Survivin Is a Master Regulator of Mesenchymal Stem Cell Functions and Potential Therapeutic Target for Mesenchymal Stem Cell Expansion

Mesenchymal stemcells (MSCs) are found throughout adult organisms and are involved in tissue maintenance and repair, as well as in the regulation of hematopoiesis and immunologic responses. Several clinical trials are currently under way to use allogeneic MSCs for enhancement of hematopoietic stem cell transplantation and treatment of graft-versus-host disease, spinal cord injury, cartilage and meniscus repair, and stroke. However, the therapeutic uses of MSCs can be limited by insufficient MSC number, their survival, and their ability to differentiate into multiple lineages, pointing to an important need to identify factors regulating MSC survival, proliferation, and differentiation. In this study, we explored the role of the anti-apototic protein Survivin in MSC functions. Survivin is an intracellular member of the inhibitor-of-apoptosis protein (IAP) family and is implicated in regulation of apoptosis, cell division, and cell cycle control of many cell types including hematopoietic stem cells, leukemic cells, and endothelial cells. However, whether Survivin is involved in MSC activity or function is unknown. Using flow cytometry and function assays, we found that adult mouse and human MSCs, identified as CD45^-Ter119^-CD51⁺PDGFR⁺Nestin⁺ cells, express high levels of Survivin, which regulates MSCs survival and expansion under normal physiological and stressful conditions. Flow cytometry analysis revealed that approximately 40.5±3.6% of mouse bone marrow MSCs, 33.6±4.2% of adult human bone marrow MSCs, and 43.6±5.3% human cord blood MSCs express Survivin. Treatment of mice in vivo with YM155 (10 mg/kg), a small molecule Survivin inhibitor, for 6 days decreased bone marrow CD45^-Ter119^-CD51⁺PDGFR⁺Nestin⁺ MSC by 2.1 fold and functional fibroblast colony formation (CFU-F) by 2.8 fold. Survivin gene deletion using Survivin-specific SiRNA decreased mouse and human bone marrow derived MSCs number and CFU-F ability by 3.2 fold and 2.8 fold respectively. Retroviral overexpression of Survivin in mouse MSCs enhanced CFU-F formation by 4 fold. In an in vitro wound healing assay YM155-treated MSCs recovered more slowly compared to control cultures. To determine underlying mechanisms involved in Survivin dependent regulation of MSC function, we measured the survival and proliferation of YM155 treated MSCs. Treatment of mouse or human bone marrow-derived MSCs with YM155 (50 nmol/l) for 48 hours enhanced the caspase 3 and 7 expression by 42.8% and 63.9% respectively, while BrdU incorporation was similar in control and YM155-treated MSCs, suggesting that Survivin is primarily involved in MSC survival. To explore whether Survivin is also involved in growth factor-mediated MSC survival and expansion, mouse bone marrow derived MSCs were cultured in the presence of platelet derived growth factor (PDGF) or basic fibroblast growth factor (b-FGF) with or without the Survivin inhibitor YM155. PDGF enhanced the Survivin expression by 52.8±2.2% and b-FGF enhanced Survivin expression by 43.8±4.5% and increased the CFU-F counts by 5.6 fold and 4.2 fold, respectively. In contrast, YM155 treated MSCs did not show any enhancement in Survivin expression, and CFU-F were substantially lower than the corresponding controls (PDGF or b-FGF treated cultures). To determine whether Survivin affects the Survival of stressed MSCs, we exposed mouse bone marrow MSCs to 4 Gy irradiation dose and followed by culture for 3 days with or without the Survivin inhibitor YM155. Irradiation alone reduced the survival of MSCs by 53.5%, however the viability of irradiated MSCs treated with YM155 was reduced by 82.8%. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We found that the exposure of MSC cultures to YM155 during the lineage differentiation process inhibits MSC osteogenic and adipogenic differentiation while sparing chondrogenic cell differentiation. In conclusion, our study demonstrates that Survivin controls basal and growth factor dependent survival and expansion of mouse and human MSCs, and protects them from irradiation induced cell death. Furthermore, our data suggest the regulating Survivin expression in MSCs would be beneficial to enhance MSC recovery for clinical utility.