Purpose Lung transplantation (LTx) is limited by the scarcity of suitable donor lungs. One option to expand the donor pool is the use of donation after circulatory death (DCD) lungs. Additionally, ex vivo lung perfusion (EVLP) allows for evaluation and treatment of donor lungs prior to LTx. There are limited data describing the cellular, specifically leukocyte populations in EVLP perfusate, and their link to donor mode of death. We hypothesized that EVLP perfusate leukocytes differ between DCD and neurological determination of death (NDD) lungs, and between lungs accepted and declined for transplant. Methods EVLP perfusates were sampled hourly from clinical EVLP cases until termination. Perfusate cells were separated and cryopreserved. The first and last available perfusate from lungs accepted (n = 10 NDD and n = 10 DCD) and declined (n = 10 NDD and n = 10 DCD) for LTx were selected and stained with flow cytometry panels to characterize major leukocyte populations. Results Lymphoid cells form the majority of perfusate cells (Fig A). B cells were significantly higher in declined lungs regardless of mode of donor death (Fig B). CD4+ memory T cells (CD3+ CD4+ CD45RO+) and activated CD8+ T cells (CD3+ CD8+ CD25+) were elevated in the DCD lung perfusates (Fig C). In all groups, CD163hi, HLA-DR+ monocytes were observed at first available perfusate and diminished at the end of EVLP, while neutrophils were low in the first perfusate sample but increased significantly by the end of EVLP (Fig D). Conclusion Interestingly, leukocyte populations in EVLP perfusate differ between mode of donor death, with lower levels of B cells associated with lungs accepted for transplant, and certain T cell subsets over-represented in DCD lungs. While no significant changes were observed in myeloid cells, their kinetics over time vary based on cell type. Future studies will be required to determine the sources of these cell populations and whether they are involved in the pathogenesis of lung injury prior to transplant.

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

During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDHhi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDHl ° and ALDHhi MSC subsets demonstrated similar expression of stromal cell (>95% CD73+ , CD90+ , CD105+ ) and pericyte (>95% CD146+ ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDHhi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDHhi MSC or CDM produced by ALDHhi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDHl ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDHhi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDHhi demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542-1553.

Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDHhi) stimulate islet regeneration after transplantation into mice with streptozotocin‐induced β cell deletion. However, ALDHhi cells represent a rare progenitor subset and widespread use of UCB ALDHhi cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDHhi cells expand efficiently under serum‐free, xeno‐free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH‐activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDHhi cells diminished as culture time progressed such that total ALDHhi cell number was maximal (increased 3‐fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH‐activity (ALDHlo subset) from less differentiated progeny with high ALDH‐activity (ALDHhi subset). The ALDHhi subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34+/CD38− cells, 37.0% ± 6.9% CD34+/CD133+ cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDHlo subset. Notably, bulk cells or ALDHlo cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin‐treated NOD/SCID mice. However, transplantation of the repurified ALDHhi subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet‐associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH‐activity after short‐term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ. Stem Cells 2016;34:873–887

This unit describes the isolation and application of human umbilical cord blood progenitor cells to modulate vascular regenerative functions using in vitro co-culture systems and in vivo transplantation models. Using aldehyde dehydrogenase as a marker of stem cell function, blood-derived progenitors can be efficiently purified form human umbilical cord blood using flow cytometry. We describe in vitro approaches to measure cell-mediated effects on the survival, proliferation, and tube-forming function of endothelial cells using growth-rate assays and Matrigel tube-forming assays. Additionally, we provide a detailed protocol for inducing acute unilateral hindlimb ischemia in immune-deficient mice to assess progenitor cell-modulated effects on vascular regeneration by tracking the recovery of blood flow using noninvasive laser Doppler perfusion imaging. Collectively, we present combined in vitro and in vivo transplantation strategies for the pre-clinical assessment of human progenitor cell-based therapies to treat ischemic disease.

Cell sorting based on high aldehyde dehydrogenase (ALDH) activity has emerged as a clinically applicable method to purify human bone marrow (BM) and umbilical cord blood (UCB) progenitors based on a conserved stem cell function. Although rare, ALDHhi cells are highly enriched for progenitors of hematopoietic, endothelial, and mesenchymal stromal cell (MSC) lineages. Transplanted ALDHhi progenitors are under investigation in clinical trials to enhance UCB engraftment in adults undergoing transplantation. Transplanted BM ALDHhi cells also recruited to areas of tissue ischemia and augment endogenous revascularization and recovery after femoral artery ligation. Moreover, ex vivo expanded MSCs from ALDH-purified cells stimulated the neogenesis of small beta cell clusters in models of pancreatic injury. Understanding how angiogenic and regenerative programs are stimulated by ALDHhi progenitor subsets may provide new approaches in progenitor cell therapy for tissue repair.

Umbilical cord blood (UCB) represents a readily available source of hematopoietic and endothelial precursors at early ontogeny. Understanding the proangiogenic functions of these somatic progenitor subtypes after transplantation is integral to the development of improved cell-based therapies to treat ischemic diseases. We used fluorescence-activated cell sorting to purify a rare (<0.5%) population of UCB cells with high aldehyde dehydrogenase (ALDH(hi) ) activity, a conserved stem/progenitor cell function. ALDH(hi) cells were depleted of mature monocytes and T- and B-lymphocytes and were enriched for early myeloid (CD33) and stem cell-associated (CD34, CD133, and CD117) phenotypes. Although these cells were primarily hematopoietic in origin, UCB ALDH(hi) cells demonstrated a proangiogenic transcription profile and were highly enriched for both multipotent myeloid and endothelial colony-forming cells in vitro. Coculture of ALDH(hi) cells in hanging transwells promoted the survival of human umbilical vein endothelial cells (HUVEC) under growth factor-free and serum-free conditions. On growth factor depleted matrigel, ALDH(hi) cells significantly increased tube-like cord formation by HUVEC. After induction of acute unilateral hind limb ischemia by femoral artery ligation, transplantation of ALDH(hi) cells significantly enhanced the recovery of perfusion in ischemic limbs. Despite transient engraftment in the ischemic hind limb, early recruitment of ALDH(hi) cells into ischemic muscle tissue correlated with increased murine von Willebrand factor blood vessel and CD31+ capillary densities. Thus, UCB ALDH(hi) cells represent a readily available population of proangiogenic progenitors that promote vascular regeneration. This work provides preclinical justification for the development of therapeutic strategies to treat ischemic diseases using UCB-derived ALDH(hi) mixed progenitor cells.

Aims/hypothesis We sought to investigate the stimulation of islet regeneration by transplanted human umbilical cord blood (UCB) cells purified according to high aldehyde dehydrogenase (ALDH) activity (ALDH hi ), a conserved characteristic of multiple progenitor lineages. We hypothesised that direct intrapancreatic (iPan) delivery of ALDH hi progenitors would augment islet regeneration via timely and localised exposure to islet-regenerative stimuli. Methods Cells were purified from UCB based on flow cytometry for low ALDH activity (ALDH lo ) vs ALDH hi . UCB ALDH lo or ALDH hi cells were compared for surface marker expression, as well as haematopoietic, endothelial and multipotent stromal progenitor content in vitro. UCB ALDH lo or ALDH hi cells were i.v. or iPan injected into streptozotocin-treated non-obese diabetic/severe combined immune-deficient mice temporally monitored for blood glucose, serum insulin and glucose tolerance. Human cell recruitment and survival in the pancreas, insulin content, islet-associated cell proliferation and islet vascularisation were documented in situ. Results UCB-derived ALDH hi cells were highly enriched for haematopoietic and endothelial progenitor frequency, and showed increased expression of progenitor and myeloid cell surface markers. Although i.v. transplantation of ALDH hi cells demonstrated low pancreas engraftment and only transient blood glucose lowering capacity, iPan injected ALDH hi cells reversed established hyperglycaemia, increased serum insulin and improved the response to a glucose challenge. iPan injected ALDH hi cells surrounded damaged islets at early time points and increased islet-associated cell proliferation, resulting in the recovery of beta cell mass. Conclusions/interpretation iPan delivery of UCB ALDH hi cells potentiated islet-associated cell proliferation, insulin production and islet revascularisation, resulting in the recovery of host islet function. Elucidation of the progenitor-specific pathways stimulated during islet regeneration may provide new approaches to promote islet expansion during diabetes.

Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (<100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.

Abstract Abstract 3042 Poster Board II-1018 Clinical application of regenerative cell-based therapies for the treatment of ischemic vascular disease has proven challenging to implement due the involvement of multiple human cell types that co-ordinate angiogenesis. Previously, transplanted human progenitor cells from hematopoietic, endothelial, and mesenchymal lineages have all been implicated in the recovery and de novo production of perfused blood vessels in vivo. Using high aldehyde dehydrogenase (ALDHhi) activity, a conserved function of these pro-angiogenic hematopoietic and non-hematopoietic progenitor cell lineages, we have recently shown that transplanted human bone marrow (BM) ALDHhi cells transiently recruit to areas of hypoxia and augment revascularization and recovery of perfusion in ischemic limbs via the stimulation of endogenous vascular regeneration. However, clinical use of autologous BM-derived cells in patients with critical limb ischemia may be limited by the availability or dysfunction of transplanted pro-angiogenic cells due to vascular disease-related pathologies. The non-invasive collection and early ontogeny of human umbilical cord blood (UCB) progenitor cells provides a promising alternative source of pro-angiogenic progenitor cells with vascular regenerative functions. Human UCB mononuclear cells (MNC) were purified using high-speed fluorescence-activated cell sorting to accrue clinically applicable cell populations with low side scatter and high (ALDHhi, 0.4±0.1% of total MNC) versus low (ALDHlo, 41.2±3.6% of total MNC) ALDH activity (n=10). Compared to UCB-derived ALDHlo cells, which were devoid of hematopoietic and non-hematopoietic progenitor function in vitro, ALDHhi cells were enriched for hematopoietic colony forming cells (1 HCFC in 3.6 cells, n=4) and endothelial colony forming cells (1 ECFC in 5.8×104 cells, n=4) enumerated after 14 days in culture. In contrast to human BM-derived ALDH-purified cells which were enriched for mesenchymal colony forming cells, analogous ALDHlo and ALDHhi cells from UCB did not establish mesenchymal-stromal colonies in vitro suggesting a comparative deficiency in mesenchymal stem cell frequency after UCB venipuncture. We investigated the vascular regenerative capacity of these human UCB-derived ALDHlo and ALDHhi cells by tail-vein transplantation into sublethally irradiated (275cGy) immune-deficient, β-glucuronidase (GUSB) deficient, NOD/SCID/MPSVII mice with acute limb ischemia induced by unilateral femoral artery ligation and transection. Using weekly laser Doppler perfusion imaging to track the kinetics of blood flow recovery indicated by the perfusion ratio (PR) in the ischemic versus non-ischemic limb, mice transplanted with 2×105-4×105 purified ALDHhi cells showed significantly improved recovery of limb perfusion by day 21 post-transplantation (PR=0.70±0.06, n=6) compared to mice injected with phosphate-buffered saline (PR=0.35±0.06, n=5, P<0.01), 10×106 ALDHlo cells (PR=0.45±0.07, n=7, P<0.01), or 20×106 unpurified UCB MNC (PR=0.40±0.06, n=7, P<0.01). Furthermore, transplantation of ALDHhi progenitor cells augmented revascularization in the ischemic limb compared to all other treatments at day 28 post-transplantation (P<0.01), quantified by immunohistochemical detection and enumeration of von Willebrand factor+ blood vessels in frozen sections from the ischemic adductor muscle. Only mice transplanted with ALDHhi cells showed retention of human hematopoietic (CD45+/HLA-A,B,C+) cells in the ischemic adductor muscle for 28 days (3.0±2.1%, n=6) quantified by flow cytometry. Ischemic muscle sections from NOD/SCID/MPSVII mice transplanted with human UCB ALDHhi cells confirmed human cell retention and engraftment adjacent to damaged vasculature and ischemic muscle fibres at day 28 post-transplantation, measured by colorimetric detection of ubiquitous GUSB activity in transplanted human cells. Human UCB-derived ALDHhi cells, which were devoid of mesenchymal-stromal cells but enriched for potentially pro-angiogenic endothelial and hematopoietic progenitor lineages, showed persistent retention in areas of hypoxia and augmented the endogenous revascularization and recovery of perfusion in ischemic limbs. Thus, ALDHhi mixed progenitor cells may prove to be a useful allogeneic alternative to human BM in the clinical treatment of patients with severe peripheral vascular disease. Disclosures No relevant conflicts of interest to declare.