Importance of the culture method for differential expression of osteogenic and stemness genes in the lineage commitment of bone marrow-derived mesenchymal stromal cells

Abstract

Bone marrow-derived mesenchymal stem or stromal cells (BM-MSCs) are considered good sources for cell therapy in clinical applications. However, BM-MSCs lose their self-renewal capacity and multi-lineage differentiation potential during prolonged cell passage in vitro. Furthermore, BM-MSCs that are cultured for a long time become committed to the osteogenic lineage as they approach senescence and lose their potential to differentiate along the other lineages. The decreased stem cell properties of MSCs pose significant limitations to their application in cell-based regenerative medicine. BM-MSCs are composed of heterogeneous cell populations. However, little is known about the effects of the interaction between the different BM-MSC populations on cellular senescence. Thus, the purpose of this study was to identify mechanisms that induce cellular senescence in BM-MSCs via cell-cell interactions between the heterogeneous BM-MSC populations. In addition, this study proposes an alternative BM-MSCs culture method, which involves repopulating a “primitive” cell population from late-passage MSCs with poor multipotentiality and low cell proliferation rate by simply altering the plating density.First, we confirmed senescence in BM-MSCs following repeated serial subculture. Late-passage MSCs include a subpopulation of more committed osteogenic cells with increased expression of osteogenic transcription factors (Runx2 and Dlx5) that is further elevated during subsequent passage and diminishing stemness with decreased expression of stemness genes (Sox2 and Nanog). Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation of BM-MSCs. This result indicated that Sox2 is important for maintaining the stemness of BM-MSCs. Next, we hypothesized that cytokines secreted by the large-cell BM-MSC population would affect the cellular senescence and osteogenic lineage commitment of primitive cells (small-cell population) in heterogeneous BM-MSCs because the large-cell population has been considered the senescent population. Indeed, senescence and osteogenic lineage commitment of BM-MSCs were strongly induced in the presence of cytokines secreted by the large-cell population. Among the cytokines, the level of interleukin-6 (IL-6) secreted by large-cell population was significantly higher than that secreted by the small-cell population. Therefore, we focused our attention on the ability of IL-6 to induce cellular senescence and osteogenic lineage commitment in the small-cell population. IL-6 induced osteogenic lineage commitment and cellular senescence in the BM-MSC population by increasing Runx2 and Dlx5 protein levels, and reduced stemness by decreasing Sox2 protein expression. Furthermore, the IL-6-induced Runx2 and Dlx5 proteins decreased the transcriptional activity of Sox2. These results indicate that IL-6, one of the cytokines secreted from the large-cell population of BM-MSCs, can induce osteogenic lineage commitment by up-regulating the expression of osteogenic transcription factors (Runx2 and Dlx5) and decrease the self-renewal capacity and multi-lineage differentiation potential of primitive cells by down-regulating the transcriptional activity of Sox2, stemness-related gene.Further, we determined the effects of low-density culture (compared to high density culture) on late-passage BM-MSCs. We repopulated “primitive” cells (small-cell population) by replating late-passage BM-MSCs at low density (10-20 cells⁄cm2) regardless of donor age. The repopulated BM-MSCs derived from low-density cultures were smaller than the cells from high-density cultures had spindle-shaped morphology, and exhibited enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potentials. The strong expression of osteogenic genes (Runx2, Dlx5, alkaline phosphatase and type I collagen) in late-passage BM-MSCs was reduced by replating at low density, whereas expression of 3 stemness markers (Sox2, Nanog and Oct-4) reverted to levels observed in early-passage BM-MSCs.In conclusion, among the cytokines secreted by the large-cell population, IL-6 may play important roles in cellular senescence and osteogenic lineage commitment of BM-MSCs during prolonged cell passage in vitro. Therefore, molecules targeting the IL-6 signaling pathway would be useful in maintaining the primitive BM-MSC population in long-term culture. In addition, plating density should be considered a critical factor in the enrichment of “primitive” cells in heterogeneous BM-MSC populations.