(176c) Modulation of Mitochondrial Activity in Human Mesenchymal Stem Cells with Biomolecules to Maintain Cell Stemness

Human mesenchymal stem cells (hMSCs) are multipotent cells that have the potential to differentiate into several tissue types including fat, cartilage, bone, and muscle. The proliferative capacity and multipotency of hMSCs have motivated great interest in the therapeutic potential of these cells in the field of regenerative medicine. However, in vitro expansion of stem cells faces significant challenges including spontaneous differentiation and premature aging. In this work, we explored the use of two biomolecules (M1 and M2) to regulate the metabolic activity, proliferation, and differentiation potential of hMSCs. Toward this end, hMSCs were cultured for 5 days in growth media supplemented with increasing levels of M1 and M2. DNA and mRNA samples were collected, and gene expression analyses for tissue specific, proliferative, and stemness markers were performed using quantitative PCR. Furthermore, changes in DNA levels were monitored to estimate the average doubling time for cells in each condition. Finally, hMSCs were stained with JC-1 and fluorescence images were acquired with a laser scanning confocal microscope. Images were analyzed using ImageJ to evaluate changes in mitochondrial abundancy and morphology. At the gene expression level, initial results for the M1 treatments showed a downregulation in the expression of the proliferation marker PCNA, which is in agreement with the quantitative DNA assessments. The overall results suggest that M1 exposure can be used to modulate the proliferation rate of hMSCs, while the range of M2 concentrations tested did not appear to have a significant effect on net cell growth. Pertaining to differentiation potential, increasing levels of M1 did not significantly impact the expression of lineage specific markers; however, the expression of the stemness marker CD105 was upregulated. These changes in differentiation and proliferative potential were correlated with a shift in the mitochondrial morphology from elongated to spherical, which further supports a potential role for M1 in maintaining hMSC stemness.