Comparison Of Different Expression Levels Of Porcine Embryonic Stem Cells And The Role Of MicroRNAs In Inducing Pluripotent Stem Cells

Embryonic stem cells(ESCs)and induced pluripotent stem cells(i PS)become a useful tool for cell therapy,drug screening,embryonic developmental mechanisms,and construction of disease models.Currently the mouse is the primary animal model on stem cells research.However,the mouse is not the best choice on the clinical application because of the short life span and their obvious differences with human physiological functions and structures.Compared with the mouse,the pig is a good human disease model,especially in xenotransplantation medicine,immune and metabolic diseases.The establishment of porcine ESCs is increasingly urgent.The authentic porcine ESCs,which are consistent with the characteristics of mouse ESCs,have not been successfully established until now.Meanwhile,the standard porcine-specific markers to examine and distinguish two states of porcine ESCs have not been elucidated.In this study,we firstly converted the na?ve-like porcine ESCs(n ESCs,established in our Lab)into primed state and proposed a set of molecular criteria for evaluating the na?ve and primed porcine ESCs by comparing the two states of cells;We compared micro RNA(mi RNA)transcriptional profiles between the primed porcine embryonic stem cells(primed ESCs),porcine inner cell mass(ICM)and pig fetal fibroblast cells(PEF),constructed a mi RNA overexpression vector and studied the effect of mi RNA on the establishment of porcine i PSCs.Together,this study might be a step forward in the knowledge of porcine pluripotency markers and provides new insights into the effects of the mi RNA on porcine PSCs induction.Objective To obtain molecular markers that can identify the pluripotency state of porcine ESCs;To study the effect of mi RNA on the establishment of porcine i PSCs.Methods1.The porcine n ESCs established in our laboratory were cultured for the first 2 days under LIF/3i conditions.And then we switched the medium to FGF/Activin medium and cultured those colonies for another 2-3 days.We tested whether rp ESCs might reacquire features of na?ve-like state pluripotency when they were cultured in LIF/3i again.Subsequently rp ESCs(reversed primed porcine ESCs,rp ESCs)were mechanically dissociated every 4-5 days which displayed monolayer and flatten clones.Alkaline phosphatase(AP)staining was performed in rp ESCs at passage 5,and karyotype analysis was taken in rp ESCs at passage 10.We confirmed the expressions of FGF/Activin and LIF/STAT3 signaling pathway-related gene in porcine ESCs by RT-PCR;In vitro differentiation potential of rp ESCs was identified by embryoid body formation and identification of genes associated with the three germ layers.The expression of cell lineage and pluripotency related factors during the conversion was analysed through RT-PCR;Subsequently,We performed Western blot and immunofluorescence staining in order to further analysis of OCT4,LIN-28,CDX2,SOX17,c-MYC,NANOG,KLF4,Nr0B1,and TBX3 expression.2.We compared mi RNA transcriptional profiles between the primed porcine embryonic stem cells(primed ESCs),porcine inner cell mass(ICM)and PEF,micro RNA-205 was expressed higher in porcine ICM instead of primed PESCs or PEF.The mi RNA-205 precursor sequence was amplified by PCR and integrated into the expression vector p CAGDNA3-h Fat1.The recombinant expression vector p CAG-mi RNA-205 was constructed.The expression vector was transfected into porcine fetal fibroblasts containing four mouse derived transcription factors(Oct4,Sox2,Klf4 and c-Myc)by using lipofectamine transfection technique.To establish porcine i PSCs,the cells were incubated and cultured in LIF/3i medium.QPCR(real-time quantitative PCR)was used to detect the expression of mi RNA-205 in the cells before and after transfection.We also analyzed the effect of mi RNA-205 on porcine i PSCs.Results1.n ESCs were converted into rp ESCs in the condition of FGF/Activin(STO).The rp ESCs could reacquire features of na?ve-like state pluripotency when they were cultured in LIF/3i again,and we named them reversed na?ve ESCs(rn ESCs).The rp ESCs are phenotypically stable and karyotypically intact.The AP-positive and the ability of embyonic body formation suggest that rp ESCs still remain the capacity of self-renew and in vitro differentiation.The signaling pathways analysis by RT-PCR showed that rp ESCs rely on FGF/Activin signaling pathway.Western-blot for further detailed analysis indicated that the phosphorylated STAT3 was nearly undetectable in rp ESCs at 6 passages,which indicated that rp ESCs was independent of LIF/STAT3 signal pathway.However,n ESCs were obviously dependent on the LIF/STAT3 signaling pathway.The expression of lineage-associated genes,such as CDX2,SOX17,EOMES,FOXA2,FGF5 and PITX2,exhibited very low or undetectable expression in n ESCs but appreciable transcription in rp ESCs;The expression of LIN28,OCT4,SOX2,NOG,DPPA5,Nr0B1,KLF4 shown dramatically downward trend in rp ESCs;However,NANOG,TBX3 and c-MYC were exactly increased in rp ESCs.Western blot and immunostaining were performed to analyse the expression of CDX2,SOX17,OCT4,NANOG,c-MYC,KLF4,Nro B1,LIN28,TBX3 in n ESCs and rp ESCs.The results were consistent with those of QPCR.2.The recombinant plasmid p CAG-mi R205 was constructed.Compared with untransfected cells,the expression level of micro RNA-205 in the transfected cells was significantly increased(P %<0.01),and the colony number was higher after being induced in stem cell culture medium.Conclusion1.It is feasible to convert n ESCs established in our laboratory to enter the primed state,and n ESCs depend on the LIF/STAT3 signaling pathway and rp ESCs rely on the FGF/Activin signaling pathway.2.A set of molecular criteria was proposed for evaluating the na?ve and porcine primed ESCs by comparing the two states of cells in the expression of pluripotency molecular markers.The expression of lineage-associated genes,such as CDX2,SOX17,EOMES,FOXA2,FGF5 and PITX2,indicated the primed state of porcine ESCs.Strikingly,the traditional na?ve marker for mouse ESCs(TBX3,Nanog and c-MYC)could depict the primed state of porcine ESCs.In addition,The pluripotency genes(LIN28,OCT4,SOX2,NOG,DPPA5,Nr0B1,KLF4)could represent na?ve state in porcine ESCs.3.micro RNA-205 overexpression vector constructed by us will facilitate the establishment of cell lines that stably express mi RNA205 in subsequent studies for further in-depth and lay a foundation for the study of the role of mi RNA205 in stem cell reprogramming and pluripotency maintenance.