Establishment And Gene Regulation On The Proliferation Of Human Spermatogonial Stem Cell Line

Background and Objective: Male fertility has been damaged remarkably due to environmental pollution,genetic factors,and bad life styles.In general,male infertility has become the third serious disease affecting human health.Spermatogonial stem cells(SSCs)are at the foundation of spermatogenesis and male fertility.SSCs can both self-renew to maintain the stem cell population and differentiate into other types of male germ cells.Recent studies have demonstrated that SSCs can acquire pluripotency to become embryonic stem(ES)-like cells that are able to differentiate into all cell lineages of three germ cell layers.Strikingly,numerous studies by peer and us have recently demonstrated that SSCs are able to directly transdifferentiate into the cells of other lineages both in vivo and in vitro.Therefore,SSCs could have significant applications in both reproductive and regenerative medicine.Although much progress has been made on genetic and epigenetic regulation of SSCs in rodents,it is hardly known about the molecular mechanisms underlying the process of human spermatogenesis.One of the urgent problems to be solved when working with human SSCs is the limited number of these cells.Additionally,human primary SSCs proliferate slowly in vitro and a long-term culture system to expand human SSCs has not yet been available.Therefore,it is essential to immortalize human SSCs to obtain sufficient cells for their basic research and clinic applications.Currently,studies on the mechanisms of spermatogenesis arerestricted to certain genes and signaling.Recent studies have demonstrated that the microarray technology could identify a large scale of gene expression profiles and functions and reveal molecular mechanisms of biological processes and disease development.In this study,we have performed the following Aims: i)to establish a human SSC line with unlimited proliferation potentials and no tumorgenesis,which could provide an abundant source of human SSCs for their basic studiesand clinic application;ii)to uncover the global gene expression patterns for the proliferation of human SSCs.Methods: We have established a human SSC line by stably expressing human SV40 large T antigen.RT-PCR,Western blots,and immunocytochemistry revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks.Immunocytochemistry and CCK-8 cell proliferation assay were performed to determine the proliferation potential of this cell line.Transplantation assay was used to determine the function of human SSC line.Moreover,karyotyping,Y chromosome microdeletions and tumor-formation potential assays of human SSC line were employed to evaluate the safety of the cell line.Finally,we stimulated the proliferation of this cell line with GDNF,EGF and b FGF,and microarray analysis were utilized to identify a number of genes and signaling pathways involved in proliferation of human SSCs.Results: The cell line developed by overexpressing human SV40 large T antigen was positive for a number of human spermatogonial and SSC hallmarks,including VASA,DAZL,MAGEA4,GFRA1,RET,UCHL1,GPR125,PLZF and THY1,suggesting that these cells are human SSCs phenotypically.Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years,and high levels of PCNA,UCHL1 and SV40 were maintained for long-term culture.Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice.Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line.Microarray assays revealed that totally 1,172 genes were differentially expressed in human SSC line with GDNF,EGF and b FGF stimulation compared to the control.KEGG pathway analysis further showed that these differentially expressed genes were enriched in several signaling pathways.Finally,we identified that ID1 as an important gene that might play a crucial role in regulating human SSC proliferation;but the molecular mechanism of ID1 need to be further explored.Conclusions: We have for the first time established a stable human SSC line that assumes the morphological,phenotypic,and functional attributes of human primary SSCs.This cell line could colonize and proliferate in recipient mouse testes after xenotransplantation.Significantly,this cell line excludes Y chromosome microdeletions of numerous genes and no tumor formation and could be expanded with a remarkable increase of cells,which could offer cells for uncovering the molecular mechanisms underlying human SSC proliferation and survival and early stages of human spermatogenesis as well as the applications of human SSCs in treating male infertility and other human diseases.We have also identified a number of novel genes including ID1 gene that may be involved in human SSCs proliferation.