Expansion of Umbilical Cord Blood Aldehyde Dehydrogenase Expressing Cells Generates Myeloid Progenitor Cells that Stimulate Limb Revascularization

David M Putman, Tyler T Cooper, Stephen E Sherman, Ayesh K Seneviratne, Mark Hewitt, Gillian I Bell, David A Hess, David M Putman, Tyler T Cooper, Stephen E Sherman, Ayesh K Seneviratne, Mark Hewitt, Gillian I Bell, David A Hess

Abstract

Uncompromised by chronic disease-related comorbidities, human umbilical cord blood (UCB) progenitor cells with high aldehyde dehydrogenase activity (ALDHhi cells) stimulate blood vessel regeneration after intra-muscular transplantation. However, implementation of cellular therapies using UCB ALDHhi cells for critical limb ischemia, the most severe form of severe peripheral artery disease, is limited by the rarity (<0.5%) of these cells. Our goal was to generate a clinically-translatable, allogeneic cell population for vessel regenerative therapies, via ex vivo expansion of UCB ALDHhi cells without loss of pro-angiogenic potency. Purified UCB ALDHhi cells were expanded >18-fold over 6-days under serum-free conditions. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, only 15.1% ± 1.3% of progeny maintained high ALDH-activity after culture. However, compared to fresh UCB cells, expansion increased the total number of ALDHhi cells (2.7-fold), CD34+ /CD133+ cells (2.8-fold), and hematopoietic colony forming cells (7.7-fold). Remarkably, injection of expanded progeny accelerated recovery of perfusion and improved limb usage in immunodeficient mice with femoral artery ligation-induced limb ischemia. At 7 or 28 days post-transplantation, mice transplanted with expanded ALDHhi cells showed augmented endothelial cell proliferation and increased capillary density compared to controls. Expanded cells maintained pro-angiogenic mRNA expression and secreted angiogenesis-associated growth factors, chemokines, and matrix modifying proteins. Coculture with expanded cells augmented human microvascular endothelial cell survival and tubule formation under serum-starved, growth factor-reduced conditions. Expanded UCB-derived ALDHhi cells represent an alternative to autologous bone marrow as an accessible source of pro-angiogenic hematopoietic progenitor cells for the refinement of vascular regeneration-inductive therapies. Stem Cells Translational Medicine 2017;6:1607-1619.

Keywords: Aldehyde dehydrogenase; Angiogenesis; Hematopoietic progenitor cell expansion; Peripheral artery disease; Transplantation; Umbilical cord blood.

© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Figures

Figure 1
Figure 1
Purification and expansion of human UCB ALDHhi cells. Representative flow cytometry plots showing the purification of ALDHhi cells from human UCB. (A): Lin– cells treated with DEAB established selection criteria for ALDHlo (R2) and ALDHhi gates (R3). ALDHhi cells represented a rare subset (2.4% ± 0.4%) of Lin– cells. (B): Representative flow cytometry plots showing expanded progeny with low versus high ALDH‐activity after culture for 3, 6, or 9 days. (C): The frequency of cells retaining high ALDH‐activity diminished as culture time progressed. (D): Fold increase in total cells, ALDHhi cells, and ALDHlo cells was calculated at days 3, 6, or 9 compared to the number of cells seeded at day 0. Cell progeny at day 6 demonstrated significant expansion (2.7 ± 0.4‐fold) of total ALDHhi cells. Data represent mean ± SEM from 15 UCB samples. Statistical analyses were performed by ANOVA with Tukey's multiple comparisons test relative to cells seeded at day 0 (*, p < .05; **, p < .01; ***, p < .001). Abbreviations: DEAB, diethylaminobenzaldehyde; ALDH, aldehyde dehydrogenase; ALDHhi, high aldehyde dehydrogenase activity; ALDHlo, low aldehyde dehydrogenase activity; Lin–, Lineage depleted; UCB, umbilical cord blood.
Figure 2
Figure 2
Expanded cells with high ALDH‐activity coexpressed primitive surface markers. (A): Representative flow cytometry plots showing CD34 and CD133 coexpression on expanded cells at 3, 6, or 9 days culture. (B): The frequency of cells that coexpressed CD34/CD133 was significantly increased in the ALDHhi cell subset. (C): Expansion kinetics for total CD34+ cells, CD133+ cells, or CD34+/CD133+ cells during culture. CD34+, CD133+, and CD34+/CD133+ cell content was significantly increased in total cells at days 6 and 9 compared to Day 0 controls. At day 6, CD34+/CD133+ cells were almost exclusively contained within the ALDHhi cell subset. However, within the ALDHhi subset there was no increase in total CD34+, CD133+, or CD34+/CD133+ cells after day 6. Data represent mean ± SEM from five umbilical cord blood samples. Statistical analyses were performed by ANOVA with Tukey's multiple comparison test (*, p < .05; **, p < .01). Abbreviations: ALDHhi, high aldehyde dehydrogenase activity; ALDHlo, low aldehyde dehydrogenase activity.
Figure 3
Figure 3
Total hematopoietic progenitor cell number was increased after expansion. Fresh umbilical cord blood ALDHhi cells or expanded cells harvested at day 6 or day 9 were plated at limiting dilution in methylcellulose media to compare hematopoietic CFU production. (A): The frequency of CFU‐G and CFU‐M colonies was decreased at 6 or 9 days culture. (B): This resulted in significantly reduced total colony formation from 1 CFU in 3.1 ALDHhi cells fresh cells (Day 0), to 1 CFU in 7.3 expanded cells at day 6, or 1 CFU in 9.4 expanded cells at day 9. (C): Taking into account total cell number was increased 18.1 ± 1.7‐fold at day 6, and 38.9 ± 6.0‐fold at day 9, total CFU content compared to fresh ALDHhi cells was increased 7.7 ± 0.7‐fold after 6 days culture and 12.8 ± 2.0‐fold after 9 days culture. Data represent mean ± SEM from 5 UCB samples. Statistical analyses were performed by ANOVA with Tukey's multiple comparison test (*, p < .05; **, p < .01). Abbreviations: ALDHhi, high aldehyde dehydrogenase; BFU, E, blast forming unit‐ erythrocyte; CFU‐G, colony forming unit‐ granulocyte; CFU‐M, colony forming unit‐ macrophage; CFU‐GM, colony forming unit‐ granulocyte and macrophage; CFU‐mixed, colony forming unit containing erythrocytes, granulocytes, and macorphages.
Figure 4
Figure 4
Transplantation of day 6 expanded cells accelerated the recovery of perfusion and improved usage of the ischemic limb. (A–D): Representative LDPI following femoral artery ligation and i.m.‐injection of PBS (n = 10), 2 × 105 fresh umbilical cord blood (UCB) ALDHhi cells (n = 9), or 5 × 105 expanded cells harvested at day 6 (n = 7) or day 9 (n = 8). (E): Transplantation of fresh ALDHhi cells or day 6 expanded cells accelerated the recovery perfusion compared to PBS‐injected controls. However, transplantation of day 9 expanded cells showed perfusion ratios equivalent to PBS‐injected controls. (F): Noldus catwalk paw print intensity at 7 days post‐transplantation showed mice transplanted with day 6 expanded cells (n = 6) regained use of their ischemic leg faster than PBS‐injected controls (n = 6). Data represent mean ± SEM from 6 UCB samples. Statistical analyses were performed by 2‐way ANOVA with Bonferroni's multiple comparisons test (*, p < .05; ***, p < .001). Abbreviation: PBS, phosphate buffered saline.
Figure 5
Figure 5
Transplantation of day 6 expanded cells increased capillary density in the ischemic hind limb. (A, B): Representative photomicrographs of CD31 staining in ischemic adductor muscle sections taken 28 days after femoral artery ligation and intramuscular injection of PBS or day 6 expanded cells. (C, D): Summary of vessel density in the ischemic or nonischemic limb at 7 or 28 days after transplantation. Mice transplanted with fresh umbilical cord blood (UCB) ALDHhi cells or day 6 expanded cells demonstrated increased capillary density in the surgical hind limb. Data represent mean ± SEM from 5 UCB samples. Statistical analyses were performed by ANOVA with Tukey's multiple comparison test (*, p < .05). Abbreviations: ALDHhi, high aldehyde dehydrogenase; PBS, phosphate buffered saline.
Figure 6
Figure 6
Transplantation of day 6 expanded cells increased endothelial cell proliferation in the ischemic hind limb. (A, B): Representative photomicrographs of CD31 cell proliferation within the ischemic adductor muscle at 7 days post‐transplantation. Mouse CD31+EdU+ cells (arrows) were detected in ischemic muscle of mice transplanted with day 6 expanded cells. (C): Compared to mice injected with PBS or day 9 expanded cell, mice transplanted with day 6 expanded cells showed increased endothelial cell proliferation. Data represent mean ± SEM from 4 umbilical cord blood (UCB) samples. Statistical analyses were performed by ANOVA with Tukey's multiple comparison test (*, p < .05; **, p < .01). Abbreviations: EdU, 5‐ethynyl‐2′‐deoxyuridine; HPC, hematopoietic progenitor cell; PBS, phosphate buffered saline.
Figure 7
Figure 7
Coculture with day 6 expanded cells increased HMVEC tubule formation and survival under serum‐free, growth factor reduced conditions. (A): Contact coculture with day 6 expanded cells in growth factor reduced Matrigel significantly increased tubule formation by HMVEC at 6 and 24 hours. (B): Noncontact (transwell) coculture with day 6 expanded cells promoted HMVEC survival under growth factor‐free, serum‐starved conditions. (C–E): Representative flow cytometry plots of apoptotic HMVEC using 7AAD and Annexin V staining. Noncontact coculture with day 6 expanded cells decreased the frequency of dead (F): and apoptotic (G): HMVEC after 24 hours under serum and growth factor‐free conditions. Data represent mean ± SEM from 4 umbilical cord blood samples. Statistical analyses were performed by ANOVA with Tukey's multiple comparison test (*, p < .05; **, p < .01). Abbreviations: EBM2, endothelial basal media; EGM2, endothelial growth media; HMVEC, human microvascular endothelial cells.

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