Aldehyde Dehydrogenase-Activity Purifies Multiple Hemangiogenic Lineages That Accelerate Vascularization of Ischemic Tissue through Paracrine Support of Neovessel Formation.
Authors: Levac Krysta D. , Robson Debra L. , Neelamkavil Marian J. , Hohm Sarah A. , Capoccia Benjamin J. , Link Daniel C. , Nolta Jan A. , Hess David A. 
Published: April 2020

Abstract
In the context of regenerative therapies, the contributions of transplanted stem cells are not limited to the direct replacement of damaged cells. Easily procured, post-natal bone marrow (BM) contains several colony-forming cell (CFC) lineages that promote repair in damaged tissues through paracrine activities. However, determination of regenerative mechanisms after transplantation has proven difficult due to the involvement of multiple cell types in angiogenesis and inflammation. To enrich for human BM cells with hemangiogenic functions, we FACS purified based on low versus high aldehyde dehydrogenase (ALDH) activity, an enzyme with increased expression in hematopoietic and neural precursor cells. In contrast to ALDHlo cells which were mainly comprised of mature T- and B-cells (&gt;90%), ALDHhi high cells were rare (8.2±1.3% vs. 0.8±0.2% of BM cells, n=8), and showed increased expression of primitive hematopoietic/endothelial (CD34, CD133, p&lt;0.01), and mature monocyte markers (CD14, p&lt;0.05), lineages previously associated with the paracrine support of angiogenesis. Similarly, the ALDHhi population possessed significantly enhanced (p&lt;0.05) hematopoietic (1 HCFC in 8 ALDHhi cells), endothelial (1 ECFC in 1283 ALDHhi cells), and mesenchymal stromal (1 MSCFC in 932 ALDHhi cells) functions in vitro, corresponding to &gt;100-fold enrichment of respective CFCs compared to unfractionated BM cells. In contrast to mature human aortic endothelial cells, secondary cultures of ECFC in matrigel matrix did not result in organized tubule formation (n=4), suggesting that ALDH-purified ECFC lacked full endothelial cell differentiation in vitro. On the contrary, ALDHhi MSCFC were highly proliferative and showed multipotent differentiation in secondary cultures for adipocyte, osteocyte, and chondrocyte formation. To assess pro-angiogenic function in vivo, we ligated the femoral artery in NOD/SCID β2M null mice, and performed i.v. transplantation without preparative irradiation within 24 hours of surgery. Compared to mice injected with unpurified BM (n=5) or ALDHlo cells (n=7), mice transplanted with ALDHhi cells (n=8) showed significantly accelerated recovery of perfusion within 7 days post-transplantation by laser Doppler perfusion imaging (p&lt;0.05), and increased collateral capillary formation at day 28. However, using the novel NOD/SCID/MPSVII mouse for sensitive tracking of donor cells in situ, long-term engrafting cells in the ischemic limb were exceedingly rare (&lt;0.1% by FACS), and did not permanently incorporate into neovessels. Cell tracking using fluorescent dextran-coated iron nanoparticles showed ALDHhi cells homed specifically to the site of vascular injury, and were only detected between 1–7 days post-injection, suggesting that transiently engrafting ALDHhi cells stimulate a localized pro-angiogenic cascade. We are currently investigating the release of trophic factors by ALDHhi cells that support the development of a pro-angiogenic regenerative niche. Nevertheless, selection of ALDHhi cells from human BM simultaneously purifies multiple hemangiogenic cellular lineages, and represents a promising population for the development of cellular therapies to promote vascular recovery during peripheral artery or ischemic disease.