Functional Characterization,Stable Gene Labeling And In Vitro Regulation Of Human Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells Against Ischemic Environment

Objective Mesenchymal stem cells(MSCs)are important seed cells in regenera tive medicine.Although there are abundant distribution in adult tissues,ad ult MSCs generally have limited proliferation ability and limited access to t hem.MSCs derived from hi PSC differentiation have high self-renewal and proliferation ability,providing a potential alternative source for adult MSC.The purpose of this study is to systematically characterize the function o f hi MSC and to explore its feasibility as an alternative source of adult MS C.Then,to explore the system and method of stable genetic markers of hi PSC,which will lay a foundation for tracking and detection of hi MSC aft er transplantation.Finally,aiming at the ischemic microenvironment,the fea sibility and effective system of regulating hi MSC’s tolerance to ischemic m icroenvironment by nanoparticles carrying mi R21 were explored,so as to establish a new method of modifying hi MSC in vitro before transplantation to improve its therapeutic potential.MethodsThe hi PSC was directly induced to differentiate into MSC cells(hi MS C)by MSC induction medium.Flow pattern analysis was used to compare the surface markers CD11 b,CD19,CD90,CD73 and CD105 of hi MSC a nd h BMSC.The morphological characteristics of early algebras of hi MSC and h BMSC were observed and compared under microscope,and the earl y and long-term proliferative ability of hi MSC and h BMSC were compared by CCK8 test.Real-time quantitative PCR was used to detect and compa re the pluripotency of hi MSC and h BMSC and the paracrine function of hi MSC and h BMSC.In order to construct stable gene-transfected i PSC,we f irst constructed double-promoter lentiviral particles to drive the expression of GFP and fluorescein(Fluc)respectively,and compared the efficiency of different methods to concentrate lentiviruses in order to establish an opti mized lentiviral preparation system.Subsequently,transfection marker hi PS C was used to detect the expression of reporter gene during the long-ter m proliferation and differentiation of hi PSC,and to determine the promoter of stable activity in hi PSC and its source cells,so as to establish a stab le marker system for hi PSC.PLGA nanoparticles were prepared by phacoe mulsification and optimized by agarose gel electrophoresis and UV spectr ophotometer to optimize the combination of PLGA nanoparticles and mi R21.The biocompatibility of PLGA nanocarriers with h MSC was evaluated by CCK8 assay to optimize the dosage of nanocarriers.PLGA nanoparticles were labeled with fluorescent dyes and then incubated with hi MSC.The l evel of nanoparticles entering cells was observed at different time points t o determine the optimal incubation time.On this basis,the optimal dosage of PLGA / mi R21 nanoparticles for regulating hi MSC was optimized and the effective time was explored to establish an optimal system for regulati ng hi MSC in vitro based on PLGA / mi R21 nanoparticles.Hydrogen peroxi de was used to simulate the oxidative stress microenvironment under isch emic conditions.Cell viability test was used to evaluate the effectiveness of these regulatory methods in improving the therapeutic potential of hi MS C and enhancing its tolerance to oxidative stress microenvironment.ResultsCD105,CD90 and CD73 were positive for hi MSC,but CD11 b,CD19,CD34,CD45 and HLA-DR were negative for hi MSC.The early algebraic cells of h BMSC were long spindle while those of hi MSC were short spind le.The cell proliferation ability of h BMSC was basically the same as that of hi MSC before P6 generation,but after P6 generation,the cell proliferat ion ability of hi MSC was stronger than that of h BMSC.Both hi MSC and h BMSC have osteogenic and adipogenic pluripotency.The ability of h BMSC to secrete PLGF,HIF-1a,SDF-1,SCF and b FGF is stronger than that of hi MSC,while the ability of h BMSC to secrete VEGF is weaker than that of hi MSC.The transfection efficiency of lentivirus GFP by ultrafiltration tub e was the highest.GFP successfully labeled human induced pluripotent ste m cell(hi PSC)and expressed stably in their differentiated mesenchymal s tem cell(hi MSC),but Fluc failed to label human induced pluripotent ste m cells.The PLGA nanoparticles coated with PEI at 100 ug can absorb all the mi R21 of 200 pmol.After incubation with hi MSC for 7 days,the cell viability detected by CCK8 was basically the same as that detected by 0 ug/ml,20 ug/ml,100 ug/ml and 200 ug/m L PEI coated PLGA nanoparticl es.The maximum phagocytosis of PLGA nanoparticles was achieved by hi MSC after 12 hours of phagocytosis.The optimal concentration of mi R21 i n PEI-coated PLGA nanoparticles and lipofectmaine RNAi MAX was 40 n M by QRT-PCR.PEI-coated PLGA nanoparticles-mediated mi R21 transfection group had better viability than lipofectmaine RNAIMAX-mediated mi R21 tra nsfection group in oxidative stress microenvironment under simulated isch emia with hydrogen peroxide.Conclusion hiPSC can be successfully induced to differentiate into hi MSC,which can be used as a good substitute for h BMSC.GFP was stably expressed in hi PSC and its differentiated hi MSC.It was successfully verified that PEI-coated PLGA nanoparticles could regulate the expression of mi R21 better than lipofectmaine RNAIMAX-mediated mi R21 transfection group in oxidat ive stress microenvironment under simulated ischemia with hydrogen pero xide.