Humoral factors by bone marrow-derived MSC enhance survival of injured β-cells by preserving AKT signalling

Introduction: Our previous work has verified in vitro that human bone marrow-derived MSC with telomerase overexpression (hMSC-TERT) migrate towards injured rat INS-1E β-cells and increase their viability. Both cell lines were considered a valid model since they closely resemble primary rat (plastic-adherent) MSC and islets. Here we investigated whether humoral factors by cocultured hMSC-TERT reduce apoptosis and induce proliferation in INS-1E. Since AKT signalling plays a very central role for survival and proliferation in β-cells, we further analysed whether humoral hMSC-TERT-derived factors rescue p-AKT levels during β-cell injury.

Methods: hMSC-TERT were cocultured in inserts with 0.4 µm pores that allow only soluble factors but not cells to pass the membrane. Apoptosis was measured by Annexin-V FACS and caspases 3/7 activity assay. Viability was measured by MTS assay. Active (phosphorylated) p-AKT levels were detected by Western blot. Proliferation was analysed by counting cell numbers and Ki-67+ nuclei.

Results: Cocultured hMSC-TERT significantly reduced ALX and STZ-induced apoptosis in INS-1E. The degree of this reduction was similar to enhancement of viability. LD₅₀ dosages of ALX (6.6 mM) and STZ (0.66 mM) completely deplete p-AKT levels within 2 and 8h, respectively. This loss of p-AKT was substantially prevented by cocultured hMSC-TERT that have been preactivated over night by injured INS-1E while hMSC-TERT without preactivation have only small effects. Furthermore, chronic coculture of hMSC-TERT enhanced proliferation of INS-1E to significant levels after 5 days. HEK293 human kidney cells and MRC5 human fibroblasts were used as controls to verify specificity of MSC-mediated effects.

Conclusions: MSC-derived factors promote survival of injured β-cells by reduction of apoptosis via protection of p-AKT levels. Modulation of AKT signalling may further be responsible for increased viability and enhanced proliferation in untreated β-cells. Identification and characterization of humoral factors secreted by MSC may provide novel therapeutic strategies for protection of endogenous insulin biosynthesis in diabetes mellitus and for enhancement of graft survival in islet transplantation.