Primordial germ cell-derived human embryonic stem cell isolation , culture and identification
|School||Chongqing Medical University|
|Keywords||Human embryonic fibroblast Isolation Appraisal Mitomycin C|
Study Background and Purpose: Human embryonic stem cells in vitro and amplification conditions are very harsh, which is a critical factor feeder layer one. Researchers now commonly used mouse-derived fibroblasts as feeder layer to support human embryonic stem cells cultured in vitro inhibition of differentiation, but the mouse and human species varies greatly, the use of murine feeder layer is likely to bring Some pathogenic microorganisms or foreign protein contamination, giving the potential clinical application of the risk. This study aimed to explore the use of human embryonic fibroblasts instead of mouse fibroblasts support human embryonic stem cells cultured in vitro inhibition of differentiation, lay the foundation for clinical application. Key Project of the Ministry of Education, Chongqing Medical University research topics lt; WP = 7 gt; Methods: isolated, cultured and purified primary human embryonic skin fibroblasts (human embryonic dermal fibroblasts, hEDFs), after repeated subculture to get in In vitro long-term survival hEDFs. hEDFs Identification: ① light and electron microscopy its shape; ② immunocytochemical staining of human fibroblasts characteristic signs - vimentin, Ⅳ collagen expression; ③ alkaline phosphatase (AKP) staining AKP expression ; ④ chicken erythrocytes phagocytosis test to detect the presence or absence of phagocytosis. By cell counting assay different planting densities Human fibroblast clonal growth capacity, study their growth characteristics and growth curve. Long-term subculture in vitro amplification and frozen proliferation was observed after recovery and biological properties of stability. By MTT assay of mitomycin C on hEDFs growth, inhibit mitosis screening the best time and concentration. Observation of human embryonic germ cells (human embryonic germ cells, hEGCs) in the preparation of hEDFs feeder layer growth. Results: The obtained long-term survival in vitro hEDFs. Tests showed that the cell system has a typical fibroblast morphology, function, and enzyme immunocytochemistry and other biological characteristics of the chemical symbols. In 75ml flasks grown to 2.5 × 105 cells better, 5 to 17 generations hEDFs lt; WP = 8 gt; vigorous growth, the most suitable hEGCs culture; by freezing, recovery hEDFs growth remains strong, the cell mass to 32 generations can still be used hEGCs training. Final concentration of mitomycin C-treated 12.5μg/ml hEDFs 3 hours to completely inhibit the mitosis hEDFs, treated fibroblasts can at least maintain the good life 7d condition. Cultured on feeder layers in hEDFs hEGCs, cell proliferation and the formation of EGCs typical colony (see second part). Conclusion: The long-term survival in vitro hEDFs system; prepared with this hEDFs feeder layer, with the use of a medium containing inhibitor of differentiation, is fully capable of supporting hEGCs survival and proliferation in vitro and maintained in an undifferentiated state.