REX-001, a BM-MNC Enriched Solution, Induces Revascularization of Ischemic Tissues in a Murine Model of Chronic Limb-Threatening Ischemia

Abstract

Background: Bone Marrow Mononuclear Cells (BM-MNC) constitute a promising alternative for the treatment of Chronic Limb-Threatening ischemia (CLTI), a disease characterized by extensive blockade of peripheral arteries, clinically presenting as excruciating pain at rest and ischemic ulcers which may lead to gangrene and amputation. BM-MNC implantation has shown to be efficient in promoting angiogenesis and ameliorating ischemic symptoms in CLTI patients. However, the variability seen between clinical trials makes necessary a further understanding of the mechanisms of action of BM-MNC, and moreover, to improve trial characteristics such as endpoints, inclusion/exclusion criteria or drug product compositions, in order to implement their use as stem-cell therapy.Materials: Herein, the effect of REX-001, a human-BM derived cell suspension enriched for mononuclear cells, granulocytes and CD34+ cells, has been assessed in a murine model of CLTI. In addition, a REX-001 placebo solution containing BM-derived red blood cells (BM-RBCs) was also tested. Thus, 24 h after double ligation of the femoral artery, REX-001 and placebo were administrated intramuscularly to Balb-c nude mice (n:51) and follow-up of ischemic symptoms (blood flow perfusion, motility, ulceration and necrosis) was carried out for 21 days. The number of vessels and vascular diameter sizes were measured within the ischemic tissues to evaluate neovascularization and arteriogenesis. Finally, several cell-tracking assays were performed to evaluate potential biodistribution of these cells.Results: REX-001 induced a significant recovery of blood flow by increasing vascular density within the ischemic limbs, with no cell translocation to other organs. Moreover, cell tracking assays confirmed a decrease in the number of infused cells after 2 weeks post-injection despite on-going revascularization, suggesting a paracrine mechanism of action.Conclusion: Overall, our data supported the role of REX-001 product to improve revascularization and ischemic reperfusion in CLTI.

Current Status of Angiogenic Cell Therapy and Related Strategies Applied in Critical Limb Ischemia

“…These models allow us to follow-up cell mobilization in response to ischemia [36][37][38][39]. Moreover, biodistribution assays are essential to determine the cell's fate [40,41] and more importantly, to evaluate the biosafety profile, being required by regulatory guidelines prior to initiating cell therapy into the clinic [42]. Furthermore, for treatments testing human components such as human cells, immunosuppressed animals (nude, athymic, etc.)…”

“…Schematic representation of femoral artery ligation (FAL) strategies usually applied to create PAD/CLI models, from the lowest (left) to the highest (right) severity models of the disease. A representative image of the FAL strategy followed in our research group is also shown [41,47]. Legend: 1 Iliac artery, 2 Iliacofemoral artery, 3 Internal iliac artery, 4 Pudendoepigastric trunk, 5 Femoral artery and its branches (lateral circumflex and proximal caudal), 6 Superficial caudal epigastric artery, 7 Popliteal artery, and 8 Saphenous artery.…”

“…Therapies employing bone marrow mononuclear cells (BM-MNCs) constitute a promising alternative for CLI patients to avoid or delay the onset of amputation [112]. BM-MNCs consist of a heterogeneous mix of multipotent stem cells working cooperatively as MSCs, HSCs, EPCs, monocytes, lymphocytes, and pluripotent stem cells [41,113]. We and other researchers have reported the beneficial effects of different combinations of BM-MNCs, representing an effective approach in promoting new vessel formation, perfusion recovery, and CLI reversal [41,100,[114][115][116][117][118][119][120][121][122].…”

“…BM-MNCs consist of a heterogeneous mix of multipotent stem cells working cooperatively as MSCs, HSCs, EPCs, monocytes, lymphocytes, and pluripotent stem cells [41,113]. We and other researchers have reported the beneficial effects of different combinations of BM-MNCs, representing an effective approach in promoting new vessel formation, perfusion recovery, and CLI reversal [41,100,[114][115][116][117][118][119][120][121][122]. In the ischemic tissue, BM-MNCs produce and secrete different cytokines and growth factors [123] and increase neovascularization and collateral vessel formation in limb ischemia [79].…”

Long Term Response to Circulating Angiogenic Cells, Unstimulated or Atherosclerotic Pre-Conditioned, in Critical Limb Ischemic Mice

“…Perfusion was expressed as the ratio of left (ischemic) vs. right (non-ischemic) limbs. In parallel, ischemic symptoms (motility changes, inflammation, ulceration, and necrosis) were registered during the entire experiment using the Tarlov score (motility changes) [28], together with ischemic and modified ischemic scores [29] used in CLI animal models, as described [31] (Supplementary Table S1).…”