Exploring the Molecular Mechanisms for Enhancing MSC Homing and Lodgement within Sites of Liver Damage/Fibrosis.

Exogenous stem cells such as human mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) have been reported to be applicable for tissue repair, as in the case of chronic liver injury. However, one of the limitations in achieving high enough level of stem cell engraftment at these damaged sites is the low level of recruitment of exogenous stem cells. Our goals in the current studies are to enhance recruitment of human MSC to sites of tissue damage and to equip the MSC with high enzymatic activity to digest through areas of fibrosis surrounding of the damaged tissue, in this case, an immune deficient mouse model of CCl4-mediated liver damage as we have previously reported (Wang et al, 2003). Pretreatment of HepG2 cells with hypoxia significantly increased the levels of the hepatocyte growth factor (HGF) receptor, c-met. Since HGF is both a growth factor and a chemotactic agent found at high levels within sites of hypoxic liver injury, we hypothesized that pre-treatment of MSC with hypoxia would increase the expression of c-met and thus, would enhance the sensitivity of MSC to the chemotactic effect of HGF. First, we demonstrated that in the absence of serum and any exogenous growth factor, a 16-hr hypoxic treatment of MSC increased the total cellular protein level of c-met, as well as the total cellular phosphotyrosine activity following HGF stimulation. 3T3 fibroblasts engineered to secrete human HGF were plated in the bottom well of the transmigration assay and human PKH26-labeled MSC pre-incubated in hypoxic vs. normoxic conditions were added to the upper transwell. Transmigrated cells were enumerated after 3 hours to measure the functional role of c-met induction. Preliminary results show enhanced directional transmigration of hypoxia pre- treated MSC toward an HGF gradient, when compared to normoxic treated MSC. Next, we addressed the issue of liver fibrosis as a possible physical barrier that could block the entry of exogenously recruited MSC into the sites of liver damage. We demonstrated that the combination of hypoxia and HGF modulated the degree of matrix metalloproteinase (mmp2 and mmp9) activity in MSC. In addition, recent studies identified the presence of urokinase plasminogen activator receptor (uPAR) on hematopoietic stem cells. We show here for the first time, that uPAR can also be found on MSC. Ongoing studies are assessing the changes in uPAR and uPA activity following hypoxic culture. We hypothesize that changes in MMP and uPA activity can affect the ability of MSC to engraft/lodge within the fibrotic tissue. In conclusion, we propose that the pre-treatment of MSC with hypoxia and HGF might not only enhance the migration of exogenous stem cells to the site of liver damage but also enhance their ability to degrade the accumulated fibrosis at sites of injury for entry into the regions of damaged tissue.