Isolation and cultivation of retinal stem cells of embryonic rat with improved method / 解放军医学杂志

ABSTRACT

Objective To isolate and cultivate the retinal stem cells(RSCs) with improved method,identify the RSCs from embryonic rats,explore the RSCs' features in growth and differentiation,and research for an effective method to cultivate retinal stem cell in vitro from rat ocular tissues.Methods RSCs were isolated from rodents and humans and characterized in vitro.RSCs were isolated from SD rat embryonic retina(gestational age 18d) by shearing,blowing,digesting,centrifuging and filtering,and amplified by using serum-free DMEM/F12 medium containing basic fibroblast growth factor(bFGF),and then the cells were cultured and sub-cultivated by floating growth in vitro.After sub-cultivation,the RSCs were gathered by centrifuging,differentiation was induced with 5% fetal bovine serum(FBS),and the differentiated cells grew and adhered to the culture medium.Immunocytochemistry was employed with specific antibodies to identify RSCs and their potentiality of differentiation.The potentiality of RSCs to replace retinal cells in retinal degenerative diseases was confirmed by transplantation test.Results The primarily cultured retinal cells could grow in a floating state to,form compact cellular spheres,and they had the potential to proliferate in vitro in subsequent cultures to form new cellular spheres.After incubation in the medium with bromodeoxyuridine(BrDU),most of both primarily cultured and sub-cultivated cells expressed the neuroectodermal marker Nestin and BrDU.Differentiated RSCs expressed CD90.1(Thy1.1) after being induced.Conclusion The isolated retinal stem cells from SD embryonic rats,which are undifferentiated,multipotent and may self-renew,have the ability of proliferation and differentiation in vitro,and the differentiated RSCs possess the characteristics of retinal neural cells,including differentiation to retinal ganglion cells.