The Differentiation Of Human Umbilical Cord Matrix Stem Cells Toward Germ Cells Under Different Conditions

Objective:1. To establish the method for Umbilical cord matrix stem cells (UC-MSCs ) culture and observe biologic characteristic and differentiation potential of UC-MSCs.2. To investigate the influence of micro-environment on the germ cells oriented differentiation of UC-MSCs in vitro.3.To observe the translocation and growth of transplanted UC-MSCs in mouse Prematur ovarian failure (POF) model, and to investigate the influence of Micro-environment on the differentiation of UC-MSCs in vivo.Methods:1. Umbilical cord from full-term normal deliveries was obtained in sterile condition. Collagenase I-digested cells were cultured in DMEM medium. Adhered cells were harvested for cell growth curve measure and immunophenotypes was determined by flow cytometry. Lipoblast,osteoblast and myoblasts were induced in different culture condition.2. Two methods were used to induce UC-MSCs differentiation . One method was embryonic body induction: UC-MSCs were cultured in hanging drop to form the embryonic body (EB), which will be co-cultured with human / mouse ovarian granulosa cells or cultured with follicular fluid to induce UC-MSCs differentiation to primary germ cells. The expression of germ cells specific marker and immuno -phenotypes on induced UC-MSCs was detectd by RT-PCR, the influence of micro- environment on the differentiation of UC-MSCs was studied by Immuno -histochemistry and Flow cytometry . The othere method was monolayer cells induction: Follicular fluid was employed as conditioned medium to induce UC-MSCs differentiation to germ cells in vitro.3. POF animal model was established by irradiation. After GFP was transfected via lentivirus, UC-MSCs were transplanted into POF mouse through caudal vein. Tissues were obtained at different time points, Cell translocation and growth were determined by laser confocal microscopy and Fluorescence in situ hybridization; Ovary follicles number counting and estrogen level measurement were used to observe the injury and repair of ovarian.Results:1. Spind-like umbilical cord cells were shown and cells in culture were extended to more than 10 passages. In passage 3, cells in G0/G1 stage were more than 80% while in S + G2 + M stage were about 20%. Bone marrow mesenchymal stem cells (BM-MSCs)-like immuophenotypes was shown: CD44, CD73,CD90 and CD105 were positive; CD31, CD34,CD45 and HLA-DR were negative. After UC-MSCs were induced in different condition culture, lipid droplet-like, bone tubercle-like, myofilament - like structure were emerged.2. EB were formed when UC-MSCs were cultured in hanging drop.The result of RT-PCR showed that day 5 EB express germline markers Oct-4, Ifitm-3, stella, vasa and DAZL ,while UC-MSCs used as control only express Oct-4, Ifitm-3 and stella. SSEA-1 positive cells accounted for 15.61% in day 5 EB detected by flow cytometry.The result of Immunohistochemistry showed that when day 5 EB were co-cultured with human or mouse ovarian granulosa cells for 10 days, germline markers stella,vasa,SCP3 and GDF-9 were expressed while there were no expression in EB cultured with follicular fluid. Organization-like structure is formed in vitro from UC-MSCs cultured with containing 5% follicular fluid medium 20 days later.3. Mouse POF model was established after irradiation by 60COγ-ray. There were no obvious injuries in brain, liver, lungs, kidneys , heart ,spleen ,intestine and spinal cord but serious injury in ovary.Transfection efficiency of GFP by lentivirus was more than 80%. After tail vein injection the transfected cells, GFP positive cells were found in harmed ovarian, but not in other organs, like brain, liver, lung,kidney , heart ,spleen,spinal cord and intestine. The repair condition of mouse ovary were better in transplantion group by the follicles number counting and the estrogen level measurement.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 myoblast. It is an ideal source of adult stem cells.2. EB were formed when UC-MSCs were cultured in hanging drop .When EB were co-cultured with human or mouse ovarian granulosa cells , germ cells specific markers were expressed which indicate that the influence of micro-environment in vitro to the germ cells oriented differentiation of UC-MSCs is significant.3. Translocation and survived in premature-failed ovarian , the number of follicles and the level of estrogen all indicated that UC-MSCs maybe have POF repair potential and the influence of micro-environment in vivo to the differentiation of UC-MSCs is significant too.