Isolation, Culture And In Vitro Differentiation Of Embryonic Stem Cells From Rabbit

The major object of this paper was to isolate and culture embryonic stem cells from rabbitembryos, and comppared the effect of different culture conditions on isolation and culture ofrabbit ES-like cells systematically, which were subcultured and identificated. We preliminarydiscussed the effect of different concentrations of retinoic acid(RA)on ES cells differentiation intoprimordial germ cells(PGCs), to identify the efficiency of germ cell differentiation derived fromrabbit ES-like cells, which can lay foundation for establishing rabbit ES-like cells lines andprimordial germ cell differentiation of rabbit ES-like cells in vitro. The main results as follows:1. To explore the effect of superovulation on developmental potential of rabbit embryos in vitro,collect embyos treatment with natural estrus and superovulation by mating after4d, and culturedon MEF feeder, rabbit blastocysts obtained by natural estrus mating as control. The results showedthat superovulation attachment rate of blastulas, ICM formation rate and F1generation formationrate of rabbit ES-like cells were no significantly difference (P>0.05), there is no significantlyeffect on developmental potential of rabbit embryos collected from superovulation-treated rabbits.But there is significantly difference in subculture of stem cells (P<0.05). Hence, in order to ensureefficiency of experiment, expecially in the cold and hot environment, the rabbit natural matingwas tricky, we can obtain enough embryos by superovulation-treated rabbits, but embryosobtained were better by natural estrus mating in spring and autumn, which were more suitable forisolation of stem cells.2. To investigate the effect of different treatments of rabbit embryos on clone and subculture ofES-like cells. Embryos were cultured on MEF feeders, which were treated with prickingzonapellucida, cutting and dispering and control respectively by natural estrus mating. The resultsshowed that the embryos which were treated with pricking zona pellucida, cutting and dispersingattached and proliferated better easily, comparing with the control group, significantlydifferent(P<0.05). The blastulas attachment rate of cutting and dispersing embryos wassignificantly higher than pricking zona pellucida(P<0.05)(85.7%vs70.0%), but ICM formation rate of the group of pricking zona pellucida was significantly higher than the group of cutting anddispersing embryos(P<0.05)(53.3%vs40.0%). The clone forming efficiency of F1and F2ofrabbit ES-like cells of the group of pricking zona pellucida was significantly higher than the othertwo groups(P<0.05)(34.7%,25.7%), and the rabbit ES-like cells obtained were subcultured to30passages in vitro. It turned out that pricking zona pellucida would be better beneficial to improvethe efficiency of embryonic stem cells isolated from rabbit embryos and suitable for keeping therabbit ES-like cells culture and proliferation in vitro.3. To compare the effect of different mediums on clone and subculture of rabbit embryos. Theresults showed that embryos culutured in medium containing15%FBS, ICM clone rate and theformation rate of F1to F5cultured in serous medium were significantly higher than another twogroups, which were cultured in medium containing15%KSR and7.5%FBS+7.5%KSR(P<0.05),and in the process of subsequent subculture, when the medium is H-DMEM+2mmolL-glutamine+0.1mmol·L-1β-mercaptoethanol+1%NEAA+15%FBS+100IU·mL-1doubleantibody, which can improve the environment of rabbit ES-cells culture in vitro and make for theformation of rabbit ES-cells colony for stable subculture.4. To compare the effect of three different methods(mechanical separation, trypsin digestion,mechanical separation and trypsin digestion)on the subculture of rabbit ES cells. The resultsshowed that the method of passage by mechanical separation and trypsin digestion was the mostbeneficial to passage and colony formation of rabbit ES-cells and to formed the best cell clonemorphology, which was the ideal method of passage of ES cells.5. When obtained rabbit ES-like cells were examined, the results showed that alkaline phosphatase(AKP) stain of rabbit ES-like cell colonies were strongly positive. RNA of amplificationpluripotent genes(GAPDH, Nanog and Sox2) of rabbit ES-like cell colonies were detected, thesize of purpose fragments were consistent as expected by RT-PCR detection. Rabbit ES-like cellsnaturally differentiated into embryoid bodies(EBs) originated from three germ layers in vitro.6. To explore the appropriate concentration and the ability of RA inducing rabbit ES-like cells intoprimordial germ cells. With different concentrations of RA (0.2μM,2μM and20μM) and themedium without RA inducing rabbit ES-like cells, we used flow cytometry instrument for DNAploidy analysis. The results showed that haploid content was54.4%by0.2μM concentration ofRA induction21days, whose induction efficiency was highest. The haploid content was12.6%by2μM concentration of RA induction14days, while other concentration of RA did not containhaploid after inducing the same days. Hence, rabbit ES-like cells can be induced to PGCs by usingRA in vitro, the0.2μM concentration of RA induction efficiency was higher than otherconcentrations, indicated that the optimal concentration of RA inducing rabbit ES-like cells intoPGCs is0.2μM.