Low Expression Of Mesenchymal Stromal Cell Surface Markers Affects Characteristics Of Nucleus Pulposus Derived Stem Cells Isolated From Human Degenerated Intervertebral Discs

AIM:The nucleus pulposus mesenchymal stem cells were isolated from human nucleus pulposus tissue by using explants culture method.Here we investigated the biological propeties of nucleus pulposus derived stem cells from human degenerated intervertebral discs of different Individual samples.METHODS:The nucleus pulposus derived stem cells were isolated from human nucleus pulposus tissue by using explants culture method.The obtained cells were passaged for purification and cultured in vitro followed by morphological observation.The third passage of well-grown cells was collected.Surface antigens were detected with flow cytometry.Cells from the third passages were induced for osteogenic,adipogenic and chondrogenic differentiation by different revulsants,and cells were examined by alizarin red,oil red O and Alcian blue staining.MSC isolated from human degenerated intervertebral discs of different Individual samples were compared regarding surface epitopes,cell cycle and apoptosis by flow cytometry,proliferative capacity,multi-lineage differentiation potentials,and analyses by qRT-PCR of expression of NP cells related markers genes(COL2A1,Aggrecan,SOX9),NP progenitor marker Tie2 and pluripotent stem cell markers genes(OCT4 and NANOG).RESULTS:NPSCs were successfully separated and expanded by explants culture method.A majority of NPSCs exhibited typical spindle-shape,while more heterogeneous morphology cells were observed.Flow cytometry results showed that NPSCs were positive for CD29,CD44,CD73,CD73,CD90,CD105 and negative for CD11 b,CD14,CD34,CD45,HLA-DR.The positive Oil red O staining,Alizarin red staining and Alcian blue staining indicated that NPSCs could be differentiated into adipose tissue,bone and cartilage.According to the expression level of MSC specific surface markers can be divided into two groups: high expression group(H-NPSCs)and low expression group(L-NPSCs).Results showed that the expression of CD29 and CD105 down regulated in L-NPSCs compared to that of H-NPSCs.The immunophenotypic data was supported by morphologic results that L-NPSCs presented heterogeneous morphology with the appearance of a varied proportion of polygonal or digital shape cells.Results showed that H-NPSCs have a higher proliferative capacity compared with the L-NPSCs,which was in alignment with the results of cell cycle analysis showing increased G0/G1 and G2/M phase arrest and decreased S phase entry in L-NPSCs compared with H-NPSCs.Furthermore,cell apoptosis analysis also confirmed this result by showing the percentage of necrosis and apoptosis in L-NPSCs is greater than H-NPSCs.Expression of NP cells related markers genes in L-NPSCs were significantly higher than those in the H-NPSCs.Expression of Tie2 gene in L-NPSCs were significantly lower than those in the H-NPSCs.However,OCT4 and NANOG expression tended to be higher in L-NPSCs than in H-NPSCs.As for multilineage differentiation potentials,L-NPSCs exhibited reduced differentiation poptentials with regard to chondrogenic,osteogenic and adipocytic differentiation.CONCLUSION:Explants culture method is simple and can isolate,purify and amplify NPSCs in vitro.The obtained cells have general biological characteristics of mesenchymal stem cells,and also have potentiality of multi-directional differentiation.Our results highlighted a lower rate of proliferation,reduced differentiation potentials in L-NPSCs,which correlated with the lower expression of multipotent markers and higher expression of NP markers.