Malysheva S. Morphological and functional characteristics of murine pluripotent stem cells, generated with Sleeping beauty transposon system.

Generation of induced pluripotent stem cells (iPSCs) and their morphological and functional characteristics is a current issue of stem cell biology. Despite the abundance of various reprogramming techniques, generating of iPSCs with transposon-based gene-delivery systems is among the most prospective approaches. Sleeping beauty transposon based gene delivery system is a promising new reprogramming tool with a range of advantages in terms of efficiency and safety. In the current studies murine embryonic fibroblasts were reprogrammed with Sleeping beauty transposon-based gene delivery system towards actively proliferating colony-like structures, from which individual clones were generated for further characterization. The clones demonstrated typical iPSCs-like morphology and proliferation rate. It was determined that the morphology of the clones is a basic feature on the initial steps of reprogramming and that morphological properties correspond to the expression of surface pluripotency marker SSEA-1. The specific reactivation of certain pluripotency markers (Oct4, Sox2, Nanog, Rex1, Utf1, Tcl, Stella, Sall4) was indicated on mRNA level in reprogrammed murine embryonic fibroblasts. Demethylation of CpG islands of key pluripotency genes' promoters Nanog and Oct4 indicates reprogramming on the epigenetic level. Functional properties of the obtained clones were demonstrated. The formation of embryoid bodies in vitro is shown, and analysis of expression of marker genes during spontaneous differentiation indicated the appearance of endodermal and mesodermal progenitor cells. The ability of directed cardiomyocyte differentiation in suspension culture and plated embryoid bodies was evaluated. It was found that plated embryoid bodies differentiate towards cardiomyocytes ten times more effective than embryoid bodies in suspension culture, though the peak of differentiation efficiency is registered 4 days later compared to suspension culture. The clones also demonstrated their differentiation potential in vivo. Reprogrammed murine fibroblasts formed solid vascularised teratomas with endodermal, mesodermal and ectodermal tissues in immunodeficient animals, but not during syngeneic transplantation. Obtained morphological and functional characteristics of the clones indicate their pluripotency. Therefore, they can be regarded as a potential new iPS cell line, and the method of their generation can be considered as perspective technique for iPSC generation.