Preliminary Investigation On The Interaction Between Somatic Cells And Preimplantation Embryo

Objective To known whether the somatic cells, such as BMMSC, HSC or MEF can dedifferentiated into pluripotent stem cells, similar to embryonic stem cells under the environmental impact of preimplantation embryos(8-cell and blastocyst)Methods 1. Isolation, cultivation and identification for the primary embryonic fibroblast(MEF), mouse bone marrow mesenchymal stem cell(BMMSC), hematopoietic stem cell(HSC) from SCE mouse which carrying report gene EGFP. The stable passaged cell lines were obtained. 2. Cultivation of MEF and BMMSC with ESC Medium after exposed to 5-azacytidine for DNA demethylation. 3. Microinjection of EGFP-MEFs/EGFP-BMMSCs before or after demethylation and EGFP-HSCs without demethylation into 8-cell embryos or blastocysts of wild-type ICR mice. The reconstructed embryos were cultured in KSOM droplets for over 48 hs and observed for embryo development and chimera formation under DIC/fluorescence microscope. 4. Transplantation of reconstructed 8-cell embryos or blastocysts injected with donor somatic cells after cultivation in KSOM into wild-type 2.5d pseudopregnant ICR females uterus.Results 1. SCE-MEF and its demethylation cell lines were established. 2. SCE-BMMSCs cell lines were obtained with conventional method and identified by directional induced differentiation to osteogenic and adipogenic with vonkossaand oil red O staining, molecular marker analysis of CD34 negative, OCT4 and NANOG positive. 3. SCE-HSCs cell lines were established by 3D-cultivation and colonies picking method utilized Mebiol Gel and identified by molecular markers analysis of CD34+, ABCG2+, CD133+ and directional induced differentiation to myeloid and erythroid cell lines. 4. SCE-MEFs / BMMSCs / HSCs were respectively injected into 8-cell embryos and blastocysts before and after demethylation. The donor cells in the reconstructed embryos were survived but chimeric embryos or offsprings were not observed.Conclusion 1. Mouse bone marrow mesenchymal stem cells, hematopoietic stem cells and embryonic fibroblasts or demethylated mesenchymal stem cells and fibroblasts can survive in reconstructed early embryos after microinjection of mouse 8-cell embryos or blastocysts, but they did not proliferated inside the embryos and did not formed chimera. It might indicate that these cells are difficult induced to change differentiation state by early embryonic cell. 2. It was found for first time that the primary embryonic fibroblasts of mouse after demethylation and cultured with ESC medium can transdifferentiaed to closely adhered epithelioid cell clonies and could be passaged. The clonies expressed a variety of characterized molecular markers of embryonic stem cells, such as OCT4, NANOG and SSEA-1. The results show that demethylation united with ESC medium cultivation can induce the mouse embryonic fibroblasts transdifferentiation in the direction for embryonic stem cells. This might provide a useful new idea to improved the i PSC technology, establishing a new method of non-gene transfection in preparation of i PSC. 3. A novel technology for isolating mouse hematopoietic stem cell(HSC) based on temperature-sensitive hydrogel 3D cultivation was successfully established for the first time. The new method is easily, quickly and efficiently in separatingHSC colonies laiding an important foundation for hematopoietic stem cell research and the further development of human HSCs separation and regenerative medicine for blood diseases therapy.