Human Umbilical Cord Matrix Stem Cells Isolation, Identification And Primary Research On Differentiation Potential To Germ Cells

Objective:1. To establish the method for Umbilical cord matrix stem cells (UC-MSCs ) culture and to observe biologic characteristic and differentiation potential of UC-MSCs.2. To investigate the germ cells oriented differentiation potential of UC-MSCs in vitro. 3. To observe the translocation and growth of transplanted UC-MSCs in mouse Premature ovarian failure (POF) model.Methods:1. Umbilical cord from full-term normal deliveries was obtained in sterile condition. Collagenase I-digested cells were cultured in DMEM. Adhered cells were harvested for cell growth curve measure and immunophenotypes was determined by flow cytometry. Lipoblast, osteoblast and chondroblast were induced in different condition culture.2. The expression of germ cellsspecific marker on UC-MSCs was determined by RT-PCR. Follicular fluid was employed to induce UC-MSCs differentiation to germ cells.3. POF animal model was established by chemotherapy. After Ad-GFP was transfected via adenovirus, UC-MSCs were transplanted into POF mouse through caudal vein and tissues were obtained for frozen section. Cell translocation and growth were determined by laser confocal microscopy.Results:1. Spind-like umbilical cord cells were shown and double time of cells was 31.11±2.45 h. Cells in culture were extended to more than 10 passages. In passage 3, cells in G0/G1 stage were 85.84% while in S + G2 + M stage were 14.16%. Bone marrow mesenchymal stem cells (BM-MSCs)-like immuophenotypes was shown: CD29,CD44, CD73(SH3),CD90 and CD105 (SH2) were positive; SSEA-4 was weakly positive; CD31,CD34,CD45 and HLA-DR were negative. After UC-MSCs were induced in different condition culture, lipid droplet-like, bone tubercle-like, cartilage tubercle- like structure were emerged and the mRNA expressions of specific gene of fat, bone and cartilage were observed.2. Germ cells markers, OCT4, Stella, Ifitm3, were expressed in UC-MSCs. After induced by 5%, 10% or 20% follicular fluid, cells aggregated and oocyte-like structure was shown.3. Mouse POF model was established after cyclophosphamide(CTX) and busulfan(BUS) treatment. Transfection efficiency of Ad-GFP by adenovirus was about 95%, After transplantion, GFP cells were found in harmed ovarian, but not in brain, liver, kidney and pancreas.Conclusions:1. Human UC-MSCs can be cultured and amplificated in vitro. Immuophenotypes of UC-MSCs is similar to that of BM-MSCs. UC-MSCs have differentiation potential to lipoblast, osteoblast and chondroblast.2. Oocyte-like structure is induced in vitro from UC-MSCs with germ cellsspecific marker. It suggests that UC-MSCs have differentiation potential to germ cells.3. Translocation and survived in premature-failed ovarian indicated that UC-MSCs maybe have POF repairment potential.