Efficient Non-Viral Reprogramming of Myoblasts to Stemness with a Single Small Molecule to Generate Cardiac Progenitor Cells 英文参考文献.docVIP

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Efficient Non-Viral Reprogramming of Myoblasts to Stemness with a Single Small Molecule to Generate Cardiac Progenitor Cells 英文参考文献.doc

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Efficient Non-Viral Reprogramming of Myoblasts to Stemness with a Single Small Molecule to Generate Cardiac Progenitor Cells 英文参考文献

EfficientNon-ViralReprogrammingofMyoblaststo StemnesswithaSingleSmallMoleculetoGenerate CardiacProgenitorCells ZeeshanPasha,HusnainKhHaider,MuhammadAshraf* DepartmentofPathology,UniversityofCincinnati,Cincinnati,Ohio,UnitedStatesofAmerica Abstract Thecurrentprotocolsforgenerationofinducedpluripotentstem(iPS)cellsinvolvegenomeintegratingviralvectorswhich may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulationforreprogrammingofskeletalmyoblasts(SMs)usingsmallmolecules. Methods and Results: SMs from young male Oct3/4-GFP + transgenic mouse were treated with DNA methyltransferase (DNMT) inhibitor, RG108. Two weeks later, GFP+ colonies of SM derived iPS cells (SiPS) expressing GFP and with morphological similarity of mouse embryonic stem (ESCs) were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity, expressed SSEA1, displayed ES cell specific pluripotency markers and formed teratoma in nudemice.Optimizationofcultureconditionsforembryoidbody(EBs)formationyieldedspontaneouslycontractingEBs havingmorphological,molecular,andultra-structuralsimilaritieswithcardiomyocytesandexpressedearlyandlatecardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicatingthatSiPSweresimilartoESCsinmiRprofile.Fourweeksaftertransplantationintotheimmunocompetentmice modelofacutemyocardialinfarction(n=12pergroup),extensivemyogenesiswasobservedinSiPStransplantedheartsas comparedtoDMEMcontrols(n=6pergroup).Asignificantreductioninfibrosisandimprovementinglobalheartfunction intheheartstransplantedwithSiPSderivedcardiacprogenitorcellswereobserved. Conclusions: Reprogramming of SMs by DNMT inhibitor is a simple, reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarctedmyocardiumwithouttumorgenesisandimproved