Studies On Optimization Of Cryopreservation System Of Human Embryonic Stem Cells And Their Differentiation Intoretinal Pigment Epithelium Cells

Human embryonic stem cells(h ESCs) have the ability to self-renew and are characterized of differentiating into any functional cell type of three germ layers, so h ESCs catch more and more attention in the basic and clinical research. h ESCs hold great promise in clinical applications, and some h ESCs-derived functional cells have been used in early stage clinical trials, so how to obtain large amount of h ESCs becomes more and more important.The present cryopreservation efficiency of h ESCs is pretty low, so the cryopreservation system of h ESCs needs to be optimized to meet the larger demand in the future. This study optimized freeze-store liquid ingredients based on the laboratory commonly used slow cryopreservation and fast recovery method. First, we compared commercially available cryoprotectants with commonly used laboratory cryoprotectants, then optimized the cryoprotectants. We found that the optimization of cryoprotectant had better cryopreservation efficiency and lower cost. Finally, the pluripotency of recovery h ESCs was examined. The h ESCs after thawing were positive for AP staining, and the pluripotent characteristics were confirmed by immunofluorescent staining and teratoma test. The above results showed that the h ESCs after freezing-thawing had normal characteristics of h ESCs, this cryopreservation system did not affect the characteristics of h ESCs.Some functional cells derived from h ESCs have been approved in clinical trials to replace conventional treatment methods for therapy of certain diseases, in the future this would have a good prospect in clinical development. Retinal pigment epithelium(RPE) was one of the functional cells, which could cure many eye diseases theoretically and had positive effect in clinical treatment. RPE cannot regenerate, but they can be obtained by h ESCs differentiation. By this time its differentiating efficiency is barely satisfactory, and the differentiation period is too long. So it’s important to improve the differentiating efficiency. In this study, Activin A was added during differentiation day 1 to 7 based on normal differentiation method. Our results showed that Activin A could promote h ESCs differentiating into RPE, and the differentiated cells had both shape and microscopic structure of RPE. Furthermore, the differentiated cells could express the specific markers and proteins of RPE and had the function of phagocytosis. This optimized differentiation method provides a reference in the future clinical application.