Differentiation Of Human Umbilical Cord-derived Mesenchymal Stem Cells Into Male Germ Cells

Infertility affects 8%–17% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected by defective spermatogenesis due to failure in germ cell proliferation and differentiation. Stem cells exist in many tissues and are responsible for generating differentiated cells during an animal's lifetime. An area of research that generates great optimism is the use of stem cells transplantation for treatment of human diseases. Recent studies have proved that not only the embryonic stem cells but also Bone Marrow-Derived Cells can differentiate into male germ cells whether put into tissue or cultured under different conditions.But this approach remains controversial for ethical,resource and immunological rejection reasons. By contrast, a population of mesenchymal stromal cells derived from the Human Umbilical Cord Wharton's jelly (Wharton's jelly cells, WJCs) are a primitive stromal population. They may be differentiated into many kinds of cells and be well-tolerated by the immune system. So the plasticity and its capacity to transdifferentiate create a huge promise for understanding and treating diseases.PartⅠBiological Characteristics of MSCs Cultured in Conditions for Spermatogonia in vitroObjective To investigate the possibility of inducing Mesenchymal Stem Cells (huMSCs) from human umbilical cord Wharton's jelly to differentiate into spermatogonia. Methods To isolate,culture and purify huMSCs with adherent method,The growth and proliferation of human umbilical cord-derived huMSCs were observed, and their immunophenotypes were determined by flow cytometry;HuMSCs of the third generation were divided into two groups to be induced and cultured , HuMSCs of the control group were cultured in basal medium, while those of the experimental group with conditional medium. The morphologic and ultrastructure changes of control group and experimental group cells were compared with phase contrast microscopy,electron microscopy(EM) and transmission electron microscope(TEM) respectively ; The spermatogonial cells differentiated were then evaluated by immunohistochemistry stained for CD117and CD49f ;The method of Western-blot was used to test if the cells induced could express CD49f. Results A population of huMSCs were isolated from human umbilical Wharton's Jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling;After induction ,the shape of MSCs changed greatly from the fibroblast to the round , even familiar to the tadpole; Expressed the known molecular markers of spermatogonial cells, such as CD49f,CD117. Conclusions The induced huMSCs not only underwent spermatogonial-cell like morphologic changes, ultramicrostructure mature with increasing cell organs, but also expressed the spermatogonial cell markers. Which showed that human umbilical cord derived MSCs were capable of differentiating into spermatogonial cell;PartⅡTransplantation of Human umbilical cord Mesenchymal stem cells into mouse seminiferous tubulesObjective: To evaluate colonizing ability of human umbilical cord Mesenchymal Stem Cells (huMSCs) into mouse seminiferous tubules and the effects of huMSCs on reproductive function of the infertile mouse. Methods: To isolate,culture and purify huMSCs;Busulfan was used to destroy endogenous spermatogenesis of the recipient mice;HuMSCs of the third generation were marked with Brdu and Hoechst33258 respectively, and then transplanted into the seminiferous tubules of the mouse by microinjection;The survival of huMSCs in recipient testes were evaluated by immunohistochemistry stained for Brdu and Fluorescent microscopy for Hoechst33258 observation at different times;The diameter of the seminiferous tubules was detected with the HMIAS-2000 high-definition colored analyzing system for medical pictures. Results: A lot of purified huMSCs were obtained at the third generation;The dosage of busulfan resulted in 25% death in the mice , the testes of survived mice showed only basilar membrane in seminiferous tubules after 4 weeks;HuMSCs of transplantation into mouse seminiferous tubules survive in mouse testes for at least 4 months and appear to migration ;The average diameter in experimental groups were higher than those in controls (p<0.01) not only on 26 days but also 120 days. Conclusions: Transplantation of human huMSCs using mice as recipients is feasible and effective. This clinically relevant finding raises the possibility for treatment of male infertility and testosterone deficiency through the therapeutic use of stem cells.