Kathy-Ann Forner

Monocyte Derivatives Promote Angiogenesis and Myocyte Survival in a Model of Myocardial Infarction

In this study, we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent, consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF, IGF-1, MCP-1, and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-α, staurosporine, and oxidative stress, and this effect was dependent upon MD-derived sTNFR-1, HGF, and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1, HGF, and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult and attenuate pathological remodeling.

Hierarchical scaffold design for mesenchymal stem cell-based gene therapy of hemophilia B.

Gene therapy for hemophilia B and other hereditary plasma protein deficiencies showed great promise in pre-clinical and early clinical trials. However, safety concerns about in vivo delivery of viral vectors and poor post-transplant survival of ex vivo modified cells remain key hurdles for clinical translation of gene therapy. We here describe a 3D scaffold system based on porous hydroxyapatite-PLGA composites coated with biomineralized collagen 1. When combined with autologous gene-engineered factor IX (hFIX) positive mesenchymal stem cells (MSCs) and implanted in hemophilic mice, these scaffolds supported long-term engraftment and systemic protein delivery by MSCs in vivo. Optimization of the scaffolds at the macro-, micro- and nanoscales provided efficient cell delivery capacity, MSC self-renewal and osteogenesis respectively, concurrent with sustained delivery of hFIX. In conclusion, the use of gene-enhanced MSC-seeded scaffolds may be of practical use for treatment of hemophilia B and other plasma protein deficiencies.

Mesenchymal Stromal Cells Engineered to Express Erythropoietin Induce Anti-erythropoietin Antibodies and Anemia in Allorecipients

Autologous bone marrow mesenchymal stromal cells (MSCs) have been successfully used for the delivery of erythropoietin (EPO) in murine models of anemia and myocardial infarction. For clinical applications where a transient effect would be adequate, such as myocardial infarction, the use of EPO-engineered universal donor allogeneic MSCs would be a substantial convenience. We thus investigated whether MSCs from C57BL/6 mice would permit robust transient EPO delivery in normal BALB/c allorecipients. Implantation of MSCs overexpressing murine EPO led to increases in hematocrit in syngeneic and allogeneic mice, but the latter eventually developed severe anemia due to acquired neutralizing anti-EPO antibodies. As MSCs constitutively produce the CCL2 chemokine which may behave as an adjuvant to the anti-EPO immune response, experiments were performed using EPO-engineered MSCs derived from CCL2(-/-) mice and similar results were obtained. In conclusion, MHC-mismatched MSCs can break the tolerance to autoantigens and lead to the development of pathogenic autoantibodies.

Development and Function of Innate Polyclonal TCRαβ+ CD8+ Thymocytes

Innate CD8 T cells are found in mutant mouse models, but whether they are produced in a normal thymus remains controversial. Using the RAG2p-GFP mouse model, we found that ∼10% of TCRαβ+ CD4−CD8+ thymocytes were innate polyclonal T cells (GFP+CD44hi). Relative to conventional T cells, innate CD8 thymocytes displayed increased cell surface amounts of B7-H1, CD2, CD5, CD38, IL-2Rβ, and IL-4Rα and downmodulation of TCRβ. Moreover, they overexpressed several transcripts, including T-bet, Id3, Klf2, and, most of all, Eomes. Innate CD8 thymocytes were positively selected, mainly by nonhematopoietic MHCIa+ cells. They rapidly produced high levels of IFN-γ upon stimulation and readily proliferated in response to IL-2 and IL-4. Furthermore, low numbers of innate CD8 thymocytes were sufficient to help conventional CD8 T cells expand and secrete cytokine following Ag recognition. This helper effect depended on CD44-mediated interactions between innate and conventional CD8 T cells. We concluded that innate TCRαβ+ CD8 T cells represent a sizeable proportion of normal thymocytes whose development and function differ in many ways from those of conventional CD8 T cells.

Induction of Cardiac Angiogenesis Requires Killer Cell Lectin-Like Receptor 1 and α4β7 Integrin Expression by NK Cells

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice, myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6, accompanied by substantial expression of IL-2, TNF-α, and CCL2. In contrast, NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo, IL-2–activated NK cells promoted TNF-α–stimulated endothelial cell proliferation, enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2–activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin–specific binding. In this study, we show that IL-2–activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases.

Adoptive transfer of mesenchymal stromal cells accelerates intestinal epithelium recovery of irradiated mice in an interleukin-6-dependent manner

BACKGROUND AIMS Apoptosis of radiosensitive cells in the bone marrow and gut is a serious, at times life-threatening, complication arising from radiation exposure. METHODS We investigated whether adoptive transfer of allogeneic bone marrow-derived mesenchymal stromal cells (MSC) could exert cytoprotective and life-sparing effects in a mouse model of sublethal total body irradiation (TBI). RESULTS We demonstrated that a single intraperitoneal injection of C57Bl/6 MSC given to major histocompatibility complex (MHC)-mismatched Balb/c mice within 24 h of sublethal TBI significantly reduced mortality in a dose-dependent manner. Histologic analysis and Ki67 immunostaining of jejunum sections collected 3 and 6 days post-TBI indicated that MSC protected the gastrointestinal epithelium from TBI-induced damage and significantly accelerated recovery of the gut by stimulating proliferation of the crypt cell pool. Using interleukin-6(-/-) (IL-6) MSC, we demonstrated that IL-6 expressed by MSC played a role in gastrointestinal epithelium regeneration. CONCLUSIONS Our results suggest that allogeneic MHC-mismatched MSC may be exploited to reduce gastrointestinal complications and mortality arising from ionizing radiation exposure.

A novel and simplified method of culture of human blood-derived early endothelial progenitor cells for the treatment of ischemic vascular disease.

Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14+ and CD14- circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectin-coated surfaces (10 μg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml)), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.