(806e) The Intracellular Mechanical Reponse of Mesenchymal Stem Cells to Oxidative Stress Used to Compare Vertebrate Animal Models of Stem Cell Homing

THE INTRACELLULAR MECHANICAL REPONSE OF MESENCHYMAL STEM CELLS TO OXIDATIVE STRESS USED TO COMPARE VERTEBRATE ANIMAL MODELS OF STEM CELL HOMING

Kevin Rodriguez, Niti Khambhati, Michelle Dawson

Human mesenchymal stem cells (hMSCs) show great potential as cellular therapeutics. These adult stem cells can be isolated from bone marrow by adherence to plastic then differentiated into a variety of tissues. Natively, MSCs migrate in response to a gradient of soluble factors to sites of inflammation, such as wounds or tumors, where they engraft to aid in regeneration. Systemic infusion of engineered MSCs offers a minimally invasive method of delivering therapeutic genes to diseased tissues; however, transport limitations, which develop with extended culture, genetic modification, and stem cell expansion, reduce the homing efficiency of stem cell-based therapeutics. Extended culture may also result in spontaneous differentiation and cellular aging (senescence)[1]. In previous studies, the intracellular mechanical response of murine and human MSCs to soluble growth factors was characterized with multiple particle tracking microrheology [1, 2]. The cytoskeleton of murine MSCs homogeneously stiffened in response to these factors; whereas, human MSCs underwent a more local stiffening response, which was lost after a few passages. This inability of human MSCs to respond mechanically to soluble growth factors was correlated with loss of other stem cell functions. Murine MSCs are often used in characterizing the homing of MSC-based therapeutics to wound and tumor tissues; however, fundamental differences in mouse and human MSCs may limit their usefulness in these applications.

The aim of this study was to examine the intracellular mechanical response of MSCs isolated from different vertebrate animals to hypoxic conditions, used to stimulate the secretion of soluble growth factors that mediate cell migration. For these studies, MSCs were isolated from mouse, rat, pig, rabbit, and human bone marrow and cultured in hypoxic (2% oxygen) or normoxic (21% oxygen) conditions for at least 24 hours. Multiple particle tracking microrheology was used to characterize the intracellular mechanical properties. Immunostaining and fluorescence microscopy was used to characterize actin stress fiber densities and cell and nuclear shape factors. Colony forming assays were used to characterize MSC regenerative capacity, and differentiation assays were used to characterize multipotency.  In normoxic conditions, rat, rabbit and pig MSCs displayed mechanical and morphological properties that were similar to human MSCs; however, only pig MSCs underwent the local stiffening response seen in human MSCs.  For all species, reduced oxygen concentrations resulted in cytoskeletal stiffening, which was correlated with increased actin stress fiber densities and cell and nuclear elongation.  These changes in cytoskeletal mechanics and morphology may be important in facilitating the migration of MSCs through in vivo transport barriers. These results may contribute to the development of new models for studying stem cell homing. 

1. McGrail, D.J., K.M. McAndrews, and M.R. Dawson, Biomechanical analysis predicts decreased human mesenchymal stem cell function before molecular differences.Exp Cell Res, 2012.

2. McGrail, D.J., et al., Differential mechanical response of mesenchymal stem cells and fibroblasts to tumor-secreted soluble factors. PLoS One, 2012. 7(3): p. e33248.