Reconstruction Of Human-Goat Interspecies Scnt Embryo And Derivation Of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into any tissue in the human body; therefore, they are a valuable resource for regenerative medicine, drug screening, and developmental studies. hESCs originate from cells of human embryo. However, the insufficiency of human embryo greatly limits the study and application. But interspecies nuclear transfer technology, which animal oocyte is as the recipient and human somatic cell as the donor, brings new approach to get hESCs. This thesis attempted to reconstruct the human-goat interspecies nuclear transfer (hgiNT) embryo using goat oocyte as recipient and human foreskin fibroblasts (HFFs) as donor, and to derivate and culture the hESCs from interspecies nuclear transfer (iNT-hESCs). The main contents include several aspects as follow:1. To choose genetic homogeneous donor cells used for nuclear transfer, single human foreskin fibroblast was cultured in conditioned medium (CM) or co-cultured in fresh medium with feeder cells, the cell in the control was cultured with fresh medium without feeder. Then karyotype of colony cells from both methods was analyzed by Giemsa staining. The results showed the survival rate of colony in co-culture group was significantly higher than that of control (31.43 % vs. 5.56 %, P<0.05), and similar to that of CM group (31.43 % vs. 27.27 %, P>0.05), the multiplicated culture rate of colony in co-culture group was significantly higher than that of control (11.43 % vs. 0 %, P<0.05), five of seven colonies had the normal human karyotype (2n=44+XY). The resulted suggested both methods used in the experiment can scan the donor using for nuclear transfer.2. To explore the technique parameters for hgiNT, in this experiment, firstly, the electrofusion parameter were tested, secondly, the interval from fusion to activation was tested, thirdly, two embryo culture system, mSOFaa and G1/G2, was compared to observe the effect on development of reconstructed embryos. In the results, the optimal electrofusion voltage, pulse duration and pulse times were 36 V, 20μs and 2 times respectively, the optimal interval time was 4 h, the cleaved and blastocyst rate in G1/G2 system were significantly higher than that in mSOFaa system (71.81 % vs. 64.78 %, 9.73 % vs. 4.76 %;P<0.05). The results suggested electrofusion parameter and the interval of human-goat interspecies NT is similar to that of oocyte provider i.e. goat SCNT, however, embryo development require closer culture condition of donor somatic cell provider i.e. human embryo culture system.3. To investigate the effect of Trichostatin A (TSA) on hgiNT embryo development, we treated HFFs for 24 h prior to nuclear transfer, or reconstructed embryos for10 h following oocyte activation with TSA. 50-75 nM TSA treatment of donor cells increased blastocyst development compared to control (9.91 % and 12.10 % vs. 8.47 %, P>0.05). Blastocyst rate of 25 nM group was significantly higher than control (16.41 % vs. 7.63 %,P<0.05). These results indicate that TSA-treatment can dramatically improve subsequent in vitro development of cloned embryos, and could be partially erase preexisting epigenetic marks of donor cells.4. To choose the appropriate treatment time, feeder cells of HFFs, MEFCs and GEFCs were treated with 10 mg/L mitomycin C for 1.5h, 2h, 2.5h or 3h. After freezing and thawing, they were cultured for 12h. Then growth situation were observed. ICMs were inoculated on different source and density feeders to compare the effect of both on growth of hESCs. Results showed two hours is the optimal time that feeder cells are treated by mitomycin C. The cell feeders originated from HFFs and MEFCs can supported the growth of hESCs, the cell density of 3×104/mL has the highest adherent rate and the lowest differentiated rate of hESCs.5. To overcome the limitation of application to clinic of hESC cultured in these medium contained by animal components, ICM derived from hgiNT embryo were cultured in HESCO system whose component is feeder-free, serum-free, animal-free and defined. The culture system that HFFs used as feeder cells was the control. The results showed that the iNT-hESCs from HESCO system had similar cell morphology, proliferation speed, alkaline phosphatase activity and genetic stability to that from HFFs system. These results suggested that the HESCO can maintain the growth and undifferentiation of iNT-hESCs, which has great significance to the application of hESCs to clinic.