Regulation Of Gelatin And SC1 On The Self-renewal Capacity And Pluripotency Of Chicken Blastodermal Cells

Chicken blastoderm cells(cBCs)as a kind of pluripotent stem cells,which can not only serve for the research of stem cell self-renewal and pluripotency and epigenetic ideal model,and has applied in develoment of transgenic chicken.However,the ability of adhering to walls is not poor,which has been the bottleneck problem that has affected its wide application.Our previous studies have shown that using human recombinant vitronectin(hVTN)for embedding culture plate can solve the problem of poor chicken blastoderm cells adherent ability.But due to hVTN is expensive,thus limitting its application in chicken blastoderm cells to some extent.Therefore,we learned from mouse embryonic stem cell culture system that 10 g·L-1 gelatin could be used as an optional embedding reagent.However,research on the adhering to well,proliferation and pluripotency of cBCs and their adverse effects about 10 g·L-1 gelatin are rarely reported.Therefore,this study will discuss the adhesion ability,cell proliferation,pluripotent maintenance and adverse effects of gelatine in cBCs.In the process of culturing embryonic stem cells,it is very important for the culture system to maintain its pluripotency,and the same is true of cBCs.growth factors and small molecular compounds are often added to maintain their pluripotency,when culturing mouse embryonic stem cells(mESCs).However,some small molecular compounds have relatively few applications in cBCs As a result,adding small molecule compounds SCI(ERK1 and RasGAP dual inhibitors,can maintain mESCs pluripotency)to the E8 medium to keep the cBCs pluripotency.Activation of the PI3K/Akt signaling pathway promotes self-renewal of mouse embryonic stem cells,while inhibiting the phosphorylation of ERK1/2 can inhibit its differentiation.Although SCI can maintain mESCs pluripotency,there are still some differences between different species.Therefore,this study investigated whether SC1 could promote the self-renewal of cBCs and maintain its pluripotency and regulatory mechanism.The main results are as follows:1.Gelatin promotes cBCs sticking to the platesWhen cultured for 12 h,cBCs was observed.In the 10 g·L-1 gelatin group and hVTN group,cBCs was around unfolded in adherent state.But in control and PBS group,cBCs grow into clumps.And slight shaking culture plate,cBCs was moving with the medium,suggesting that cBCs barely attached to the plate.After 48 h,10 g·L-1 gelatine group cBC convergence was about 90%,slightly higher than hVTN group(about 70%).The results showed that 10 g·L-1 gelatin could promote the adherent plates of cBCs.2.The pluripotent state of the cBCs in the gelatinized plateIn order to detect whether there is any effect on cBCs pluripotency.We used alkaline phosphatase(AP)staining and SSEA-1 immunofluorescence staining to detect pluripotent status.After staining with alkaline phosphatase,undifferentiated cBC was blue-purple,and the differentiated cells was not stained.After 48 hours of culture,both gelatine and hVTN groups were dyed blue-purple.Immunofluorescence results showed that SSEA-1 was expressed as a pluripotent marker of the embedded culture plate.The results showed that there was no adverse effect on the pluripotent state of cBCs in the gelatinized plate.3.Gelatin can promote the proliferation of cBCsThe cBCs adhered to the wall and began to proliferate rapidly.EdU dyeing can be effectively detection rate of positive cells in S phase,so we adopt EdU dyeing testing proliferation cases of chicken blastoderm cell in different treatment group,the results showed that EdU-positive cells rate in 10 g ·L-1 gelatin group(76.94±3.411)%is significantly higher than PBS group(55.95±1.96)%(P<0.05),and gelatin group is slightly higher than the hVTN group(72.84±4.35)%,but there was no significant difference.The results showed that the gelatinized plate could promote the proliferation of cBCs.4.Gelatin promotes the up-regulation of mRNA levels of the pluripotent genes and inhibits mRNA expression of cBCs apoptotic genes.In addition,we also use qRT-PCR to detect mRNA expression level of pluripotent genes(Nanog,PouV)and apoptosis gene(P21,Fas)in different groups,the results have showed that gelatin can significantly promote the pluripotent gene expression(P<0.05),it's interesting that hVTN expression levels higher than the gelatin group.In addition,the expression of P21 and Fas in the gelatine group was down-regulated at the mRNA level.The results showed that there was no adverse effect on the growth of cBCs.5.SCI promotes the expression of pluripotent genes.After treated for 12 h by 1 ?M SC1,mRNA expression levels of Sox2,PouV,Nanog rose up significantly(P<0.05).In addition,western bloting display Sox2 protein levels also rose up significantly(P<0.05).As a result,the addition of SC1 to the E8 medium promoted the expression of the pluripotent gene.6.SCI inhibits the down-regulation of pluripotent genes caused by RA.Retinoic acid(RA)can be used as inducer to induce differentiation of stem cells into neuronal cells.therefore,we have set up the control group(DMSO),leukemia inhibitory factor(LIF)group,RA,and RA with SCI four groups,respectively,testing its pluripotent related genes in the mRNA expression level(treated for 6 h and 12 h)and the change of protein levels(6 h).The results showed that the mRNA expression levels of Nanog,PouV and Sox2 were significantly reduced(P<0.01)by adding RA alone for 6 h and 12h.At the same time,western blotting results showed that the expression level of the Sox2 protein decreased with the addition of RA alone.Pluripotent gene expression level was down-regulated,indicating that the cBCs were reduced in pluripotency and showed signs of differentiation.However,both SC1 and RA were added,and the expression of the pluripotent gene(Nanog,PouV,Sox2)was significantly increased at the mRNA level(P<0.05).In addition,western blotting results showed that SC1 and RA jointly processed the expression of Sox2,which was consistent with the results of mRNA level detection.Therefore,we supposed that SC1 can inhibit RA and induce the down-regulation of pluripotent genes.7.SC1 activates the PI3K/Akt signaling pathway.For stem cells,the PI3K/Akt signaling pathway is associated with its pluripotent maintenance and self-renewal.Therefore,we use the PI3K phosphorylation inhibitor,LY294002,to inhibit this pathway.After processing for 6 h with 3 ?M and 10 ?M Y294002,respectively,western blotting results showed that the expression quantity of Sox2 decreased significantly in the 10 ?M treatment group,and there was no significant difference in the 3 ?M treatment.The results show that the PI3K/Akt signaling pathway is correlated with cBCs' pluripotency.In addition,we also tested the phosphorylation level of Akt in the treatment of cBCs for 20 min by 1?M and 3 ?M SC1 and 3 ?M LY294002.The results showed that compared with the control group,and the phosphorylation of Akt decreased treated by the inhibitor LY294002 for 20 min,indicating that the PI3K/Akt signaling pathway was inhibited by LY294002.Phosphorylation level of Akt in 1 pM SCltreatment group was not significantly increased,while 3 microns Akt phosphorylation in treatment group were significantly increased,show that SCI can activate the PI3K/Akt signal pathway,and promote the multifunctional gene expression.8.SC1 inhibits the phosphorylation of ERK1In order to explore whether SCI can affect the phosphorylation of ERK1/2,we also treated cBCs20 min with 1 ?M and 3 ?M,and the results showed that the level of ERK1 phosphorylation was significantly reduced treated by 1 ?M and 3 ?M SCI.LIF did not affect the phosphorylation level of ERK1,which indicates that SCI inhibits the phosphorylation of ERK1.We hypothesized that the addition of SCI in the E8 medium might inhibit the differentiation of cBCs.Above all,10 g·L-1 gelatin embedding culture plate,can improve the adherent ability and promote chicken blastoderm cell proliferation,versatility and maintain and no adverse effect of cBCs.10 g·L-1 gelatin reflect high cost performance,and further enrich the cBCs with serum-free and feeder-free culture system.On the other hand,small molecular compound SC1 can activate the PI3K/Akt signaling pathway to promote the expression of pluripotent genes.SCI inhibits the down-regulation of pluripotent genes caused by RA.SCI can inhibit the phosphorylation of ERK1,which may inhibit the differentiation of cBCs.This regulation mechanism may be conservative in the embryonic stem cells of different species,and provides a new theoretical basis for the development and maintenance of its pluripotency in vitro.