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Background Recent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs).However,the immunophenotypic variation of NP-MSCs in vitro was unclear.The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell.Methods Tissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures.Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1).Results More than 90％ of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs,such as CD44,CD105 and CD29,but the marker of mature NP cells characterized as CD24 was negative.In continuous cultures,the proportion of mesenchymal stem cells which were expressing CD44,CD105 and CD29 in nucleus pulposus gradually decreased.The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29,with slight positivity for CD44.The CD24 expression gradually increased in proliferation.Biparametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently,and only a small part of cells expressed both CD105 and CD24 simultaneously.TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24.Conclusions Non-degenerative and degenerative NP contains mesechymal stem cells.The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.