Isolation, Cultureand In Vitro Primordial Germ Cells Differentiation Of Embryonic Stem Cells From Kunming Mouse

Kunming mouse is widely used in the lab of our country, While most of theestablished mouse embryonic stem cells (ESCs) lines were from129, C57BL/6J andBALB/C mouse, but very few reports have showed the establishment of KM mouseES cells lines. So it is very important to establish the ESCs lines of KM mouse forstem cell research in our country. The SC1small molecule functions through dualinhibition of Ras-GAP and ERK1to maintain ESCs self-renewal state. Recently,several groups have reported that ES cells differentiated into primordial germ cells（PGCs）, and further into mature gametes in appropriate differentiation conditions invitro, but the efficiency of ES cells differentiating into germ cells is still low. Inthis study, we have compared the effection of different culture conditions on isolateand culture ESCs from KM mouse, and study the effect of SC1in maintaining ESCsundifferentiation. Furthermore, retinoic acid (RA) concentration was initially screened,to identify the efficiency of germ cell differentiation derived from mES cells, whichcan offer reference for establishing KM mouse ES cells lines and primordial germ celldifferentiation of mouse ES cells in vitro. The results are as follows:1. The vitality of (Mouse embryonic fibroblast, MEF) treated by mitomycinC(10μg/mL final conc.) was detected by MTT method to compare the maintaining timeof MEF vitality in different disposal time. The result showed that the MEF vitalitycould keep about7days stably under this condition when MEF within3passageswere used to prepare feeder layer, treating MEF with mitomycin C(10μg/mL final conc.)for2~2.5h. This was the optimical condition to preparing MEF feeder layer.2. In order to isolate the ES cells efficiently, the embryoes of3.5~4dpc from KMmouse, cultured on different concentrations of1×104、1×105and1×106cells/mL asthe feeder layers, the growth of the ES cells were observed. The results showed thatthe formation rates of ES cells′s first and second passage on1×105/mL MEF werehigher than those cultured on the other two groups(P<0.05), the passage rate of blastocysts were better than that of morula,and it had improved the attachment rateand passage rate when they cultured in serum-free medium added with Stem cellfactor(SCF) and insulin(P<0.05). Examined by AKP strain, immunofluorescenceanalysis showed the isolated ES cells bear a series of characters of mice ES cells.These results suggest that blastocysts on the feeder cell of MEF with a sensity of1×10~5cells/mL is better for isolation ES cells, and add SCF and insulin in the culturemedium is more suitable for the isolation and passage of ESCs.3. Inorder to study the effection of SC1in maintaining ESCs self-renewal state,the experiment investigated formation of ICM outgrowths and isolation of ESCsfrom mouse embryos of KM specie while mitomycin mitomycin C-inactivated MEFas the feeder layer cells. In the serum-free medium added with differentconcentration of300nM、1μM、3μM SC1instead of LIF and LIF added as control.The results demonstrated that the formation rates of ICM outgrowths significanthighter than control group, the ES cells had maintained undifferentiating for7passages, and it had maintained undifferentiating for6passages without feederlayer cells. And the second passage rate of ES cells in the medium added with300nM SC1were higher than added with1μM and3μM, The isolated cells werepositive for AKP stain and also immunofluorescence analysis positive againstantigens of Oct4、Nanog and Sox2. In conclusion, SC1can maintain the pluripotencyand undifferentiation of ES cells from KM mouse without LIF and feeders.4. To investigate the method by which KM mouse ES cells could be induced toPGCs, EBs formation from the third passage ES cells were cultured by hanging dropmethod. After three days, produced EBs were transferred onto48-well plates with fineEBs per well. A series of RA concentration (0.2μM、1μM、5μM) and control culturemedium were added. Immunofluorescent staining analysis at day10indicated theefficiency of1μM RA inducing mESCs toward PGCs was apparently higher than theother groups. After10days, the rates of SCP3and VASA positive cells were31.8%and30.4%, being highest in each set. These results indicate that ESCs from KMmouse can be induced to PGCs by RA in vitro, and the optimal concentration of RAinducing ESCs to PGCs is1μM.