The comparison of biological characteristics of three kinds of stem cells in human degenerative intervertebral disc

Abstract

Objective To compare the biological characteristics of nucleus pulposus-derived stem cells (NPSCs), annulus fibrosus-derived stem cells (AFSCs) and cartilage endplate-derived stem cells (CESCs) from the same patient who had degenerative disc. Methods The human discs isolated from 7 patients with intervertebral disc herniation were separated carefully and digested and filtered before getting the original NPSCs, AFSCs and CESCs. When cultivated to third passage, the cell activity of stem cells was analyzed by using cell counting kit-8 (CCK-8); multilineage (adipogenic, osteogenic and chondrogenic) differentiation potential was analyzed to define different stem cells. The total RNA of third-passage-cells was extracted, and then reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the relative expression of adipogenic, osteogenic and chondrogenic differentiation-related genes. Results Cell morphology of NPSCs, AFSCs and CESCs from the same patient showed no significant difference. The growth curve showed that proliferative ability of CESCs (A value at day 1, 3, 5, 7, 9, 11, 13, 15 was 0.35±0.01, 0.52±0.02, 0.64±0.08, 0.80±0.19, 0.79±0.2, 1.06±0.11, 1.37±0.09, 1.31±0.41) and AFSCs (0.38±0.01, 0.50±0.03, 0.72±0.06, 0.82±0.13, 0.87±0.08, 0.87±0.09, 1.20±0.05, 1.43±0.05)was more active than that of NPSCs (0.32±0.02, 0.42±0.04, 0.59±0.01, 0.64±0.17, 0.65±0.19, 0.68±0.21, 0.67±0.12, 0.59±0.12). Multilineage differentiation results showed adipocyte-like red by oil red O, osteocyte-like cells were stained red by Alizarin red, and chondrocyte-like cells blue by toluidine blue. The RT-PCR results showed that AFSCs was better than NPSCs and CESCs in the aspect of the expression of adipogenic [the relative expression of peroxisome proliferator-activated receptor gamma (PPARγ) gene was 6.85±0.32 vs. 6.63±0.50 and 7.21±0.21; the relative expression of lipoprotein lipase (LPL) gene was 13.72±0.37 vs. 8.41±0.42 and 10.62±0.37] and chondrogenic [the relative expression of type 2 collagen (COL-2) gene was 10.45±1.10 vs. 3.53±0.62 and 8.26±0.49; the relative expression of sex determining region Y-box 9(SOX-9) gene was 14.39±1.22 vs. 7.66±0.68 and 13.70±1.10] differentiation-related genes, whereas the expression of osteogenic differentiation-related genes was best in CESCs[the relative expression of runt related transcription factor-2(RUNX-2) gene was 7.30±1.1 vs. 2.61±0.06 and 6.20±0.92; the relative expression of COL-2 gene was 8.22±0.84 vs. 7.05±0.82 and 4.94±0.90] and the expression of adipogentic differentiation-related genes, particularly LPL gene was worse in NPSCs. Conclusion The cell morphology was similar among NPSCs, AFSCs and CESCs in vitro, but biological characteristics of those were different. Induced differentiation and RT-PCR results both showed that AFSCs had more advantages than NPSCs and CESCs in the field of intervertebral disc tissue engineering. Key words: Intervertebral disc degeneration; Nucleus pulposus stem cells; Annulus fibrosus stem cells; Cartilaginous endplate stem cells; Biological characteristics