Augmented Revascularization and Recovery From Acute Limb Ischemia After Transplantation of Human Umbilical Cord Blood Progenitor Cells with High Aldehyde Dehydrogenase Activity.

Abstract 3042

Poster Board II-1018

Clinical application of regenerative cell-based therapies for the treatment of ischemic vascular disease has proven challenging to implement due the involvement of multiple human cell types that co-ordinate angiogenesis. Previously, transplanted human progenitor cells from hematopoietic, endothelial, and mesenchymal lineages have all been implicated in the recovery and de novo production of perfused blood vessels in vivo. Using high aldehyde dehydrogenase (ALDH^hi) activity, a conserved function of these pro-angiogenic hematopoietic and non-hematopoietic progenitor cell lineages, we have recently shown that transplanted human bone marrow (BM) ALDH^hi cells transiently recruit to areas of hypoxia and augment revascularization and recovery of perfusion in ischemic limbs via the stimulation of endogenous vascular regeneration. However, clinical use of autologous BM-derived cells in patients with critical limb ischemia may be limited by the availability or dysfunction of transplanted pro-angiogenic cells due to vascular disease-related pathologies. The non-invasive collection and early ontogeny of human umbilical cord blood (UCB) progenitor cells provides a promising alternative source of pro-angiogenic progenitor cells with vascular regenerative functions. Human UCB mononuclear cells (MNC) were purified using high-speed fluorescence-activated cell sorting to accrue clinically applicable cell populations with low side scatter and high (ALDH^hi, 0.4±0.1% of total MNC) versus low (ALDH^lo, 41.2±3.6% of total MNC) ALDH activity (n=10). Compared to UCB-derived ALDH^lo cells, which were devoid of hematopoietic and non-hematopoietic progenitor function in vitro, ALDH^hi cells were enriched for hematopoietic colony forming cells (1 HCFC in 3.6 cells, n=4) and endothelial colony forming cells (1 ECFC in 5.8×10⁴ cells, n=4) enumerated after 14 days in culture. In contrast to human BM-derived ALDH-purified cells which were enriched for mesenchymal colony forming cells, analogous ALDH^lo and ALDH^hi cells from UCB did not establish mesenchymal-stromal colonies in vitro suggesting a comparative deficiency in mesenchymal stem cell frequency after UCB venipuncture. We investigated the vascular regenerative capacity of these human UCB-derived ALDH^lo and ALDH^hi cells by tail-vein transplantation into sublethally irradiated (275cGy) immune-deficient, β-glucuronidase (GUSB) deficient, NOD/SCID/MPSVII mice with acute limb ischemia induced by unilateral femoral artery ligation and transection. Using weekly laser Doppler perfusion imaging to track the kinetics of blood flow recovery indicated by the perfusion ratio (PR) in the ischemic versus non-ischemic limb, mice transplanted with 2×10⁵-4×10⁵ purified ALDH^hi cells showed significantly improved recovery of limb perfusion by day 21 post-transplantation (PR=0.70±0.06, n=6) compared to mice injected with phosphate-buffered saline (PR=0.35±0.06, n=5, P<0.01), 10×10⁶ ALDH^lo cells (PR=0.45±0.07, n=7, P<0.01), or 20×10⁶ unpurified UCB MNC (PR=0.40±0.06, n=7, P<0.01). Furthermore, transplantation of ALDH^hi progenitor cells augmented revascularization in the ischemic limb compared to all other treatments at day 28 post-transplantation (P<0.01), quantified by immunohistochemical detection and enumeration of von Willebrand factor⁺ blood vessels in frozen sections from the ischemic adductor muscle. Only mice transplanted with ALDH^hi cells showed retention of human hematopoietic (CD45⁺/HLA-A,B,C⁺) cells in the ischemic adductor muscle for 28 days (3.0±2.1%, n=6) quantified by flow cytometry. Ischemic muscle sections from NOD/SCID/MPSVII mice transplanted with human UCB ALDH^hi cells confirmed human cell retention and engraftment adjacent to damaged vasculature and ischemic muscle fibres at day 28 post-transplantation, measured by colorimetric detection of ubiquitous GUSB activity in transplanted human cells. Human UCB-derived ALDH^hi cells, which were devoid of mesenchymal-stromal cells but enriched for potentially pro-angiogenic endothelial and hematopoietic progenitor lineages, showed persistent retention in areas of hypoxia and augmented the endogenous revascularization and recovery of perfusion in ischemic limbs. Thus, ALDH^hi mixed progenitor cells may prove to be a useful allogeneic alternative to human BM in the clinical treatment of patients with severe peripheral vascular disease.