| Objective To observe the developmental competence of the human oocytes matured in vitro after parthenogenetic activation of electric pulsing and 6-DMAP.We did the basic research for establishment and qualification of human parthenogenetic embryonic stem cells.Methods Our experiment included the immature oocytes at stage of germinal vesicle or metaphase of first meiosis.They were cultured in vitro.Following in vitro maturation,the number of the mature oocytes at metaphase of second meiosisthe is 117.The number of handling group that human oocytes were activated by the parthenogenetic activation of electric pulsing and 6-DMAP is 59. The number of control group that human oocytes were not activated by artifical method is 58. The activation rate and developmental potential of the parthenogenetic embryos were observed. With keeping on observing and screening the blastocysts developed from parthenogenetic embryos,we made that inner cell mass were isolated by mechanical dissection and primary generation was in clonal culture.The culture condition of human parthenogenetic embryonic stem cells is seeding the human parthenogenetic embryonic stem cells on Mitomycin C treated human foreskin fibroblasts. The medium contains 80%Knock-Out DMEM,20% Knock-Out serum replacement,8ng/mL bFGF and so on. The human parthenogenetic embryonic stem cells colonies were passaged after being digested by 0.25% trypsin-EDTA. We got the results of the staining of specific markers and karyotype detection for the identification of human parthenogenetic embryonic stem cells.Results1. The activation rate in the handling group is 32.20% after parthenogenetic activation of pulsing and 6-DMAP on the human oocytes matured in vitro,and they were 12.07% respectively in controlled group.The activation rate was significantly higher in treated group than that of controlled group.2. The cleavage rate of handling group is 21.10%,and the blastulation rate of handling group is 25.00%. The cleavage rate of controlling group is 57.10%,and the blastulation rate of controlling group is 0.00%.Compared with the cleavage and blastulation rate of the two groups,the statistics analysis was not statistically significant.3. One parthenogenetic embryo of the handling group after parthenogenetic activation of pulsing and 6-DMAP developed to the stage of blastula.We vaccinated inner cell mass which was isloated on the feeder layer of human foreskin fibroblasts. The human parthenogenetic embryonic stem cells named hPESCs-081012 were passaged more than 20 generations in vitro. With our culture method, human parthenogenetic embryonic stem cells formed colonies, and expressed cell markers that characterize undifferentiated primate embryonic stem cells.4. The immunohistochemisty staining of specific markers of human parthenogenetic embryonic stem cells.The colonies of human parthenogenetic embryonic stem cells which were seeding on the feeder layer of human foreskin fibroblasts were immunoreactive for octamer-binding transcription factor 4,stage-specific embryonic antigen 3 and stage-specific embryonic antigen 4.But there was absence of expression of negative markers such as stage-specific embryonic antigen 1.5. The colonies of human parthenogenetic embryonic stem cells which were seeding on the feeder layer of human foreskin fibroblasts were immunoreactive for Alkaline Phosphatase,but the cells of feeder layers were negative.6. hPESCs-081012 had a normal chromosome karyotype (46, XX) and could be maintained in vitro in an undifferentiated state for extended periods of time.Cone I us i ons The combination of electric pulsing with 6-DMAP on the human oocytes matured in vitro proved to be an effective method of parthenogenetic activation. We successfully established the culture system of human foreskin fibroblasts and human parthenogenetic embryonic stem cells, which can maintain hPESCs-081012 in a highly proliferative and non-differentiating state. |
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