Isolation And Culture Of Ovine Skin-derived Precursor Cells

Sheep Skin-derived precursor Cells(SKPs)are a kind of precursor cells,that located in the dermis and have the ability to self-renew,express stem cell markers,as well as exhibit the potential for multidirectional differentiation.Previously,researchers have uccessfully and quickly acidulated the fibroblasts to obtain SKPs,termed p H-SKPs.This separation method provided a new way to more simply and quickly obtain SKPs of sheep.However,the isolation and culture of p H-SKPs still remained poorly understood.Therefore,the fibroblasts were isolated and obtained by using tissue anchoring method from sheep ear tissues in the present study;followed by acidulated the third generation fibroblasts for 15 min in the Hanks’ Balanced Salt(p H 5.7)to obtain p H-SKPs;.Identification of fibroblasts and p H-SKPs were carried out by using reverse transcription-polymerase chain reaction(RT-PCR)and immunofluorescence.In addition,p H-SKPs were incubated in Dulbecco’s Modified Eagle’s Medium-high glucose(DMEM)with 15% FBS and differentiated into fibroblasts;Finally,based on the 10×Genomics platform,using sc RNA-seq technique to construct and sequence the following heterogeneity analysis of p H-SKPs.1.The sheep fibroblasts obtained by the tissue anchoring method exhibited a typical spindle shape,the p H-SKPs obtained from acidified fibroblasts growing spherically in culture medium.It was found that fibroblasts express fibroblast-specific gene Vimentin and p H-SKPs express pluripotency marker genes NANOG,OCT4 and SOX2,neural crest marker genes PAX3 and Nestin and fibroblast marker gene Vimentin by RT-PCR.As well as,fibroblast marker proteins Fibronectin and Vimentin were positively expressed in fibroblasts by immunofluorescence,indicating that the isolated cells have fibroblast characteristics;stem cell marker proteins SOX2,NANOG and OCT4 were positively expressed in p H-SKPs,which revealed that p H-SKPs have the characteristics of stem cells;the differentiated cells of p H-SKPs expressed the Vimentin gene and the fibroblast marker protein Vimentin,which demonstrated that the differentiated cells might be fibroblasts.2.The pH-SKPs were clustered into seven cell subgroups.SKPs marker genes SNAI1,SNAI2 and SOX9 showed low expression pattern in cluster0,cluster1,cluster2,cluster4,cluster5 and cluster6.The fibroblast marker genes FAP and FBLN1 were highly expressed in cluster3,and the myoblast marker gene ID1 was highly expressed in cluster6.These results speculated that cluster1,cluster2 and cluster5 were p H-SKPs that had strong stemness,cluster3 begins to differentiate into fibroblasts,and cluster6 has the potential to differentiate into myoblasts or begins to differentiate into myoblasts.The KEGG of the differentially expressed genes of the fibroblasts and p H-SKPs were mainly enriched in the ECM-receptor interaction pathways,focal adhesion pathways,and apoptosis pathways,predicted the regulation of ECM-receptor interaction pathways,focal adhesion pathways and apoptosis pathways were through gene COL1A1,MYLK,FN1,IFI30 and ATF4,that inhibit the growth of fibroblasts and accelerate the proliferation and growth of a small amount of SKPs in fibroblasts.Presumably regulated ECM-receptor interaction pathways,focal adhesion pathways and apoptosis pathways inhibit the growth of fibroblasts and accelerate the proliferation and growth of a small amount of SKPs in fibroblasts by gene COL1A1,MYLK,FN1,IFI30 and ATF4.Taken together,this study successfully established an in vitro culture system for fibroblasts and p H-SKPs of sheep,and they were stablly cultured in good condition.In addition,p H-SKPs were found to be heterogeneous,which further clarified the characteristics of p H-SKPs.It laid the foundation forpreferable for stem in vitro differentiation,somatic cell nuclear transfer and rapid propagation of superior varieties in sheep.