmium chloride abolished spontaneous electrical activity. Confocal microscopy analysis of intracellular Ca 2ϩ in maGSC-derived cardiomyocytes showed that calcium increased periodically throughout the cell in a homogenous fashion, pointing to a fine regulated Ca 2ϩ release from intracellular Ca 2ϩ stores. By using line-scan mode, we found rhythmic Ca 2ϩ transients. Furthermore, we transplanted maGSCs into normal hearts of mice and found that maGSCs were able to proliferate and differentiate. No tumor formation was found up to 1 month after cell transplantation. Taken together, we believe that maGSCs provide a new source of distinct types of cardiomyocytes for basic research and potential therapeutic application. (Circ Res. 2007;100:1615-1625.) Key Words: spermatogonial stem cells Ⅲ cardiac differentiation Ⅲ gap junction Ⅲ L-type Ca 2ϩ channels Ⅲ cell transplantation W hen heart muscle is damaged with myocyte apoptosis or necrosis, functional contracting cardiomyocytes are replaced by nonfunctional scar tissue. Experimental evidence suggests that the heart harbors a resident population of stem cells able to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells. 1,2 A subset of native cardiac progenitors has been identified in hearts of newborn mice, rats, and humans. 3 Despite the possible existence of these populations and their ability to contribute to cardiac repair, these intrinsic mechanisms are inadequate to restore the cardiac function of a failing heart. Therefore, a potential therapeutic approach for treatment of heart failure is to replace lost cardiomyocytes with new functional ones as an alternative to whole-heart transplantation.Several groups have shown that functional cardiomyocytes can be differentiated from embryonic stem cells (ESCs), derived from the...
Transplantation of stem cells represents an upcoming therapy for many degenerative diseases. For clinical use, transplantation of pluripotent stem cell-derived cells should lead to integration of functional grafts without immune rejection or teratoma formation. Our previous studies showed that the risk of teratoma formation is highly influenced by the immune system of the recipients. In this study, we have observed a higher teratoma formation rate when undifferentiated so-called multipotent adult germline stem cells (maGSCs) were transplanted into the heart of T, B, and natural killer (NK) cell-deficient RAG2−/−γc−/− mice than in RAG2−/− mice, which still have NK cells. Notably, in both strains, the teratoma formation rate was significantly reduced by the immunosuppressive drug cyclosporine A (CsA). Thus, CsA had a profound effect on teratoma formation independent of its immunosuppressive effects. The transplantation into RAG2−/− mice led to an activation of NK cells, which reached the maximum 14 days after transplantation and was not affected by CsA. The in vivo-activated NK cells efficiently killed YAC-1 and also maGSC target cells. This NK cell activation was confirmed in C57BL/6 wild-type mice whether treated with CsA or not. Sham operations in wild-type mice indicated that the inflammatory response to open heart surgery rather than the transplantation of maGSCs activated the NK cell system. An activation of NK cells during the transplantation of stem cell-derived in vitro differentiated grafts might be clinically beneficial by reducing the risk of teratoma formation by residual pluripotent cells.
Pluripotent stem cells hold great promise for the treatment of cardiovascular disease. We previously described multipotent adult germline stem cells (maGSCs) from mouse testis with differentiation potential similar to embryonic stem cells. The aim of this work was to differentiate maGSCs into functional endothelial cells and to study their potential for vasculogenesis. MaGSCs were cocultivated with OP9 stromal cells to induce differentiation into cardiovascular progenitors, i.e. fetal liver kinase 1-positive (Flk-1<sup>+</sup>) cells. Five days later, Flk-1<sup>+</sup> cells were separated using fluorescence-activated cell sorting, followed by cultivation on collagen type IV under endothelial differentiation conditions. At different time points, maGSC-derived endothelial-like cells were characterized using RT-PCR, flow cytometry, immunofluorescence and functional assays. Cultivation of Flk-1<sup>+</sup> cells resulted in the progressive upregulation of endothelial cell markers, including VE-cadherin, von Willebrand factor and endothelial nitric oxide synthase. Moreover, Flk-1<sup>+</sup> maGSC-derived endothelial-like cells were able to branch and form networks in vitro and promoted functional blood vessel formation in vivo. Importantly, Flk-1<sup>+</sup> cells retained their potential to proliferate and could be continuously expanded, while the ability of contact inhibition was preserved. Thus, maGSCs may provide a useful source of endothelial-like cells to study the basic mechanisms of vasculogenesis or endothelial differentiation.
AMI acute myocardial infarction, akuter Myokardinfarkt (auch MI genannt) bidest. bidestilliert Bp Basenpaare ß-ME ß-Mercaptoethanol BSA Bovines Serumalbumin cDNA complementary DNA, komplementäre DNA CM-DiI Chlormethylbenzamido-Derivate des 1,1'-Dioctadecyl-3,3,3'3' tetramethylindocarbocyanin-perchlorat CsA Ciclosporin A Cy3 Indocarbocyanin d day, Tag DAPI 4', 6-Diamidino-2-phenylindoldihydrochlorid
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