| Stem cell researches in recent years holds considerable medical promise in the generation of differentiated cells for cell replacement. The effective and safe clinical application of stem cells needs mechanism researches and abundant in vivo and in vitro experiments, which needs materials of stem cells from human resources. Human embryonic stem cells (hESC) are undifferentiated totipotent cells isolated from inner cell mass of human blastocysts. Infinite proliferation and undifferentiated status are symbols of hESC. Various types of cells can be obtained from hESC after induction. Human ESCs is ideal materials for studies on medicine, biology, pharmaceutical sciences, etc.The limiting fator in hESC studies is the resource of human embryos. Embryos with fragmentation are aften observed in the process of human in vitro fertilization-embryo transfer (IVF-ET) with rare implantation and usually abandoned by the patients. Such embryos can be used in the stem cell researches.The developmental potential of human embryos with low morphological scores can be improved by the removal of fragments. The epigenetic status is related to the developmental potential of the blastomeres. The higher levels of H3arginine methylation predispose blastomeres to contribute to the pluripotent cells of the ICM.The human embryos with fragments over50%were used in this study. The zona pellucida was digested by protease and the fragments were removed. The developmental potential of manipulated embryos was observed. The blastocysts obtained were used to establish the hESC lines. EGFP-mRNA was synthesized in vitro and injected into the blastomeres of mouse embryos. The green fluorescence was observed to determine the mRNA expression in the embryos.Compared with the successes in animal cloning, progress in human SCNT studies has been slowed due to the legal and social considerations that limit the availability of human oocytes for research. In addition to oocytes, mitotic zygotes can support nuclear reprogramming. Polyspermic zygotes from human in vitro fertilization programs, which are routinely discarded, could be used in NT studies as a comprehensive source of NT recipients. A study was conducted with this goal, but the preimplantation developmental potential of the SCNT embryos from polyspermic zygotes was limited to the8-cell stage.Incomplete reprogramming is, at least in part, responsible for the limited developmental potential of cloned embryos. The partial erasure of pre-existing epigenetic markers by a DNA methyltransferase inhibitor (such as5-aza-dC) would be beneficial to cloned embryos.In the present study, we used mitotic human polyspermic zygotes as recipients for nuclear transfer. The effects of CB treatment on the survival rate of the manipulated zygotes and the effects of5-aza-dC treatment on the cleavage rate of the reconstructed embryos were observed.Part I The removal of fragments of human embryosMethods:(1) The removal of fragments. The zona pellucida of human embryos was digested by protease and the blastomeres were scattered in the calcium/magnesium free medium. The blastomeres were treated with PHA for2hours and then cultured in regular medium until the blastocyst stage.(2) The establishment of hESC lines. The ICM of the blastocysts were isolated and cultured for continuous passages. Results:(1) Blastocysts were obtained in every experimental group. There was no significant difference of the blastocyst developmental rates among the control groups and PHA treatment group (30.0%,35%, and36.4%, respectively). The blastocyst developmental rate of fragment-removal group without PHA treatment decreased significantly (11.5%).(2) No hESC line was obtained. Conclusions:(1) Highly qualified blastocysts could be obtained after fragment removal.(2) PHA treatment was benificial to the development of the scattered blastomeres.Part Ⅱ mRNA microinjection to mouse embryosMethods:(1) Construct the pcDNA3.0-EGFP recombinant plasmid.(2) Synthesize the EGFP-mRNA in vitro.(3) Collect mouse embryos in the2-cell stage and inject the EGFP-mRNA into one of the blastomere. Observe the development of the manipulated embryos and the fluorescence expression. Results:(1) Compared to the control group, there was no significant difference in the blastocyst developemental rate in the microinjection groups, whether or not the polyA was added the EGFP-mRNA (Control group:64.41%; Injection group without polyA (R):48.98%; Injection group with polyA (RA):55.74%).(2) The mouse embryos did not express the green fluorescence after the injection of EGFP-mRNA without polyA. After injection of EGFP-mRNA with polyA,36.07%of the mouse embryos expressed the green fluorescence. Conclusions:(1) The mRNA micro injection itself did not compromise the developmental potential the manipulated embryos.(2) The successful tailing of polyA was the key fator of the expression of the synthesized mRNA.Part Ⅲ Nuclear transfer using human polyspermic zygotes as recipientsMethods:Six experimental groups were included, as follows:PSZ-C, cultured without any manipulation to investigate the efficiency of the culture system; PSZ-H, stained with Hoechst33342and exposed briefly to ultraviolet (UV) light to eliminate any adverse effects of DNA staining and UV exposure on embryo development; EO, one of the three pronuclei was extracted in interphase to investigate the effects of micromanipulation on embryo development; NT-CB, polyspermic zygotes were treated with7.5μg/ml Cytochalasin B during the enucleation process; NT-Aza and NT, nuclear transfer with or without5-aza-dC treatment, respectively, for the comparison of the cleavage rate.(1) Nuclear transfer. The nucleus of the human polyspermic zygote was removed the metaphase and the nucleus of the human cumulus cell was microinjected into the denucleated zygote. The reconstructed embryos were treated with or without5-aza-dC for the comparison of the cleavage rate.(2) The human polyspermic zygotes in cleavage stage (PSZ-C), the reconstructed embryos treated with or without5-aza-dC (NT-AZA-C and NT-C) were collected for5-methylcytosine immunodetection. The confocal microscopy and quantitative analysis were conducted. Results:(1) The cell cycle distribution of the human cumulus cells.72.6±6.0%of the human cumulus cells were in the G0/G1stage.(2) Four blastocysts were obtained in the PSZ-C group (11.1%). One blastocyst was obtained in the PSZ-H group (7.7%).。 Four blastocysts were obtained in the EO group (12.9%). There was no significant difference of the8-cell and blastocyst developmental rates among the above groups.(3) The nuclear transferred embryos did not develop beyond the8-cell stage. The survival rates, cleavage rates, and8-cell developmental rates in the NT-CB, NT, and the NT-Aza groups were (48.3%,21.4%,0%),(73.8%,48.9%,11.1%), and (81.3%,65.4%,32.7%), respectively. The survival rate, cleavage rate, and8-cell developmental rate in the NT-CB group were significantly lower than those in the NT and NT-Aza groups. There was significant difference of the8-cell developmental rate between the NT and the NT-Aza groups.(4) There was significant difference of the5-methylcytosine fluorescence intensities between the NT-AZA-C and the NT-C groups. The fluorescence intesity of the NT-AZA-C group was lower than that in the NT-C group, but still higher than than in the PSZ-C group.(5) The abnormal nuclear division patterns were found in the NT embryos.Conclusions:(1) The cell cycle of the human cumulus cells was suitable for the nuclear transfer.(2) The culture system was suitable for the embryo culture.(3) The Hoechst33342staining and the transient exposure to UV light did not affect the embryo development.(4) The5-aza-dC treatment was benificial to the development of the reconstructed embryos.(5) The abnormalities in the epigenetics and nuclear division patterns might contribute to the limited developmental potential of the reconstructed embryos.Conclusions1. Highly qualified blastocysts could be obtained after fragment removal. PHA treatment was benificial to the development of the scattered blastomeres.2. The mRNA microinjection itself did not compromise the developmental potential the manipulated embryos. The successful tailing of polyA was the key fator of the expression of the synthesized mRNA.3. The5-aza-dC treatment was benificial to the development of the reconstructed embryos. The abnormalities in the epigenetics and nuclear division patterns might contribute to the limited developmental potential of the reconstructed embryos. |
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