Establishment Of Human Embryonic Stem Cells Line And Human Parthenogenetic Embryonic Stem Cells Line

Objective Embryonic stem cells are plouripotent cells derived from human primordial germ cells and inner cell mass of mammalian blastocyst. They are characterized by high proliferative and self renewal potential and ability to differentiate into many kinds of functional cell types. Parthenogenetic ES cells are derived from the ICM of a parthenogenetic blastocyst have developed from a single MⅡ oocyte and they only contain the maternal genome. As a result, these cells were regarded as tremendous potential resources for regenerative medicine, human developmental biology and cell-based therapies. The main goal of this study is to establish a stable and standard culture protocol of human ES cells in our own faculty. We support the prolonged undifferentiated growth of hES cells. To this end, the effects of three types of feeder cells for human ES maintenance were analyzed, in order to optimize the culture condition. At the same time, we also try to establish the clinical human embryonic stem cells line using the optimal culture condition and immature oocytes resources which could facilitate studies of therapeutic cloning for research and clinical applications.Materials and methods The donated immature oocytes and discarded embryos were collected from the Center for Reproductive Medicine, Tianjin Central Hospital for Obstetrics and Gynecology. The egg donors was clearly informed of all the study details and they signed detailed informed consent documents voluntarily. The culture condition of human ES cells is seeding the human ES cells on Mitomycin C treated human fibroblasts feeder layers. The medium contains85%KnockOut DMEM,15%KnockOut serum replacement,4ng/mL bFGF. We established human ES cells using the human feeder layers and the medium. The primary antibodies included SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81and AP and ability of differentiation in Vitro and in Vivo was identified. Then, human ES cells were seeded on mouse embryonic fibroblasts, human foreskin fibroblasts and human embryonic pulmonary fibroblasts cells respectively. The proliferation of human ES cells, such as morphology of colonies, colony number, the ratio of undifferentiated colonies and characterization of the human embryonic stem cell line were observed. Furthermore, based on the experience from human ES cell culture, using the human oocytes activated by the parthenogenetic activation, we did the basic research for establishment and qualification of human parthenogenetic embryonic stem cells. Meanwhile, we improved the culture system in order to facilitate the further study and application of such cells in research and clinical settings.Results With our culture method, human ES cell (H-TJ1&H-TJ2) and human parthenogenetic embryonic stem cell (P-TJ) form colonies, and express cell markers that characterize undifferentiated primate ES cells. All of the three feeder layers can ensure human ES colonies formed, maintain pluripotency, yet the morphology of colonies on different feeder layers varies obviously. There were more AP positive colonies and more cell quantity on mouse embryonic fibroblasts. As homologous feeder cells, the cloning efficiency on the human foreskin fibroblasts feeder layers was enough to enable to assure cell survival, and the differentiation rates were low enough to enable prolonged undifferentiated culture without continuous removal of the differentiated colonies from the culture. Human foreskin fibroblast feeder layer was better than the other two kinds of feeder cells.Conclusions We successfully established the culture system of human embryonic stem cells and human parthenogenetic embryonic stem cells. We also isolated human embryonic stem cells lines and human parthenogenetic embryonic stem cells line using the discarded embryos and in vitro maturation of MⅡ, GV oocytes, and maintained their characteristics of human embryonic stem cells and human parthenogenetic embryonic stem cells characteristics. Human foreskin fibroblasts could maintain the characters of the human embryonic stem cells and the long-term cultivation as homologous feeder cells. The modified cultural system could maintain human parthenogenetic embryonic stem cells long-term cultivation. The human parthenogenetic embryonic stem cells is close to clinical grade human embryonic stem cell line standards. This should facilitate studies of therapeutic and laid the foundation for the application.