Brian H. Johnstone

Secretion of Angiogenic and Antiapoptotic Factors by Human Adipose Stromal Cells

Background—The delivery of autologous cells to increase angiogenesis is emerging as a treatment option for patients with cardiovascular disease but may be limited by the accessibility of sufficient cell numbers. The beneficial effects of delivered cells appear to be related to their pluripotency and ability to secrete growth factors. We examined nonadipocyte stromal cells from human subcutaneous fat as a novel source of therapeutic cells. Methods and Results—Adipose stromal cells (ASCs) were isolated from human subcutaneous adipose tissue and characterized by flow cytometry. ASCs secreted 1203±254 pg of vascular endothelial growth factor (VEGF) per 106 cells, 12 280±2944 pg of hepatocyte growth factor per 106 cells, and 1247±346 pg of transforming growth factor-&bgr; per 106 cells. When ASCs were cultured in hypoxic conditions, VEGF secretion increased 5-fold to 5980±1066 pg/106 cells (P =0.0016). The secretion of VEGF could also be augmented 200-fold by transfection of ASCs with a plasmid encoding VEGF (P <0.05). Conditioned media obtained from hypoxic ASCs significantly increased endothelial cell growth (P <0.001) and reduced endothelial cell apoptosis (P <0.05). Nude mice with ischemic hindlimbs demonstrated marked perfusion improvement when treated with human ASCs (P <0.05). Conclusions—Our experiments delineate the angiogenic and antiapoptotic potential of easily accessible subcutaneous adipose stromal cells by demonstrating the secretion of multiple potentially synergistic proangiogenic growth factors. These findings suggest that autologous delivery of either native or transduced subcutaneous ASCs, which are regulated by hypoxia, may be a novel therapeutic option to enhance angiogenesis or achieve cardiovascular protection.

A Population of Multipotent CD34-Positive Adipose Stromal Cells Share Pericyte and Mesenchymal Surface Markers, Reside in a Periendothelial Location, and Stabilize Endothelial Networks

It has been shown that stromal–vascular fraction isolated from adipose tissues contains an abundance of CD34+ cells. Histological analysis of adipose tissue revealed that CD34+ cells are widely distributed among adipocytes and are predominantly associated with vascular structures. The majority of CD34+ cells from freshly isolated stromal–vascular fraction were CD31−/CD144− and could be separated from a distinct population of CD34+/CD31+/CD144+ (endothelial) cells by differential attachment on uncoated plastic. The localization of CD34+ cells within adipose tissue suggested that the nonendothelial population of these cells occupied a pericytic position. Analysis of surface and intracellular markers of the freshly isolated CD34+/CD31−/CD144− adipose-derived stromal cells (ASCs) showed that >90% coexpress mesenchymal (CD10, CD13, and CD90), pericytic (chondroitin sulfate proteoglycan, CD140a, and CD140b), and smooth muscle (α-actin, caldesmon, and calponin) markers. ASCs demonstrated polygonal self-assembly on Matrigel, as did human microvascular endothelial cells. Coculture of ASCs with human microvascular endothelial cells on Matrigel led to cooperative network assembly, with enhanced stability of endothelial networks and preferential localization of ASCs on the abluminal side of cords. Bidirectional paracrine interaction between these cells was supported by identification of angiogenic factors (vascular endothelial growth factor, hepatocyte growth factor, basic fibroblast growth factor), inflammatory factors (interleukin-6 and -8 and monocyte chemoattractant protein-1 and -2), and mobilization factors (macrophage colony-stimulating factor and granulocyte/macrophage colony-stimulating factor) in media conditioned by CD34+ ASCs, as well a robust mitogenic response of ASCs to basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor-BB, factors produced by endothelial cells. These results demonstrate for the first time that the majority of adipose-derived adherent CD34+ cells are resident pericytes that play a role in vascular stabilization by mutual structural and functional interaction with endothelial cells.

Radiolabeled Cell Distribution After Intramyocardial, Intracoronary, and Interstitial Retrograde Coronary Venous Delivery Implications for Current Clinical Trials

Background—Several clinical studies are evaluating the therapeutic potential of delivery of various progenitor cells for treatment of injured hearts. However, the actual fate of delivered cells has not been thoroughly assessed for any cell type. We evaluated the short-term fate of peripheral blood mononuclear cells (PBMNCs) after intramyocardial (IM), intracoronary (IC), and interstitial retrograde coronary venous (IRV) delivery in an ischemic swine model. Methods and Results—Myocardial ischemia was created by 45 minutes of balloon occlusion of the left anterior descending coronary artery. Six days later, 107 111indium-oxine–labeled human PBMNCs were delivered by IC (n=5), IM (n=6), or IRV (n=5) injection. The distribution of injected cells was assessed by γ-emission counting of harvested organs. For each delivery method, a significant fraction of delivered cells exited the heart into the pulmonary circulation, with 26±3% (IM), 47±1% (IC), and 43±3% (IRV) of cells found localized in the lungs. Within the myocardium, significantly more cells were retained after IM injection (11±3%) compared with IC (2.6±0.3%) (P<0.05) delivery. IRV delivery efficiency (3.2±1%) trended lower than IM infusion for PBMNCs, but this difference did not reach significance. The IM technique displayed the greatest variability in delivery efficiency by comparison with the other techniques. Conclusions—The majority of delivered cells is not retained in the heart for each delivery modality. The clinical implications of these findings are potentially significant, because cells with proangiogenic or other therapeutic effects could conceivably have effects in other organs to which they are not primarily targeted but to which they are distributed. Also, we found that although IM injection was more efficient, it was less consistent in the delivery of PBMNCs compared with IC and IRV techniques.

Robust functional vascular network formation in vivo by cooperation of adipose progenitor and endothelial cells.

Rapid induction and maintenance of blood flow through new vascular networks is essential for successfully treating ischemic tissues and maintaining function of engineered neo-organs. We have previously shown that human endothelial progenitor cells (EPCs) form functioning vessels in mice, but these are limited in number and persistence; and also that human adipose stromal cells (ASCs) are multipotent cells with pericytic properties which can stabilize vascular assembly in vitro. In this study, we tested whether ASCs would cooperate with EPCs to coassemble vessels in in vivo implants. Collagen implants containing EPCs, ASCs, or a 4:1 mixture of both were placed subcutaneously into NOD/SCID mice. After a range of time periods, constructs were explanted and evaluated with regard to vascular network assembly and cell fate; and heterotypic cell interactions were explored by targeted molecular perturbations. The density and complexity of vascular networks formed by the synergistic dual-cell system was many-fold higher than found in implants containing either ASCs or EPCs alone. Coimplantation of ASCs and EPCs with either pancreatic islets or adipocytes produced neoorgans populated by these parenchymal cells, as well as by chimeric human vessels conducting flow. This study is the first to demonstrate prompt and consistent assembly of a vascular network by human ASCs and endothelial cells and vascularization by these cells of parenchymal cells in implants. Mixture of these 2 readily available, nontransformed human cell types provides a practical approach to tissue engineering, therapeutic revascularization, and in vivo studies of human vasculogenesis.

IFATS Collection: Human Adipose Tissue‐Derived Stem Cells Induce Angiogenesis and Nerve Sprouting Following Myocardial Infarction, in Conjunction with Potent Preservation of Cardiac Function

The administration of therapeutic cell types, such as stem and progenitor cells, has gained much interest for the limitation or repair of tissue damage caused by a variety of insults. However, it is still uncertain whether the morphological and functional benefits are mediated predominantly via cell differentiation or paracrine mechanisms. Here, we assessed the extent and mechanisms of adipose‐derived stromal/stem cells (ASC)‐dependent tissue repair in the context of acute myocardial infarction. Human ASCs in saline or saline alone was injected into the peri‐infarct region in athymic rats following left anterior descending (LAD) coronary artery ligation. Cardiac function and structure were evaluated by serial echocardiography and histology. ASC‐treated rats consistently exhibited better cardiac function, by all measures, than control rats 1 month following LAD occlusion. Left ventricular (LV) ejection fraction and fractional shortening were improved in the ASC group, whereas LV remodeling and dilation were limited in the ASC group compared with the saline control group. Anterior wall thinning was also attenuated by ASC treatment, and post‐mortem histological analysis demonstrated reduced fibrosis in ASC‐treated hearts, as well as increased peri‐infarct density of both arterioles and nerve sprouts. Human ASCs were persistent at 1 month in the peri‐infarct region, but they were not observed to exhibit significant cardiomyocyte differentiation. Human ASCs preserve heart function and augment local angiogenesis and cardiac nerve sprouting following myocardial infarction predominantly by the provision of beneficial trophic factors. STEM CELLS 2009;27:230–237

Synthetic Zinc Finger Transcription Factor Action at an Endogenous Chromosomal Site ACTIVATION OF THE HUMAN ERYTHROPOIETIN GENE

We have targeted the activation of an endogenous chromosomal locus including the human erythropoietin gene using synthetic transcription factors. These transcription factors are targeted to particular DNA sequences in the 5′-flanking region of the erythropoietin gene through engineering of a zinc finger DNA binding domain. The DNA binding domain is linked to a VP16 transcriptional activation domain. We find that these synthetic transcription factors invariably activate transiently transfected templates in which sequences within the 5′ flank of the erythropoietin gene are fused to a luciferase reporter. The efficiency of activation under these circumstances at a defined site is dependent on DNA binding affinity. In contrast, only a subset of these same zinc finger proteins is able to activate the endogenous chromosomal locus. The activity of these proteins is influenced by their capacity to gain access to their recognition elements within the chromatin infrastructure. Zinc finger transcription factors will provide a powerful tool to probe the determinants of chromatin accessibility and remodeling within endogenous chromosomal loci.

IFATS Collection: The Conditioned Media of Adipose Stromal Cells Protect Against Hypoxia‐Ischemia‐Induced Brain Damage in Neonatal Rats

Adipose tissue stroma contains a population of mesenchymal stem cells, which support repair when administered to damaged tissues, in large part through secreted trophic factors. We directly tested the ability of media collected from cultured adipose‐derived stem cells (ASCs) to protect neurons in a rat model of brain hypoxic‐ischemic (HI) injury. Concentrated conditioned medium from cultured rat ASCs (ASC‐CM) or control medium was infused through the jugular vein of neonatal Sprague‐Dawley rats subjected to HI injury. The ASC‐CM was administered either 1 hour before or 24 hours after induction of injury. Analysis at 1 week indicated that administration at both time points significantly protected against hippocampal and cortical volume loss. Analysis of parallel groups for behavioral and learning changes at 2 months postischemia demonstrated that both treated groups performed significantly better than the controls in Morris water maze functional tests. Subsequent post‐mortem evaluation of brain damage at the 2‐month time point confirmed neuronal loss to be similar to that observed at 1 week for all groups. We have identified several neurotrophic factors in ASC‐CM, particularly insulin‐like growth factor‐1 and brain‐derived neurotrophic factor, which are important factors that could contribute to the protective effects of ASCs observed in studies with both in vitro and in vivo neuronal injury models. These data suggest that delivery of the milieu of factors secreted by ASCs may be a viable therapeutic option for treatment of HI, as well as other brain injuries. STEM CELLS 2009;27:478–488

Suppression of Hepatocyte Growth Factor Production Impairs the Ability of Adipose‐Derived Stem Cells to Promote Ischemic Tissue Revascularization

The use of adipose‐derived stem/stromal cells (ASCs) for promoting repair of tissues is a promising potential therapy, but the mechanisms of their action are not fully understood. We and others previously demonstrated accelerated reperfusion and tissue salvage by ASCs in peripheral ischemia models and have shown that ASCs secrete physiologically relevant levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor. The specific contribution of HGF to ASC potency was determined by silencing HGF expression. RNA interference was used to downregulate HGF expression. A dual‐cassette lentiviral construct expressing green fluorescent protein (GFP) and either a small hairpin RNA specifically targeted to HGF mRNA (shHGF) or an inactive control sequence (shCtrl) were used to stably transduce ASCs (ASC‐shHGF and ASC‐shCtrl, respectively). Transduced ASC‐shHGF secreted >80% less HGF, which led to a reduced ability to promote survival, proliferation, and migration of mature and progenitor endothelial cells in vitro. ASC‐shHGF were also significantly impaired, compared with ASC‐shCtrl, in their ability to promote reperfusion in a mouse hindlimb ischemia model. The diminished ability of ASCs with silenced HGF to promote reperfusion of ischemic tissues was reflected by reduced densities of capillaries in reperfused tissues. In addition, fewer GFP+ cells were detected at 3 weeks in ischemic limbs of mice treated with ASC‐shHGF compared with those treated with ASC‐shCtrl. These results indicate that production of HGF is important for the potency of ASCs. This finding directly supports the emerging concept that local factor secretion by donor cells is a key element of cell‐based therapies.

Adipose Stem Cell Treatment in Mice Attenuates Lung and Systemic Injury Induced by Cigarette Smoking

RATIONALE Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery. OBJECTIVES We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema. METHODS Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors. MEASUREMENTS AND MAIN RESULTS Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro. CONCLUSIONS These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.

Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing.

Background: The authors examined the efficacy of adipose stem cells, when supplied either alone or in platelet-rich fibrin gels, to improve wound healing. Methods: A porcine full-thickness wound model was used to compare six topical treatments: platelet-poor plasma; platelet-rich plasma; autologous adipose stem cells plus platelet-poor plasma; autologous adipose stem cells plus platelet-rich plasma; allogeneic adipose stem cells containing green fluorescent protein plus platelet-poor plasma; and saline (control). One week after isolation, adipose stem cells were applied to full-thickness wounds on the paraspinal and thoracic regions of three pigs (44 wounds per pig; each treatment was applied to eight separate wounds). Each wound was monitored over 21 days for closure, cosmesis, and histopathology. Results: There was no significant difference in the reepithelialization rate, but treatments containing adipose stem cells demonstrated increased microvessel densities (31.75 ± 5.73 vessels/cm2 versus 7.93 ± 3.61 vessels/cm2) compared with groups without adipose stem cells. Wound cosmesis was improved in the adipose stem cell plus platelet-rich plasma group compared with other treatment groups (p < 0.05). Vascular endothelial growth factor levels detected in matrices containing adipose stem cells were approximately 7-fold higher compared with platelet-rich plasma or platelet-poor plasma (p < 0.05). Localization of transgenic green fluorescent protein plus adipose stem cells indicated incorporation near neovasculature. Conclusions: In normal healing wounds, adipose stem cells appear to enhance the healing process only when provided in a fibrin gel vehicle containing a number of complementary wound-healing trophic factors. Perivascular adipose stem cell localization suggests a function in enhancing blood supply through providing physical and paracrine support to newly forming vessels.