Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc

Abstract

Background: Human adipose-derived mesenchymal stem cells (ADMSCs) may be ideal source of cells for intervertebral disc (IVD) regeneration, but the harsh chemical microenvironment of IVD may significantly influence the biological and metabolic vitality of ADMSCs and impair their repair potential. This study aimed to investigate the viability, proliferation and the expression of main matrix proteins of ADMSCs in the chemical microenvironment of IVD under normal and degeneration conditions.

Methods: ADMSCs were harvested from young (aged 8-12 years, n = 6) and mature (aged 33-42 years, n = 6) male donors and cultured under standard condition and IVD-like conditions (low glucose, acidity, high osmolarity, and combined conditions) for 2 weeks. Cell viability was measured by annexin V-FITC and PI staining and cell proliferation was measured by MTT assay. The expression of aggrecan and collagen-I was detected by real-time quantitative polymerase chain reaction and Western blot analysis.

Results: IVD-like glucose condition slightly inhibited cell viability, but increased the expression of aggrecan. In contrast, IVD-like osmolarity, acidity and the combined conditions inhibited cell viability and proliferation and the expression of aggrecan and collagen-I. ADMSCs from young and mature donors exhibited similar responses to the chemical microenvironments of IVD.

Conclusion: IVD-like low glucose is a positive factor but IVD-like high osmolarity and low pH are deleterious factors that affect the survival and biological behaviors of ADMSCs. These findings may promote the translational research of ADMSCs in IVD regeneration for the treatment of low back pain.

Figure 1

Characterization of isolated human ADMSCs by osteogenic and adipogenic differentiation. A: Human ADMSCs were expanded in monolayer culture (×40). B: ADMSCs were cultured in osteogenic media for 21 d and stained positive with alizarin red. Red staining marked mineral deposition in their newly formed extracellular matrix (×100). C: ADMSCs were cultured in adipogenic media for 14 d and stained positive with Oil red O staining. The lipid drop appeared red under fluorescence microscope (×100).

Figure 2

The viability of ADMSCs under IVD-like chemical conditions. The numbers of viable, apoptotic, and secondary necrotic cells were determined by annexin V-FITC and PI (AV-PI) staining. Compared to the standard condition, IVD-like glucose condition slightly decreased the number of viable cells while IVD-like osmolarity, IVD-like pH and combined conditions significant decreased the number of viable cells and increased the number of apoptotic cells.

Figure 3

Percentage of apoptotic and necrotic cells of ADMSCs under IVD-like chemical conditions. Flow cytometry analysis showed a slight increase in the number of apoptotic cells under IVD-like low glucose condition, a decrease in the number of vital cells and an increase in the number of apoptotic and necrotic cells under IVD-like osmolarity, IVD-like pH as well as combined IVD-like conditions. Data were presented as mean ± SD (n = 6). Statistically significant differences were indicated with asterisks, *** P < 0.001.

Figure 4

The proliferation of ADMSCs under IVD-like chemical conditions. MTT assay of ADMSCs cultured for 1 week or 2 weeks under IVD-like chemical conditions. Data were presented as mean ± SD with P < 0.05 for n = 6 (young males) and n = 6 (mature males). Statistically significant differences were indicated with asterisks, *** P < 0.001.

Figure 5

mRNA expression of aggrecan and collagen-I in ADMSCs under the IVD-like chemical conditions. RT-PCR analysis of aggrecan and collagen-I mRNA levels in ADMSCs cultured for 1 week or 2 weeks under IVD-like chemical conditions. Statistically significant differences were indicated with asterisks, * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 6

Protein expression of aggrecan and collagen-I in ADMSCs under the IVD-like chemical conditions. Western blot analysis of aggrecan and collagen-I protein levels in ADMSCs cultured for 1 week or 2 weeks under IVD-like chemical conditions. Shown were representative blots from six independent experiments with similar results. β-actin served as loading control. Statistically significant differences were indicated with asterisks, ** P < 0.01, *** P < 0.001.