IPS Cells Reprogrammed From Mouse Sertoli Cells

Somatic cells could switch its fate to induced pluripotent stem cells (iPS cells) by transmitting a sum of transcription factors. iPS cells, like embryonic stem (ES) cells, have the capacity of maintaining self-renewal and differentiating into various cells. Furthermore, iPS cells could avoid immune rejection and ethical issues in the usage of ES cells. Hence, this technology brought broad prospect in basic scientific research and regenerative medicine field. At present, although varieties of iPS cells could be induced from different sources, the quality of iPS cells changed a lot, which showed difference in the aspect of the gene expression, epigenetic modification and differentiation. Thus, to get high qualities of iPS cells, the first step is to choose proper cell origin according to the experiment purpose.Sertoli cells from testis have the abilities of immune privilege and maintaining the stable microenvironment for sperm cells. Sertoli cells proliferate fast and have a good growth status in vitro. Moreover, Sertoli cells could express Klf4and c-Myc, which may promote the reprogramming process. The high purity of Sertoli cells could eliminate the interference of heterogeneous cell populations in the induction process. Therefore, Sertoli cells are ideal materials for iPS cells induction.Nitric oxide (NO) is involved in the survival and proliferation regulation of stem cells as an intracellular signaling molecule. The regulation of NO is concentration dependent; high NO levels promote stem cells differentiation, while low levels could contribute to maintain the pluripotency of stem cells. An understanding about the functions of NO to iPS cells is important for the long-term culture of iPS cells.The main purpose of this study is to induce mouse Sertoli cells to iPS cells. The research mainly contains the following parts:the amplification and detection of retroviral vectors; separation and purification of Sertoli cells; retrovirus package and infection; production and identification of iPS cells; the effects of low concentration NO to iPS cells. The main conclusions are as follows:1. We confirm the accuracy of the retroviral vectors by enzyme digestion and DNA Sequencing.2. We obtain high purity Sertoli cells from mouse testisSertoli cells were separated by two-step enzyme digestion and purified by density gradient centrifugation and differential adhesion. Sertoli cells could proliferate in vitro and keep good morphology. By RT-PCR, we confirmed the specific markers of Sertoli cells SGP-1, SGP-2, Transferrin, ABP and AMHII were expressed, Klf4and c-Myc were weakly expressed, and the pluripotent genes Nanog, Sox2and Oct4were not expressed.3. We successfully establish the iPS cell lines from mouse Sertoli cellsSertoli cells were infected with retroviral vectors that led to the expression of Oct4, Sox2, Klf4and c-Myc. GFP was tranduced to monitor the infection efficiency as well as an indicator of exogenous gene in part of induction experiments. As early as3days post-infection, cells started to express GFP and morphologically differed from uninfected Sertoli cells. As cell culture prolonged, the single cell was getting smaller, more and more cells were aggregating to form colonies. At day18, GFP were silenced, compact and round-shaped colonies formed. The colonies we got could be renewed for a long period of time (more than20passages) and the iPS cells kept a normal karyotype of40chromosomes, including X and Y.4. The identification of iPS cellsThe iPS cells showed AKP positive and expressed the majority of ES cell-marker genes, Nanog, Oct4, Sox2, Rexl, Fbxl5, E-cad and Fgf4. Real time PCR showed the levels of Oct4and Fgf4had no significant difference compared with those in ES cells. By immunoflurscence, these iPS cells were positive for Nanog and Oct4. In the reprogrammed iPS cells, the exogenous genes were silenced and the endogenous genes were reactivated. Moreover, iPS cells have similar cell cycle distribution with ES cells. iPS cells have differentiation potential, embryoid bodies (EBs) in vitro and teratoma in vivo was formed. We obtained EBs by hanging drops and then differentiated into cells with three primary germ layers. For in vivo differentiation, the teratoma form iPS cells contained a wide variety of tissues derived from all three germ layers.5. Low concentration of NO could promote the expression of Nanog in iPS cells and maintain the expression levels of Oct4, Sox2, Bcl2, Bcl2lll and Bac, decrease the expression levels of Bakl and Casp7. It demonstrates low concentration of NO could maintain the pluripotency of iPS cells and delay the iPS cells apoptosis without LIF in the medium.