Isolation,Purification And Dedeifferentiation Of Mouse Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs)are the only stem cells in mammals that can transmit genetic information to the next generation.Isolation and culture of SSCs in vitro are prerequisite for exploring their biological characteristics,proliferation,differentiation and dedifferentiation mechanisms.Additionally,SSCs are unipotent stem cells.Under culture conditions supplemented with specific small molecule compounds,mSSCs could be induced to pluripotent state without transgenes.Also,pluripotent stem cells can differentiate to a variety of cells and tissues required to form a body,which implies great potential applications in biomedical and animal breeding.Nevertheless,induced pluripotent stem cells(iPSCs)can differentiate into three germ layers in vivo and in vitro,the process,however,requires specific transgenes,which could limit their potential applications.Therefore,compared to iPSCs,SSCs seem to be more suitable for biomedical research,male infertility and animal breeding applications.The objectives of the project were to isolate,purify mouse SSCs and to dedifferentiate the SSCs into pluripotent state through small molecules.Experiments were divided into two series.The first series of experiments were to use mouse as an animal model to isolate,purify and maintain mouse spermatogonial stem cells(mSSCs)in vitro.mSSCs-like cells were isolated and purified from testis of Oct4/GFP × CD1 hybrid F1 male mice testicles by a two-step method,namely enzymatic digestion combined with continuous differential adherence.When the cells grew stably,cell morphological assessment,alkaline phosphatase staining,immunofluorescence staining,RT-PCR and RT-qPCR were carried out and the results showed that the isolated cells met the standards of the mSSCs.Also,the second series of the experiments focused on dedifferentiating the mSSCs into pluripotent stem cells using small molecule compounds.The stable clones of the cells were obtained and maintained in the culture conditions,the pluripotent gene markers were identified and analyzed at the gene expression level such as Oct4,Sox2,Nanog and Rex1.Cell morphological assessment,GFP expression,alkaline phosphatase staining,immunofluorescence staining,RT-PCR and RT-qPCR were performed.The preliminary results indicated that small molecule compounds could induce the reprogramming of mSSCs-like cells into pluripotent state.However,germline transmission of the dedifferentiated cells needs to be proved by blastocyst-injections and embryo transfer.This study may provide not only an alternative method to derive pluripotent stem cells through mSSCs and to further investigate the mechanism of mSSCs reprogramming,but also could path a way forstudying male infertility in humans.