Abstract
Disorders such as peripheral artery disease cause hypoxic areas in tissues. Work from our group and others shows that stem cells appear to have innate mechanisms to respond to hypoxic conditions by migrating to the region of damage, and releasing trophic factors which initiate regeneration. Many tissues activate hepatocyte growth factor (HGF) as a response to ischemic injury. Multiple progenitor cell types express cMet, an HGF receptor. Mesenchymal stem cells (MSC) have been shown to improve regeneration of injured tissues in vivo, but their mechanisms of homing to the site of injury remain unclear. In the current studies we examined the potential for human MSC to repair injury caused by hind limb ischemia in immune deficient mice. We observed that hypoxic pre-conditioning of MSC upregulated expression of cMet, which could render the MSC more responsive to active HGF present at the site of ischemic injury. We first analyzed muscle lysates from mice that had undergone hind limb ischemia, vs sham-operated controls. ELISA results demonstrated that although a sham surgery caused a slight elevation in HGF levels 12 to 48 hours post surgery, ischemia caused a steady increase in HGF secretion from 12 hours to 48 hours post surgery. These data suggested that HGF might play a role in recruiting c-met+ MSC to the injury area. We next subjected primary human MSC to a 24-hr preconditioning in hypoxic (2 to 3% oxygen- actually tissue normoxia) vs. normoxic (21% oxygen, most commonly used in the incubator) conditions. MSC upregulated cMet in hypoxic conditions and then responded more robustly to HGF stimulation by signaling through cMet. Hypoxic pre-conditioning also caused signaling through a pro-survival Akt pathway, possibly improving the survival potential of MSC as they migrate in vivo. We next asked whether MSC are more motile in hypoxia. MSC were cultured in hypoxic or normoxic conditions +/− 25ng/ml HGF. While both HGF alone and a combination of hypoxia and HGF increased the cell migration capacity, treatment with hypoxia alone caused MSC to be the most migratory. These results suggest that hypoxic pre-conditioning may help MSC to migrate to the site of injury, while high active HGF levels in the tissue will hold the stem cells at the site of damage. Finally, to address the question of whether hypoxic pre-conditioning of MSC improves their tissue regeneration ability, we cultured them in hypoxic vs. normoxic conditions for 24 hrs and then transplanted them into NOD/SCID/B2m null mice that had undergone hind limb ischemia surgery one day prior to the transplant. Laser Doppler imaging showed significantly better blood flow recovery in the limbs of injured mice that were treated with pre-conditioned MSC, as compared to the saline control group. Mice that had received hypoxic pre-conditioned MSC improved bloodflow to the injured limb more rapidly than those transplanted with normoxic MSC, with a significant difference observed at day 5, demonstrating that hypoxic pre-conditioning increased the therapeutic potential of MSC. In summary, our data confirm that a 24 hour hypoxic pre-conditioning in vitro prior to transplantation improves the therapeutic potential of MSC, through activation of the pro-survival Akt pathway, upregulation of cMet, which allows them to be more responsive to the HGF activated at the site of ischemic injury, and an increased motility that allows them to more rapidly reach the area of injury.
Hypoxia-Regulated miRNAs in Human Mesenchymal Stem Cells: Exploring the Regulatory Effects in Ischemic Disorders