Cellular mechanical properties reflect the differentiation potential of nucleus pulposus-derived progenitor cells

Abstract

Mechanical properties of cells reflect differences in cellular subpopulations, differentiation potency, and cell behaviors. Previous study has revealed that intervertebral disc (IVD) degeneration leads to alterations in cell behavior and differentiation potency. Human nucleus pulposus-derived progenitor cells (NPPCs) are an attractive cell sources for IVD regeneration. However, the relationship between mechanical properties and differentiation potential in different NPPC subpopulations is few known. In this study, mechanical properties of different NPPC subpopulations were measured via atomic force microscopy (AFM) and correlated with differentiation potential of NPPCs. We found that elastic modulus, relaxed modulus, and instantaneous modulus were positively correlated with osteogenic potential of NPPCs. And apparent viscosity was correlated with chondrogenic potential of NPPCs. These results indicated that the mechanical properties were predictive markers for differentiation potential of NPPC subpopulations, and could be used for enrichment based on differentiation potential, which could significantly improve the outcome of IVD regeneration.

Keywords: Cellular mechanical properties; intervertebral disc degeneration; nucleus pulposus; stem cell.

Figure 1

Characterization of NPPCs from human degenerated nucleus pulposus. Single NP cells were inoculated into methylcellulose medium in 96-well culture plates, and were observed on day 0 (A), day 7 (B), and day 14 (C). The colonies derived from human primary NP cells in methylcellulose medium at 14 days (D). Immunophenotypic profile of nucleus pulposus-derived progenitor cells (NPPCs) from colony-forming assay (CFA) analyzed by FACS (E). The blue lines represent the fluorescence intensity of cells stained with the indicated antibodies and the black lines represent the negative control cells, which were stained with a non-immunoreactive isotype control antibody.

Figure 2

Cell stiffness varies in different clonal population. The mechanical properties of NPPCs were heterogeneous, which suggested a possible means to identify lineage-specific subpopulations. Elastic and viscoelastic properties of 30 NPPCs clones were measured via AFM indentation and stress relaxation tests, respectively. Within each clonal population, an average of 25 cells was tested. The following cellular mechanical properties were measured: elastic modulus (A), relaxed modulus (B), instantaneous modulus (C), and apparent viscosity (D). Elastic and viscoelastic data fit well to Hertzian mathematical models (R²=0.8736 ± 0.052 and R²=0.943 ± 0.027, respectively). Data are presented as scatter dot plot overlaid with the individual geometric means of each clone.

Figure 3

Cell stiffness is correlated with the differentiation potential of NPPCs. NPPCs treated with growth culture medium alone showed negative alizarin red staining (A), negative alcian blue staining (B), negative oil red O staining (C). NPPCs treated with osteogenic medium for 14 days showed mineral deposition around the cells to form nodular aggregates (D). NPPCs treated with chondrogenic medium for 21 days showed positive alcian blue staining (E). NPPCs treated with adipogenic medium for 4 days showed negative oil red O staining (F). Bar=100 μm. Each differentiation lineage showed extensive variability in differentiation potential, suggesting heterogeneity among the clonal populations. Results were used to determine the multipotentiality of clones investigated in this study (G).