| The establishment of models studying germ cells development and differentiation in vitro based on ESCs differentiating into germ cells have high repeatability and good controllabilities, being of great significance to investigate germ cell development, cell proliferation regulation and treatment of human infertility. Recently, several groups have reported that ESCs differentiated into primordial germ cells (PGCs) and further into mature gametes in appropriate differentiating conditions in vitro. But the efficiency of ESCs differentiating into germ cells is still low.In this study, retinoic acid (RA)concentration was initially screened, in combination with testicular extracts, testicular cell conditioned medium, estrogen (E2) and gonadotropin (FSH), to identify the efficiency of germ cell differentiation derived from mESCs. mESCs was transfected with pStra8-EGFP in which GFP was regulated by Stra8 promoter (Stra8-EGFP). The cells were induced to form EBs and cultured in medium containing 0.2μM RA, 40% testicular cell conditioned medium, 1μg/mL E2 and 0.1 IU/mL FSH, in combination with co-culturing with Sertoli cells. The aim is to establish a stable system of male germ cell differntiation and a reliable model for the research of mammalian male germ cell specification.1. EB formation with mESCs was performed by hanging drop method. Compared with the quality of EBs with a series of cell concentrations, this study showed that EBs formed with a concentration of 1200 cells were superior to 800 and 400 cells per well. After three days, induced EBs were transferred onto 24-well plates treated with 0.1% gelatin with 10 EBs per well. For induction a series of RA concentration (20μM, 2μM and 0.2μM) and control were added. Both RT-PCR analysis and immunofluorescent staining analysis at day 4 and day 7 indicated the efficiency of 0.2μM RA inducing mESCs toward PGCs was apparently higher than the others. At day 4 and day 7, the rates of VASA positive cells were 36% and 52%, being highest in each set. Also, the rates of SCP3 positive cells at day 4 and day 7 were 32% and 45%, respectively. In addition, preliminary study results showed that the testicular cell conditioned medium, estrogen (E2) and gonadotropin (FSH) had obvious effects of inducing mESCs toward germ cells, but testicular extracts did not. 2. Plasmids expreesion GFP under the control of Stra8 promoter were transfected into mESCs using electric transfection technology to act as a germ cell differentiation marker. After two weeks of 200μg/mL G418 selection, cells stable transfected were obtained and had been cultured for 22 passages. Transfected mESCs were shown to express pluripotency markers Oct4, Nanog and Sox2 by RT-PCR analysis and immunofluorescent staining analysis, being able to differentiate into three germ layers. Spontaneously differentiation showed expression ofβ-ⅢTUBLIN, ISLET1and AFP by immunohistochemistry staining analysis, indicating transfected mESCs could be employed to differentiate toward germ cells.ES-Stra8 were induced to form EBs, transferred onto mitomycin C treated Sertoli cells and induced toward male germ cells adding 0.2μM RA, 40% testicular cell conditioned medium, 1μg/mL E2 and 0.1 IU/mL FSH. Under this inducing condition, GFP positive cells were detectable from day 7. For RT-PCR analysis and immunofluorescent staining analysis, germ cell specific markers OCT4, VASA, FE-J1and SCP3 were shown to express in both RNA and protein level. Cells in G1 phase of cell cycle increased obviously reaching G1%=91.118, approaching spermatogonia G1%=91.334. Anline blue staining also indicated occurrence of nuclear concentration. At day 31, sperm-like cells were found.3. mESCs induced by the method after 7d, labeled with DAPI for 12h after the transplantation of spermatogenic defects in mice testis. The transplanted cells were tested for the proliferation after 4 weeks and the seminiferous tubules which were damaged has been repaired to some extent.
|
PDF Full Text Request