Study On Embryonic Stem Cells From Reconstructed Embryos In Mice

Therapeutic cloning can be used restoring human pathological tissue and organ by somatic cell nuclear transfer and ES cell biotechnology, which not only can avoid graft versus host reaction, but also provide lots of seed cells used in cell engineering, tissue engineering and organ transplant. In order to provide knowledge and experience in clinic application of human therapeutic cloning, feasible reconstructed condition, activation method of reconstructed embryos, application of different donor cells in somatic cell nuclear transfer, suitable isolation method of embryonic stem cells, isolation and cultivation of ES cells from reconstruction embryos and others were studied in this paper. 1. Mice embryos had normal image and synchronous development by ethanol or SrCl2 activation and co-culture with mice oviduct epidermal cells; Mice activation rate of parthenogenetic embryos in ethanol plus feeder cells group was highest 97.15%; Mouse 2-cell rate of parthenogenetic embryos in ethanol plus feeder cells group was highest 93.14%,; Mice morula rate of parthenogenetic embryos in ethanol plus feeder cells group was highest 80.38%; Mice blastocyst rate of parthenogenetic embryos in SrCl2 plus feeder cells group was highest 76.21%; Mice hatched inner cell mass rate of parthenogenetic embryos in SrCl2 plus feeder cells group was highest 75.28%; Mice embryonic stem cell hatched rate of parthenogenetic embryos with SrCl2 plus feeder cells group was highest 29.21%, significant difference with ethanol plus feeder cells group, and SrCl2 group (p<0.01). It was indicated that Kunming species mice parthenogenetic embryos activated with SrCl2 have stronger development ability in vitro. 2. Isolation rate of mice inner cell masses with cardiomyocyte media conditioned was 76.30 %, leukemia inhibitor factor media was 59.35 %, and difference between two medias was significant (p<0.01); Isolation of mice inner cell masses with leukemia inhibitor factor media was earlier than that with cardiomyocyte media conditioned, mean time difference was 11.2 h; Differentiation rate of embryonic stem cell masses of 48th h with cardiomyocyte media conditioned was 51.55 %, leukemia inhibitor factor media was 31.69 %, and difference between two medias was significant (p<0.01); Normal karyotype rate of mice embryonic stem cells with cardiomyocyte media conditioned 78.6 % was slightly higher than that with leukemia inhibitor factor media 76 %. It was indicated that rat cardiomyocyte media conditioned is more feasible for isolation and cultivation of Kunming species mouse embryonic stem cells than leukemeia inhibitor factor media conditioned. 3. Encleation rate of mice oocytes 14~16 h after hCG injection was highest 80.09%; Activation rate of embryos reconstructed during 20~22 h was highest 85.14 %; Blastocyst rate of mice oocytes during 14~16 h was highest 13.94%, significant difference with 16~18 h (p<0.05), 18~20 h, 20~22 h (p< 0.01). It was indicated that encleation time of mice oocytes 14-16 h after hCG injection is good. 4. 195 of 325 mice reconstructed embryos with cumulus cells in SrCl2 plus feeder cells group were developed to 2-cell embryo, 71 to morula, 38 to blastocyst, 9 to embryonic stem cells, in which 5 embryonic stem cells could be passaged and cultivated successfully; Embryonic stem cell colonies isolated were with island-like image, strong positive by AKP staining, could become embryoid bodies and spontaneously differentiate into epidermal-like cells around them in vitro; 162 of 289 mice reconstructed embryos in SrCl2 group were developed to 2-cell embryo, 10 to morula, 3 to early blastocyst. It is approved that feeder cells, modified media, and cardiomyocyte media conditioned play a key role to development of reconstructed embryos in vitro, and mice cumulus cells as donor cells of somatic cell nuclear transfer can be applied in therapeutic cloning field. 5. 227 of 420 mice reconstructed embryos with skin fibroblast as donor cells in SrCl2 plus feeder cells group were developed to 2-cell embryo, 72 to morula, 29 to blastocyst, 6 to embryonic stem cell, in which 3 embryonic stem cell-like colonies could be passaged and cultivated successfully; Karyotype analysis to 5th passage embryonic stem cells was researched, normal rate of karyotype was 77.84 %, 75.18 %, 77.20 % respectively; Embryonic stem cells isolated were island-like, positive by AKP staining, and could spontaneously differentiate into epidermal or shuttle-like cells around them in vitro; In addition, after frozen and thawed routinely, embryonic stem cells were with strong proliferation and previous image; 184 of 372 mice reconstructed embryos with skin fibroblasts in SrCl2 group were developed to 2-cell embryo, 5 to morula. It is indicated that feeder cells, modified media, and cardiomyocyte media conditioned are key to development of reconstructed embryos in vitro, and mice skin fibroblasts as donor cells of somatic cell nuclear transfer can be feasible in therapeutic cloning field. 6. 128 of 312 mice reconstructed embryos with blood lymphocytes as donor cells inSrCl2 plus feeder cells group were developed to 2-cell embryo, 69 to morula stage, 6 to blastocyst stage, 3 to inner cell mass, which had similar image with inner cell masses from embryos produced in vivo; 121 of 343 mice reconstructed embryos with blood lymphocytes in SrCl2 group were developed to 2-cell embryo, 2 to morula. It was indicated that feeder cells, modified media, and cardiomyocyte media conditioned have a key role to development of reconstructed embryos in vitro, and it is feasible to construct embryos with peripheral blood lymphocytes in mice, warranting further study in this area. 7. Nuclear transfer embryonic stem cells from reconstructed embryos with cumulus cells and skin fibroblasts were induced and differentiated into epithelial cells, which had high purity and cell proliferation capability, cytokeratin identification of epithelial cells was positive, so epithelial cells can be used as seed cells in clinic application. 8. Cardiomyocytes with rhythmical beat were induced and differentiated from nuclear transfer embryos reconstructed with cumulus cells and fibroblasts as donor cells, which still had the rhythmical synchronization beat after passaged; Immunocytochemistry identification of cardiomyocytes a-sarcmeric actin and cardiac troponin-T was positive, and lots of glucogen were in cardiomyocyte cytoplasma; Cardiomyocytes had very strong division potential in vitro. It was indicated that cardiomyocytes could be induced and differentiated from nuclear transfer embryos reconstructed, and applied in clinic as seed cells.