Bone Marrow-Derived Aldehyde Dehdrogenase Expressing Cells Possess Endothelial Progenitor Function in Addition to Hematopoietic Repopulating Ability and Aid in Blood Flow Recovery after Acute Ischemic Injury.
Authors: Capoccia Benjamin J. , Wirthlin Louisa , Shepherd Rebecca , O’Dell Kevin , Nolta Jan A. , Link Daniel C. , Hess David A. 
Published: April 2020

Abstract
Human bone marrow (BM) is a heterogeneous compartment of stem and progenitor cells from hematopoietic, endothelial, and mesenchymal lineages. These cell types may be recruited to sites of new blood vessel formation including ischemic tissues and tumor microenvironments. Prospective isolation of cells with neoangiogenic function has proven difficult due to the lack of surface markers specific to endothelial precursors. We have previously characterized a novel population of reconstituting hematopoietic stem and progenitor cells from human umbilical cord blood by selection of cells with high aldehyde dehydrogenase (ALDH) enzyme activity (Hess, Blood 2004). In the current study, we isolated ALDHhi and ALDHlo cells with low side scatter properties from human BM, and assayed for hematopoietic and angiogenic function. The ALDHhi subset represented 0.8±0.2% of total nucleated BM cells whereas ALDHlo cells were 10-fold more abundant (8.2±1.3%, n=5). ALDHhi cells highly expressed cell surface molecules associated with hematopoietic (89.1±1.3% CD45+, 70.5±7.5% CD117+) and endothelial progenitor function (76.5±1.8 CD34+, 72.0±7.6% CD133+). ALDHhi cells also expressed mature macrophage/monocyte markers (24.2±3.9% CD11b+), whereas mature lymphocyte markers (3.5±0.5% CD3+, 5.1±0.3% CD19+), and mature endothelial cell markers (2.6±0.8% VEGFR2+) were rare. The corresponding ALDHlo population demonstrated a significantly reduced frequency of cells expressing a primitive phenotype (8.6±1.5% CD34+, 1.4±0.4% CD117+, 2.2±0.6% CD133+, p&lt;0.05), and a concurrent increase in contaminating lymphocytes (19.8±3.6% CD3+, 10.8±1.0% CD19+, p&lt;0.05). Injection of 104-2x105 BM-derived ALDHhi cells consistently produced hematopoietic repopulation in sublethally irradiated NOD/SCID (n=9) and NOD/SCID beta-2 microglobulin (B2M) null mice (n=13). Equivalent doses of ALDHlo cells produced no human engraftment. Culture of ALDHhi cells in endothelial growth media supplemented with angiogenic growth factors (VEGF, hFGF, IGF, EGF) produced colonies of cells that expressed CD34 and the mature endothelial cell marker CD144 (VE-cadherin). To test the neoangiogenic function of ALDHhi cells in vivo, we performed femoral artery ligation in the hind limb of NOD/SCID B2M null mice, and transplanted purified ALDHhi or ALDHlo cells via the tail vein within 24 hours of ischemic injury. The recovery from acute ischemic injury was calculated by the ratio of blood flow in the ischemic versus the non-ischemic leg by laser Doppler perfusion imaging, performed twice weekly over 28 days. In comparison to PBS-injected control mice (n=6) and mice transplanted with 5x105 ALDHlo cells (n=6), mice transplanted with 105 ALDHhi cells (n=7) showed enhanced blood flow to the ischemic limb by day 7 post-injection (p&lt;0.05). This enhanced recovery of blood flow was maintained throughout the 28-day monitoring period. We are currently investigating the phenotype(s) of human cells recruited to the ischemic area and implicated in the regeneration of vascular function. In summary, the ALDHhi population from human BM is functionally heterogeneous and contains cells with both hematopoietic and endothelial activities. These findings may have implications in the development of cellular therapies for the treatment of patients with acute vascular injury or peripheral vascular disease.