Purification And Identification Of Human Nucleus Pulposus Mesenchymal Stem Cells

Objectives:To investigate the Flow Cytometry Cell Sorting of Human Nucleus Pul-posus Mesenchymal Stem Cells(NPMSCs). Estimate its biological activity of NPMSCs from morphology, proliferation, differen- tiation potential,and identified the NPMSCs according to the ISCT.Methods: Nucleus Pulposus cells were isolated from human nucleus pulposus using enzyme digestion methods and then nucleus pulposus cells were cultured in standard MSC culture medium, After the nucleus pulposus cells reached 80-90% fusion, the cells were trysinized and obtained a single cell suspension.NPMSCs were obtained by Flow Cytometry Cell Sorting accoding to expression of surface CD73,CD90 and CD105.Furthermore, NPMSCs were culture in vitro,then observing the cellular morphology under the microscope, detecting the ability of cell proliferation by using cell counting kit-8(CCK-8), identifying the multiple differentiation(Osteogenic, chondrogenic and adipogenic) potential of NPMSCs. alizarin red staining was used to observe the osteogenesis, oil red O staining was used to observe the adipogenic, toluidine blue staining was used to observe the chondrogenic.Results: FACS use the PE-CD73,APC-CD90,V450-CD105 as positive surface marker,get an average of(3.53±0.78)×106 cells, the proportion of positive cells were(89.67±2.52)%. Expression of the surface marker CD73,CD90,CD105 of second generation nucleus pulposus cells were(89.79±2.40)%,(94.07±2.31)%,(90.49±1.63)% respectively. Primary nucleus pulposus cells were adherence after 5 to 7 h, Primary cells are short shuttle-like, as fusion with 80-90%,the cells orderly growed as radioactive,blaze or helix in shape. NPMSCs were adherence after 4 to 6h, the cells growed as long fusiform shape, single adherent cell was infrequent. NPMSCs were fusion with 80-90% after 12 to 15 days. In the 4h to1 d, Proliferation activity of NPMSCs was inferior to nucleus pulposus cells(P<0.05). In the 3rd day, there was no notable difference between the two(P>0.05). On the contrary, Proliferation activity of NPMSCs was apparently higher than nucleus pulposus cells(P<0.05). Immunophenotyping of NPMSCs tests revealed, expression of the surface marker CD73,CD90,CD105 were(98.55 ± 0.35)%,(98.47 ± 0.57)%,(98.20 ± 1.24)% respectively. Expression rate of NPMSCs was apparently higher than nucleus pulposus cells. Expression of the surface marker of hematopoietic stem cell CD34,CD45,HLA-DR was lower than 4%. Induction of 28 days, black opaque areas were oberserd in the NPMSCs and the P2 nucleus pulposus cells, The NPMSCs and the P2 nucleus pulposus cells formatted lots of red stained calcium salts after alizarin red staining,the NPMSCs was more higher than nucleus pulposus cells in staining area percentage(P<0.05). Induction of 28 days, light circular lipid droplets were oberserd in the NPMSCs and the P2 nucleus pulposus cells, The NPMSCs and the P2 nucleus pulposus cells formatted lots of red dye lipid drops of cavitation after Oil red "O" staining,the NPMSCs was more higher than nucleus pulposus cells in staining area percentage(P<0.05). Induction of 28 days, milky cartilage microspheres were oberserd in the NPMSCs and the P2 nucleus pulposus cells, The NPMSCs and the P2 nucleus pulposus cells formatted lots of aizen cartilage cells of cavitation after toluidine blue staining,the NPMSCs was more higher than nucleus pulposus cells in staining area percentage(P<0.05).Conclusions:This research indicates that through Flow Cytometry Cell Sorting,a large proportion of NPMSCs can be obtained,and following culture research would be making, for the study of the NPMSCs of the biological characteristics provide reliable cell separation and purification methods.The NPMSCs grew with adherence,with multiple differentiation potential, proving that the presence of Mesenchymal Stem Cells again in human nucleus pulposus.