Studies on the differentiation of urine-derived stem cells into nucleus pulposus cells induced by nucleus pulposus cells

English Keywords:Urine-derived stem cells  Nucleus pulposus cells  Co-culture  Differentiation

English Abstract:

  【Abstract】 Objectives: To investigate the role of human nucleus pulposus cells(NPCs) in inducing the differentiation of human urine-derived stem cells(USCs) into nucleus pulposus cells. Methods: The nucleus pulposus tissue of L4/5 from patients with lumbar disc herniation was obtained during surgery, and NPCs were isolated and cultured in vitro by adherent method. The USCs were obtained from the urine of healthy adults and cultured in vitro. Cell morphology was observed by inverted microscope. Morphology, differentiation potential and cell surface marker proteins of USCs obtained were identified by osteogenic, lipogenic and chondrogenic differentiation induction and Western blot (WB). The Transwell chamber was used to culture P3 NPCs with USCs to establish a co-culture system. The experimental group, control group and NPCs group were set up. The experimental group was the co-culture group of NPCs and USCs, while the control group was of single USCs and the NPCs group was of single myeloid nucleus cells. After 14 days of culture, the morphology of the cells was observed by inverted microscope. The mRNA and protein expressions of aggrecan (ACAN), SOX-9 (SRY-related high mobility groupbox gene 9), collagen type Ⅱ (COL2) and hypoxia-inducing factor 1α (HIF-1α) were detected by fluorescence quantitative PCR (qRT-PCR) and WB, respectively. Immunofluorescence staining was used to observe the fluorescence expressions of COL2A1 and ACAN in the 3 groups. Results: The results of osteogenic, lipogenic, and chondrogenic differentiation of USCs were all positive. The CD29, CD44, CD73 and CD90 were highly expressed in USCs, while CD34 and CD45 were not detected. After 14 days of culture, the morphology of USCs group and NPCs group remained unchanged under inverted microscope, while the morphology of USCs cells in the experimental group differentiated into nucleus pulposus cells, and the morphological changes were obvious. The mRNA and protein expressions of ACAN, SOX-9, COL2A1 and HIF-1α genes in NPCs group and experimental group of USCs cells were significantly higher than those in the control group after 14 days of coculture induction(P<0.05). Immunofluorescence showed that the fluorescence intensity of ACAN and COL2A1 was significantly higher in the NPCs group and the experimental group than in the control group. There was no statistical difference between the experimental group and the NPCs group in terms of mRNA, protein expression and fluorescence intensity. Conclusions: In in vitro experiments, human NPCs can induce human USCs to differentiate into nucleus pulposus cells by co-culture, which can provide a source of NPCs for disc tissue engineering research.