Biological characteristics of human nucleus pulposus mesenchymal stem cell under different oxygen concentrations

English Keywords:Intervertebral disc degeneration  Nucleus pulposus mesenchymal stem cell(NPMSCs)  Oxygen concentrations  Biological characteristics

English Abstract:

  【Abstract】 Objectives: To observe the biological effects of different oxygen concentrations on human nucleus pulposus mesenchymal stem cells(NPMSCs) in vitro, and to explore the mechanism of intervertebral disc degeneration. Methods: MSCs obtained in this study were isolated from non-degenerative nucleus pulposus(Pfirrmann Ⅰ or Ⅱ) which were harvested from 4 patients with congenital scolisis, and then were cultured and passaged in vitro. The immunophenotypes(CD90, CD73, CD105, CD44, CD31, CD34, CD45) of the third generation cells were identified by flow cytometry, and then were induced into osteogenesis, adipogenic and chondroblast cells which were identified by alizarin red, oil red O and toluidine blue staining, respectively. Briefly, the cells should be identified by the criteria of the International Society for Cellular Therapry (ISCT). The 2% oxygen concentration was set to mimic oxygen level of intervertebral disc degeneration and 20% oxygen concentration was used as normoxic oxygen concentration and then the cells were further cultured in different oxygen concentration(2% O2, 5% CO2, 37℃; 20% O2, 5% CO2, 37℃; respectively). The morphology of the cells was observed by inverted microscope at different time points and the growth curve was analyzed by cell counting. The pH and osmotic pressure of the culture medium at each time point were measured by using the Architect c8000 automatic biochemical detector. The mRNA expression levels of stemness-related genes(POU5F1, NANOG, SOX2), proliferation-related genes(CCND1, MYC), hypoxia-induced genes(EPAS1), Energy related gene(adenosine triphosphate synthetase, ATP5A1) and mitochondria -related genes[cytochrome c oxidase Ⅳ subunit 1 isoenzyme(COX4I1), mitochondrial transcription factor A(TFAM), mitochondrial coding for cytochrome c oxidase I(MT-CO1), MT-CO2 were evaluated by QRT-PCR. Results: Nucleus pulposus mesenchymal stem cells(NPMSCs) grew into typical, adherent monolayer cells, with high expressions of CD90(80.4%), CD73(99.9%), CD105(99.8%), CD44(95.9%) and low expressions of CD31(5.3%), CD34(4.4%), CD45(6.8%). Alizarin red staining showed deposition of large amounts of calcium nodules in NPMSCs. Oil red O staining displayed a large amount of lipid droplets deposited in cells. Chondrogenic differentiation was positive for alcian blue staining. Based on the above results, the cells obtained in this study met the criteria of ISCT and these cells were absolute mesenchymal stem cells. NPMSCs that were cultured in a hypoxic atmosphere(2% O2) became comparatively smaller in size, with an original spindle-shaped morphology. The growth curve assays showed that the doubling time of NPMSCs at hypoxia was significantly earlier than that of cells grown at normoxia (31.22±1.98h, 39.56±2.02h, respectively, P<0.05). The pH and osmolality from different conditions were within limits to promote normal cell growth(P>0.05). The major markers of primitive stem cells, POU5F1(Oct4), NANOG and SOX2 increased in NPMSCs grown at 2% O2 compared with NPMSCs cultured in 20% O2(P<0.05). Under the condition of hypoxia, the proliferation-related genes CCND1(CyclinD1) and MYC(c-Myc) significantly increased(P<0.05), and the hypoxia-inducible factor HIFα-related gene EPAS1 was also significantly elevated(P<0.05). The relative mRNA expression levels of ATP5A1 and nuclear-encoded energy-related gene, were significantly elevated under hypoxia(P<0.05). However, nuclear coding mitochondria -related genes, the mRNA expression levels of COX4I1 and TFAM significantly decreased(P<0.05) in hypoxic conditions. Still, the mRNA expression levels of MT-CO1 and MT-CO2 also significantly decreased under hypoxic conditions(P<0.05). Conclusions: Hypoxic atmosphere(2% O2) favored a more primitive morphology, higher NPMSC proliferation rate, and better stemness-related genes expression.