Purification and identification of human nucleus pulposus mesenchymal stem cells

English Keywords:Nucleus pulposus mesenchymal stem cells  Fluorescence activated cell sorting  Purification  Biological activity

English Abstract:

  【Abstract】 Objectives: To explore a method for purifying human nucleus pulposus mesenchymal stem cells(NPMSCs), and to identify its biological activity. Methods: Degenerative nucleus pulposus tissue(Pfirrmann Ⅳ) from 3 patients with lumbar disc heriniation was collected, cells were isolated from human nucleus pulposus tissue by using enzyme digestion. Adherent method and fluorescence activated cell sorting(FACS) method were used for purifying NPMSCs, NPMSCs were obtained by FACS accoding to the expression of surface markers including CD73, CD90 and CD105. Then, the obtained NPMSCs by two methods were cultured in vitro and the cellular morphology was observed under the microscope, cell proliferation tests were performed by using Cell Counting Kit-8(CCK-8). Immunophenotyping of NPMSCs of adherence group was detected by FACS, and NPMSCs of FACS group were detected after the fusion rate reaching 80%-90%. Differentiated potential of NPMSCs was investigated by multiple differentiation(Osteogenic, chondrogenic and adipogenic). Alizarin red staining was performed to observe the osteogenesis potential, oil red O staining was used to observe the adipogenic potential, toluidine blue staining was used to observe the chondrogenic potential. Imag J software was used to test the stained area percentage of differentiated NPMSCs under two methods. NPMSCs of both groups were compared. Results: NPMSCs of both groups showed spindle shape, single cell was observed in the adherence group. NPMSCs of FACS group showed longer shuttle and rare single adherent cell. After FACS, the ratio of CD73+, CD90+, CD105+ NPMSCs from NPMSCs reached(89.67±2.52)%, typically long spindle shape could be observed, 80%-90% confluence was reached after 12-15 days. Expressons of CD73, CD90 and CD105 of FACS were higher than those of adherent method(P<0.05), more than adherent method 5 days later(P<0.05). The multiple differentiation potentials(Osteogenic, chondrogenic and adipogenic) of NPMSCs by FACS were superior than those of adherent method(P<0.05). Conclusions: Large proportion of NPMSCs can be obtained through FACS, which provides a reliable cell separation and purification method. Compared with method of adherence, NPMSCs obtained by FACS show better proliferation and multiple differentiation potential compared with adherent method.