In vitro isolation, culture and characteristics identification of rat stem cells derived from potential stem cell niches of the intervertebral disc

English Keywords:Intervertebral disc  Stem cell niches  Mesenchymal stem cells  Cell differentiation  Cell immunophenotype

English Abstract:

  【Abstract】 Objectives: To investigate the isolation, culture and identification of rat stem cells derived from potential stem cell niches of the intervertebral disc(ISN-SCs). Methods: Sprague Dawley rats(male, 10 weeks old) were used as the experimental animals in this study. Tissues of potential stem cell niches of the intervertebral disc(ISN) were separated from the anatomical regions(the perichondrium areas outside of the epiphyseal plate) before being digested by type Ⅱ collagenase. After being harvested, the cells were cultured and expanded in vitro, and passage 4 populations were used for further analysis. The flow cytometry was used to detect the cell cycle and immunophenotypes of ISN-SCs. The MTT method was used to determine proliferation curve of the cells. qPCR analysis was used to determine the relative mRNA expression level of stem cell-related genes. After being cultured in the differentiation medium, the cells were stained by alizarin red and calcium cobalt for osteogenesis, alcian blue for chondrogenesis, and oil red O for adipogenesis. Also, the relative expression of multipotential differentiation-related genes were determined by qPCR. Results: Rat ISN-SCs were fibroblast-like cells with multi-tentacles forming vortexes partially. They were slow-cycling cells with the ability of adherence to plastic. Meanwhile, high expression levels of stem cell-related positive antigen molecules(CD29, CD90, CD44) and low expression levels of negative antigen molecules(CD34, CD45, CD19, CD11b) were observed. Besides, positive results were detected in alizarin red and calcium cobalt staining for osteogenesis, alcian blue staining for chondrogenesis, and oil red O staining for adipogenesis. Also, the differentiation-related gene(osteogenesis: Runx2, OPN and OCN; chondrogenesis: SOX-9, COL2a1 and ACAN; adipogenesis: PPARγ and C/EBPα) expression levels significantly increased in the corresponding induction group compared with the controls. Moreover, ISN-SCs expressed comparable stem cell-related genes(NANOG, SOX-2, OCT-4) with bone marrow mesenchymal stem cells(BMSCs). Conclusions: ISN-SCs belong to the MSC family and possess multipotent differentiation capacities. These findings will contribute to further studies on the migration of ISN-SCs into intervertebral disc(IVD) and its self-repair effect. Meanwhile, a new perspective on biological strategy for IVD self-regeneration is proposed.