The Role Of Reversine On Dedifferentiation Of Fetal Porcine Muscle Satellite Cells And Differentiation Into Female Germ Cells

Muscle satellite cells, which function as myogenic precursors, are believed to form astable, self-renewing pool of stem cells in adult muscle where they function in tissue growthand repair. These cells shared features of Mesenchymal stem cells and pluripotent stem cells,which had the ability to differentition into all three germ cells and female germ cells. Germcells, as special adult cells in mammals, take the responsibility of transferring geneticmaterials to the next generation. By now, there are many researches on induction of MSCsand skin derived stem cells into female germ cells, but with a low efficiency, and long curve.Reversine was a purine derivative small heterocyclic compound, which can promote theplasticity of lineage-committed progenitor cells and differentiation into other lineages whencultured with the appropriate medium. Now, there are few researches on how to improve theefficiency of the mesodermal lineages to differentiate into germ cells.In this study, the primary muscle satellite cells were isolated from fetal porcine skeletalmuscle. The cells can be purified through repeated differential adhesion, using a culturecondition described in this report. Compared the purified cells with the first adhesion cellsand analysised their characters; Treated the purified cells with Reversine and analysised theirchanges in cell biology; To study the effect of follicular fluid on satellite cells differentiationinto female germ cells, further more, treated the cells with Reversine to study the efficiencyof differentiation into female germ cells.1. Study on the isolation and biological properties of muscle satellite cells derivedfrom fetal porcine skeleton muscle (PMDSCs)In this study, we dissociated PMDSCs from60d old fetal porcine skeletal muscle with1mg/mL collagenase Ⅰ and0.25%(w/v) trypsin, purified through repeated differentialadhesion. The PMDSCs had a stronger proliferative capacity than the first adhension cells bycell growth curve, RT-PCR and FACS. The biological characteristics of the purified cellswere identified by flow cytometry analysis of surface antigens, immunofluorescence stainingand differentiation into three grem cells, myotubule and neural-like cells in vitro. 2. The effects of Reversin on PMDSCsAfter treated the purified cells with Reversine in vitro, the pluripotency of cellsincreased, whilst the proliferation reduction by immunofluorescence staining, QRT-PCR,Western-blotting and BrdU proliferation assay. The efficiency of differentiation intoadipocyte-like cells improved after treated with Reversine by QRT-PCR assay and oil red-Ostatistics.3. Differentiation of PMDSCs into female germ cells in vitroThese results strongly indicated that a small fraction of PMDSCs were able todifferentiate into female germ-like cells in the presence of20%FF, and the large round cellswere positive for Vasa, Dazl, Scp3, Stra8, Zp2and Zp3; Furthermore, we found thatReversine can promote the efficiency of PMDSCs differentiation into female germ cells;There were large female germ-like cells positive for Figla after overexpression of Figla geneand promoter; We found that the ovary of the transplanted side was recovered bettercompared to the other side ovary after transplanted with the induced PMDSCs under thekidney capsule of recipient mice for30days by H.E. staining. Furthermore, garland-likestructure was seen in the grafts, and immunological staining revealed that the cells coulddifferentiate into germ cells in vivo.These results indicated that PMDSCs has strong proliferation and differentiationcapacity in vitro, Reversine promotes PMDSCs differentiation into female germ-like cells andadipocyte-like cells, which can provide adequate cell resources for tissue engineering andregenerative medicine.