| Embryonic stem cells (ESCs) have the characteristics including the infinite proliferation, self-renewal and multipotent differentiation in vitro. Embryonic stem cells can be induced to differentiate into almost all the types of the cells either in vitro or in vivo. Parthenogenetic activation refers to the early embryonic development process which results from the mitotic divisions of the oocyte in MII state. These mitotic divisions happen in the oocyte in MII state which stays diploid by the stimulation of some physical and chemical factors without fertilization. Parthenogenetic blastocyst is the one which is stimulated by the parthenogenetic activation of the oocyte. Pharthenotic embryonic stem cells line is separated from the inner cell mass of the parthenogenetic blastocyst. The pharthenotic embryonic stem cells have the potential of differentiation as the embryonic stem cells. Besides, the mammal parthenogenetic embryos come from haploid gametes, so they cannot develop into complete individual.The utilization of the pharthenotic embryonic stem cells not only provides new source for the research of the stem cells but also averts many ethical problems. The retinal pigmented epithelium cells which control the chemical composition in the subretinal space and nourish the retina by offering the nutrition and removing waste locate between the nerve of retina and the choroid, thus they are the outer layer of the blood-retina barrier. The abnormal proliferation, structural damage and loss of function of the retinal pigmented epithelium can lead to many diseases of the retina. The literatures in recent years report that there are many advantages of the retinal pigmented epithelium cells derived from human embryonic stem cells as a cell transplant donor source.Compared to the the ARPE-19(the cell lines originated from the human RPE),the cell form, gene expression and immune histologic features of the RPE cells derived from the human embryonic stem cells are closer to the human retinal pigmented epithelium cells and they have the function of phagocytosis, besides, the cells have shown some effects of improving the visual function in the animal experiments.In the experiment we adopt the cultured system from the the parthenogenetic embryonic stem cells line originated from the human. We make a tentative exploration of the stable passage and identification of human parthenogenetic embryonic stem cells and the related content of the RPE cells induced by the cytokines. The genotype of the RPE cell is the same as the one of the donor that provides the oocyte; there is completely no rejection effect of the RPE cells in the retina regeneration treatments. At the same time we confirm that the there is differentiation potential in the parthenogenetic embryonic stem cells just as the human embryonic stem cell lines which are derived from normal fertilization embryos. All of these above not only lay the experimental basis for the clinical treatment of parthenogenetic embryonic stem cells, but also provide the cell source and theoretical basis for the application of the regeneration treatments in clinical in the future.Objective The human parthenogenetic embryonic stem cells which have been established and stably passaged are thawed and recovered, and then the human parthenogenetic embryonic stem cells are induced to differentiate into the retinal pigmented epithelium cells. The purified pigments cells are tested by immunohistochemical analysis. We can see the thawed human parthenogenetic embryonic stem cells line have the same characteristic of stably passaging and efficient differentiation potential as the human fetal retinal pigment epithelium cells.Methods The established human parthenogenetic embryonic stem cells line from the Reproductive Medicine Center in Tianjin Central Hospital of Gynecology Obstetrics are used. The16th and18th generations of the human parthenogenetic embryonic stem cells are thawed and recovered, the human foreskin fibroblasts is used as the feeder layer to establish the human parthenogenetic embryonic stem cells line and then it is passaged stably,the medium is consisted of80%KnockOut DMEM,20%KnockOut KSR,2mM L-glutamine,0.1mM p-mercaptoethanol,8ng/ml bFGF. The specific markers such as SSEA-3, SSEA-4, SSEA-1, TRA-1-60, TRA-1-81, transcription factor Oct-4and alkaline phosphates are identified, the ability of the differentiation potential of the human parthenogenetic embryonic stem cells line is completely evaluated.Finally, the human parthenogenetic embryonic stem cells are induced to differentiate by the four steps:the multiplication of the human parthenogenetic embryonic stem cells, differentiation and amplification of the pigment epithelial cells, differentiation into retinal pigment epithelial cell. Immunohistochemical stains are used for the cells in each phage to observe the results of the differentiation.Results Using the culture method of human parthenogenetic embryonic stem cells line in our laboratory and the refined homology culture system, the thawed hPESCs-100616and hPESCs-100618are both passaged more than five generations, maintaining the characteristic of human parthenogenetic embryonic stem cells and showing the fine performance of proliferation and self-update. In the process of differentiation into human retinal pigment epithelial the human parthenogenetic embryonic stem cells line can differentiate into pigmented cells after3-4weeks in the culture medium including the bFGF. The clusters of the pigment cells grow in size and number after an additional2-3weeks time culture. Granules of brown pigment appear in the edges of the cytoplasm of cells in4-6weeks. The similar result can also be seen in the culture without bFGF. This process is consistent with the one in which human fetal retinal pigment epithelium cells acquire pigmentation. Epithelial morphology and the expression of pigment suggest this kind of cells is the RPE cells are originated from the human parthenogenetic embryonic stem cells.Conclusions The established human foreskin fibroblasts culture layer and the culture system of hPESCs are completely suitable for the culture of thawed human parthenogenetic embryonic stem cells, which can keep the human parthenogenetic embryonic stem cells in a high degree of proliferation but without spontaneous differentiation characteristics. The cytokines can induce the hPESCs to differentiate into a large number of human retinal pigment epithelial cells, which provides safer cell source and a more solid theoretical foundation for the application of the retinal pigment epithelial cells in the clinical research. |
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