Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity

Abstract

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.

Figures

Figure 1

Purification and cell surface marker expression of ALDHlo and ALDHhi cells isolated from human BM. (A) Human BM cells were selected for low side scatter and low (R2 = 8.2% ± 1.3%) or high (R3 = 0.8% ± 0.2%) Aldefluor fluorescence (n = 11). (B,C) ALDHhi cells showed increased forward scatter mean fluorescence intensity compared with ALDHlo cells. (D,E) Representative FACS of ALDHlo and ALDHhi cells for human cell surface markers expressed on primitive progenitors (CD34, CD133), monomylocytic cells (CD14, CD33), ECs (CD31, CD144), B lymphocytes (CD20, CD19), and T lymphocytes (CD4, CD8).

Figure 2

Transplantation of BM-derived ALDHhi cells improved perfusion and capillary density in ischemic limbs. (A-D) Representative LDPI after right femoral artery ligation and tail vein injection of PBS (n = 8), 50 × 106 BM MNCs (n = 6), 5 × 105 ALDHlo (n = 8), or 1 to 2 × 105 ALDHhi cells (n = 8) monitored weekly for 21 days. Numbers in the lower left of each LDPI image indicate PR of the ischemic versus the nonischemic limb. (E) Summary of PRs for all mice transplanted as indicated in panels A through D. Transplantation of BM ALDHhi cells augmented perfusion in ischemic limbs from 7 to 21 days after transplantation (*P < .05). (F) Capillary density at days 21 to 28 was increased in mice transplanted with ALDHhi cells (*P < .05). All micrographs were viewed with an Olympus (Hamburg, Germany) BX50 microcoscope using air lenses and betaglucuronidase and hematoxylin stains. All images were taken with a Hitachi HV-F2S CCD camera using Northern Eclipse version 7.0 software. The following numeric aperture (NA) air objectives were used: panels C and F, 10×/0.30; panels E and G, 20×/0.50.

Figure 3

Transplanted BM-derived ALDHhi cells are recruited to the ischemic limb. (A,B) Representative FACS showing removal of noncellular events (R1) and selection of ALDHhiFe[750]lo (R2 = 47.3% ± 9.9%) and ALDHhiFe[750]hi (R3 = 22.5% ± 6.5%) nanoparticle-labeled cells (n = 5). (C) Prussian blue staining of sorted ALDHhiFe[750]hi cells shows cytoplasmic accumulation of iron nanoparticles. (D) Recruitment of ALDHhiFe[750]hi cells to the site of ischemic injury 24 to 48 hours after transplantation demonstrated by Kodak multimodal imaging. Intense signal beside mouse limb represents internal standard for Fe[750] fluorescence. (E) Two days after MNC injection, CD45+ cells (brown) were detected near arterioles (, inset), and GUSB+ human cells (red) that were negative for CD45 expression were detected adjacent to muscle fibers () in the ischemic limb. (F) Human cells were not detected in the ischemic limbs of mice transplanted with BM ALDHlo cells. (G) After injection of ALDHhi cells, GUSB+CD45− human cells were detected adjacent to vascular structures and muscle fibers as early as 2 days after transplantation and remained for up to 30 days after transplantation (). (H,I) Transplantation of ALDHhiFe[750]hi cells produced increased fluorescence in the ischemic limb at 1 and 7 days. Fluorescent signal was cleared from the ischemic limb by 14 days after transplantation (n = 3).

Figure 4

Human BM ALDHhi cells are enriched for multipotent HCFCs and MCFCs. (A) ALDHhi cells cultured in methylcellulose media established hematopoietic colonies (1 HCFC in 8 cells) at an increased frequency compared with ALDHlo cells (1 HCFC in 2500 cells; *P < .05, n = 6). (B) ALDHhi cells cultured in Amniomax media established mesenchymal colonies (1 MCFC in 1370 cells) at an increased frequency compared with ALDHlo cells (1 MCFC in 2.5 × 104 cells; *P < .05, n = 5). ALDHlo- and ALDHhi-derived MCFCs (C,D) were differentiated into adipocytes (E,F), osteocytes (G,H), or chondrocytes (I,J). ALDHhi-derived MCFCs demonstrated multilineage differentiation forming adipocytes (Oil Red O+), osteocytes (Alizarin Red+), and chondrocytes (Safranin O+) in secondary cultures. ALDHlo-derived MCFCs showed reduced differentiative capacity (n = 3).

Figure 5

Human BM ALDHhi cells cultured in mesenchymal- or endothelial-supportive conditions form cellular networks in secondary Matrigel cultures. (A) Human BM ALDHlo and ALDHhi cells cultured in complete EGM-2 media formed colonies at 9 to 14 days of culture. (B) Only ALDHhi colonies established proliferative outgrowth. (C) ALDHhi cells showed an increased frequency of colony formation (1 CFC in 1464 cells) compared with ALDHlo cells (1 CFC in 4.2 × 105 cells; *P < .05, n = 4). (D-F) HAECs cultured in EGM-2 media, ALDHhi cells cultured in Amniomax media, and ALDHhi cells cultured in EGM-2 were tested for spontaneous tubule forming capacity in secondary Matrigel assays. (G) Mature HAECs formed patterned multinucleated tubule networks. (H) ALDHhi MCFCs grown in Amniomax aggregated into organized cellular networks with elongated tubule-like cellular morphology. (I) ALDHhi cells grown in EGM-2 media also aggregated into cellular networks forming tubule-like structures with more diffuse cellular connections (n = 3).