Regulation Of Murine Primordial Germ Cells Development By Nanog

Derived from the inner cell mass (ICM) of the blastocyst and the primordial germ cells of the genital ridge, embryonic stem (ES) cells and embryonic germ (EG) cells have been the focus of the life sciences’ attention over years, due to their capacity to proliferate indefinitely in vitro and to differentiate into the cells of all three germ layers. Murine embryonic stem cells have wide research values, especially in the research field of mammalian development. Murine embryonic stem cells are the best model in vitro to study those key factors and genes for lineage determination and differentiation during mammalian embryogenesis, which is the important fundamental research work for clinical treatment via stem cell engineering. Besides, PGCs and EG cells as the origin of stem cells play a critical role in studying the regulation of normal germ cells development and abnormal germ lineage tumorigenesis.The biology of development and the biology of stem cell supplement each other, and share their common focus on self-renewal and differenti-ation regulation of multipotent stem cell. For the cells of ICM, PGCs in vivo and the ES cells, EG cells in vitro; self-renewal molecular mechanism of which has always been the highlight in their research field, yet it has not been clear till today. There are fewer perspectives on PGCs and EG cells due to the difficulty in culture. Up to now, several transcriptional factors have been identified to be essential for the ES cell identity, including Oct4, Nanog and an HMG factor Sox2. They cooperate to regulate the core downstream targets which are related to pluripotency, self-renewal and differentiation. Functionally, external sigaling eventually leads to distinct regulation of these genes to maintain a multipotent cell phenotype or not.Prior to entering gonadal ridges and contacting intimately with somatic cells to become authentic germ cells, PGCs behave transiently as diploid precursors for germ cells. PGCs can be considered as the precursors for germ cells which develop into gametes in vivo, and they also can be considered as multipotent stem cells because they can be long-term cultured in vitro as EG cells. Therefore it has double meanings to explore the regulation of PGCs development concerned with the diagnosis, treat of germ lineage disorder and stem cell engineering. Nanog is a newly highlight in the biology of development and the biology of stem cell. Yet most of the studies in existence have focused on its influence on the early embryo and ES cells pluripotency maintainance.In order to make a further study on the regulation of ES cells, and to explore the influence of Nanog on PGCs development, we studied as follow:1. Compared the biologic characteristics between mouse embryonic fibroblasts (MEFs) and STO cell line in order to establish an optional feeder layer system.2. Established a long-term culture system for ES and EG cells.3. Explored the temporal and spatial expression of Nanog in developing PGCs of mouse.4. Explored the influence of RNA interference-mediated silencing of Nanog on EG cells. A brief presentation about the studies is given as follow:1. Establishment of MEFs and STO feeder layers:the MEFs were successfully isolated from the mouse embryos at 13.5-14.5dpc (days post-coitus) through primary tissue digestion and tissue piece culture. STO cells proliferated more rapidly than MEFs showed by MTT growth curves and FASC cell cycle assay, but they needed a passage in a shorter period. MTT showed that MEFs mitosis could be efficiently repressed after being treated with mitomycin C 10μg/ml for 2.5-3.5 hours or 20μg/ml for 1.5-2.5 hours, and the MEFs layer could maintain at least for 7 days. While the optional condition of STO treated by mitomycin C was 5-10 μ g/ml for 1.5-2.5 hours, and the feeder layer could maintain almost for 5 days. Considered the growth character, MEFs feeder layer was optional for primary ES cells. We considered STO feeder layer was better for EG cells, because of stronger fluorescence intensity showed by STO detected by FASC; however it related to abnormal karyotype. Our study optimized the feeder layer condition for ES cells and EG cells.2. Isolation, purification, culture and identification of mouse ES and EG cells in vitro:In our study,3.5dpc blastocyst was fed on MEFs layer, and the blastocyst had attached then spread out with a visibly growing ICM derivative five days later. The cells of ICM were passaged onto a new feeder layer, then ES colonies could be obtained and passaged. Those cells expressed SSEA-1 with high level of ALP activity; however they could not be amplificated largely.11.5dpc mouse genital ridges were taken as material to isolate and purify PGCs. PGCs could proliferate to form typical EG colonies which were passaged stably. PGCs-derived EG cells maintained undifferentiated with high ALP activity and with the expression of SSEA-1, Oct4 and Nanog detected by histochemistry, immunohistochemistry, immunofluoresence, RT-PCR respectively. Also the EG cells could differentiate into the cystic embryoid body demonstrating their muliti-differential potential. Chromosome analysis revealed a high level of normal karyotype. The successful EG cell culture system had made foundation for further exploration on embryonic germ cells regulation by self-renewal related factors. However, more were needed to be done for ES cells amplification.3. The temporal and spatial expression of Nanog in developing PGCs of mouse:Parafin section and immunofluorescence techniques were taken to qualitatively analyze the expression of Nanog in PGCs of 7.5dpc-15.5dpc mouse embryo, the result demonstrated the PGCs in mouse allantois、hindgut、dorsal mesentery and genital ridges were Nanog positive, and the fluoresence intensity of Nanog in 12.5dpc-14.5dpc gonadal ridges became weaker and weaker till disappeared in 15.5dpc gonadal ridges. The further quantitative analysis of Nanog expression change in PGCs of 11.5dpc-15.5dpc mouse was detected by SSEA-1、 Nanog double labelling immunofluorescence staining using confocal microscopy. Both of the highest positive ratio and the highest fluorescence intensity were appeared in PGCs of 11.5dpc mouse embryo. For the female mice, Nanog expression droped dramatically after 12.5dpc, and for the male mice, a noticeable decline of Nanog expression was occurred after 13.5dpc. Both of the results revealed that Nanog expressed stably in the proliferating PGCs and down-expressed in differentiated gonadal ridges. Maybe its expression relates to the undifferentiated or differentiated state of PGCs. Nanog expression overlaps with Oct4 expression hints the functional relativity between these two transcriptional factors.4. Study on the influence of RNA interference-mediated silencing of Nanog on EG cells:On the basis of Nanog expression analysis of PGCs in vivo, RNAi assay was taken for further exploration on the regulation of EG cells by Nanog. Immunofluoresence and semiquantitative RT-PCR analysis indicated that chemically composed siRNA could efficiently repress Nanog expression in 48 hours. After 48h of transfection with Nanog siRNA, the number of typically undifferentiated EG colonies decreased dramatically, demonstrating that Nanog was the key factor to maintain self-renewal of EG cells. Showed by Semiquantitative RT-PCR analysis, with down-regulation of Nanog expression, the expression of vasa, the germ cell specific gene, was up-regulated dramatically, and the meiosis-related molecule SCP3 expressed positively, which indicated that down-regulated expression of Nanog may be related to the initiation of PGCs differentiation. Transmission electron microscope and TUNEL assay revealed the apoptosis of PGCs, which hinted that maybe multipotent stem cell specific internal transcriptional factors could repress the apoptosis of multipotent stem cells. Combined with the results in vivo, our study demonstrate that Nanog had a key role in maintaining self-renewal of PGCs and in initiating the differentiation of PGCs.To sum up, the ES and EG cells culture systems were set up on the basis of optimized feeder layers. The Nanog expression pattern in vivo was analyzed, on the other hand, as the model of PGCs, the EG cells in vitro were taken as experimental subjectcs treated by RNA interference assay to discuss the possible way of Nanog regulation on developing PGCs. The whole study will be a meaningful exploration on self-renewal and differentiation regulation of PGCs and EG cells.