Stem cells play an important role in restoring cardiac function in the damaged heart. In order to mediate repair, stem cells need to replace injured tissue by differentiating into specialized cardiac cell lineages and/or manipulating the cell and molecular mechanisms governing repair. Despite early reports describing engraftment and successful regeneration of cardiac tissue in animal models of heart failure, these events appear to be infrequent and yield too few new cardiomyocytes to account for the degree of improved cardiac function observed. Instead, mounting evidence suggests that stem cell mediated repair takes place via the release of paracrine factors into the surrounding tissue that subsequently direct a number of restorative processes including myocardial protection, neovascularization, cardiac remodeling, and differentiation. The potential for diverse stem cell populations to moderate many of the same processes as well as key paracrine factors and molecular pathways involved in stem cell-mediated cardiac repair will be discussed in this review.

We assessed the feasibility of dynamic 3-dimensional (3D) PET/ CT tracking of 18 F-FDG-labeled circulating progenitor cell (CPC) therapy during intracoronary injection, using a porcine model of acute myocardial infarction (MI). Methods: Human and porcine CPC were radiolabeled with 18 F-FDG, with variation in temperature and incubation time to determine optimal conditions. For in vivo experiments, CPC were harvested before induction of infarction (using 90-min coronary balloon occlusion). At 48 h, animals underwent cardiac MRI to assess infarct size. A balloon catheter was placed in the infarct artery at the same location as that used for induction of MI, and during dynamic 3D PET/CT 3 · 10 7 autologous 18 F-FDG progenitor cells were injected through the central lumen using either (a) 3 cycles of balloon occlusion and reperfusion or (b) high-concentration, single-bolus injection without balloon occlusion (n 5 3 for both protocols). Peripheral blood was drawn at 1-min intervals during cell injection. Results: Labeling efficiency was optimized by 30-min incubation at 37°C (human CPC, 89.9% 6 4.8%; porcine CPC, 91.6% 6 6.4%). Cell-bound activity showed a nonsignificant decrease at 1 h (human, 74.3% 6 10.7%; porcine, 77.7% 6 12.8%; P . 0.05) and a significant decrease at 2 h (human, 62.1% 6 8.9%; porcine, 68.6% 6 5.4%; P 5 0.009). Mean infarct size was similar for both injection protocols (16.3% 6 3.4% and 20.6% 6 2.7%; P . 0.05). Dynamic scanning demonstrated a sharp rise in myocardial activity during each cycle of balloon-occlusion cell delivery, with a significant fall in activity (around 80%) immediately after balloon deflation. The latter was associated with a transient spike in peripheral blood 18 F-FDG activity, consistent with the first pass of labeled cells in the systemic circulation. A single spike and gradual fall in myocardial activity was observed with high-concentration, single-bolus therapy. At 1 h, myocardial activity was 8.7% 6 1.5% of total injected dose for balloon-occlusion delivery and 17.8% 6 7.9% for high-concentration, single-bolus delivery (P 5 0.08). Conclusion: Dynamic tracking during intracoronary injection of 18 F-FDG-labeled CPC is feasible and demonstrates significant cell washout from the myocardium immediately after balloon deflation. High-concentration, single-bolus therapy may be as effective as balloon-occlusion delivery. This tracking technique should facilitate development of improved delivery strategies for cardiac cell therapy.

Attenuated measles viruses (MVs) propagate selectively in human tumor cells, and phase I clinical trials are currently underway to test their oncolytic activity. A major theoretical impediment to systemic MV application is the presence of pre-existing antiviral immunity. We hypothesized that autologous MV-infected cells might be a more reliable vehicle than cell-free virions to deliver the infection to tumor cells in subjects with neutralizing titers of anti-measles antibodies. Our in vitro studies, using a dual-color fluorescent model, demonstrated efficient cell-to-cell transfer of infection via heterofusion. In contrast to infection by naked virions, heterofusion between infected cell carriers and tumor cells was more resistant to antibody neutralization. Infected monocytic, endothelial, or stimulated peripheral blood cells could deliver oncolytic MV to tumor lesions in vivo, after intravenous (i.v.) or intraperitoneal (i.p.) administration. Single or repeated i.p. injections of monocytic carriers significantly improved survival of animals bearing human ovarian cancer xenografts. Systemic or i.p. injection of MV-infected cells successfully transferred infection by heterofusion to Raji lymphomas or hepatocellular carcinoma tumors in the presence of neutralizing antibodies. These results suggest a novel strategy for systemic delivery of oncolytic virotherapy in cancer patients that can "bypass" the pre-existing humoral immunity against MV.

Rational The regenerative capacity of the heart is markedly diminished shortly after birth coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is therefore of high interest and has been the focus of extensive investigation over the past years. Objective We have recently identified C3orf58 as a novel Hypoxia and Akt induced Stem cell Factor (HASF) secreted from mesenchymal stem cells that can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. Methods and Results Neonatal ventricular cardiomyocytes were stimulated in culture for seven days with purified recombinant HASF protein. Compared to control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by BrdU incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50–100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a PI3K-AKT-CDK7 pathway as revealed by the use of PI3K pathway specific inhibitors and silencing of the Cdk7 gene. Conclusion Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a PI3K-AKT-CDK7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.

Mesenchymal stem cells (MSCs) transplanted into injured myocardium promote repair through paracrine mechanisms. We have previously shown that MSCs overexpressing AKT1 (Akt-MSCs) exhibit enhanced properties for cardiac repair. In this study, we investigated the relevance of Abi3bp towards MSC biology. Abi3bp formed extracellular deposits with expression controlled by Akt1 and ubiquitin-mediated degradation. Abi3bp knockdown/knockout stabilized focal adhesions and promoted stress-fiber formation. Furthermore, MSCs from Abi3bp knockout mice displayed severe deficiencies in osteogenic and adipogenic differentiation. Knockout or stable knockdown of Abi3bp increased MSC and Akt-MSC proliferation, promoting S-phase entry via cyclin-d1, ERK1/2 and Src. Upon Abi3bp binding to integrin-β1 Src associated with paxillin which inhibited proliferation. In vivo, Abi3bp knockout increased MSC number and proliferation in bone marrow, lung, and liver. In summary, we have identified a novel extracellular matrix protein necessary for the switch from proliferation to differentiation in MSCs.

Administration of endothelial progenitor cells (EPC) is a promising therapy for post-infarction cardiac repair. However, the mechanisms that underlie apparent benefi cial effects on myocardial remodeling are unclear. In a porcine model of acute myocardial infarction, we investigated the therapeutic effects of a mixed population of culture modifi ed peripheral blood mononuclear cells (termed hereafter porcine EPC). Porcine EPC were isolated using methods identical to those previously adopted for harvest of EPC in human cell therapy studies. In addition the therapeutic effects of paracrine factors secreted by these cells was evaluated in vitro and in vivo. Intracoronary injection of autologous porcine EPC was associated with increased infarct territory mass and improved regional ventricular systolic function at 2 months compared to control. Treatment with conditioned media derived from autologous EPC was associated with similar improved effects on infarct territory mass and function. Histologic analysis of the infarct territory revealed signifi cantly increased cardiomyocyte size in EPC and conditioned media treated groups, when compared to controls. A paracrine EPC effect was also verifi ed in a pure myocardial preparation in which cardiomyocytes devoid of fi broblast, neuronal and vascular elements directly responded by increasing cell mass when exposed to the same conditioned media. Analysis of conditioned media revealed elevated levels of TGFβ1 (human 267.3±11.8 pg/ml, porcine 57.1±6.1 pg/ml), a recognized mediator of hypertro-phic signaling in the heart. Neutralizing antibodies to TGFβ1 attenuated the pro-hypertrophic effect of conditioned media, and use of recombinant TGFβ1 added to fresh media replicated the pro-hypertrophic effects of conditioned media in vitro. These data demonstrate the potential of paracrine factors secreted from endothelial progenitor cells to induce cardiomyocyte hypertrophy contributing to increased infarct territory LV mass, with favorable medium term effects on regional function following myocardial infarction. STEM CELLS AND DEVELOPMENT 17:941-952 (2008)

The renin-angiotensin-aldosterone system (RAAS) regulates BP and salt-volume homeostasis. Juxtaglomerular (JG) cells synthesize and release renin, which is the first and rate-limiting step in the RAAS. Intense pathologic stresses cause a dramatic increase in the number of renin-producing cells in the kidney, termed JG cell recruitment, but how this occurs is not fully understood. Here, we isolated renal CD44 + mesenchymal stem cell (MSC)-like cells and found that they differentiated into JG-like renin-expressing cells both in vitro and in vivo. Sodium depletion and captopril led to activation and differentiation of these cells into renin-expressing cells in the adult kidney. In summary, CD44 + MSC-like cells exist in the adult kidney and can differentiate into JG-like renin-producing cells under conditions that promote JG cell recruitment.

Rationale: Smooth muscle precursor cells have previously been reported to reside in bone marrow and in the circulation, but little is currently known regarding the proximate stimuli for smooth muscle cell differentiation of these putative progenitors. Objective: Because local thrombin generation occurs as an initial response to vascular injury, we hypothesized that thrombin may influence the differentiation of circulating smooth muscle progenitor cells. Methods and Results: Peripheral blood mononuclear cells were cultured on type I collagen using a protocol optimized to stimulate smooth muscle cell outgrowth. Thrombin-stimulated upregulation of the transcription factor myocardin and smooth muscle myosin heavy chain, and both were inhibited by hirudin or the RhoA inhibitor Y27632. After 10 days of culture, smooth muscle outgrowth colonies formed, which stained positive for ␣-smooth muscle actin, smooth muscle myosin heavy chain, and calponin, in addition to having a contractile response to 100 nmol/L angiotensin II. Coincubation of peripheral blood mononuclear cells with thrombin, 10 mol/L protease-activated receptor-1, but not protease-activated receptor-4 activating peptide significantly increased the number of smooth muscle outgrowth colonies formed. Thrombin-induced enhancement of smooth muscle outgrowth colony formation was inhibited by hirudin, Y27632, and an antibody against protease-activated receptor-1. Conclusions: These data illustrate a novel thrombininduced pathway for smooth muscle differentiation from putative smooth muscle progenitors in peripheral blood. W e have previously described circulating smooth muscle outgrowth cells (SOCs). 1,2 Moreover, we have shown in human subjects who have undergone bone marrow transplantation, smooth muscle cells (SMCs) of donor origin are markedly enriched in coronary atherosclerotic plaque compared to the nondiseased vessel wall. 3 A number of potential agents may mediate SMC differentiation from blood borne progenitors including transforming growth factor-␤; plateletderived growth factor-BB 4 ; and sphingosine-1-phosphate, which induces RhoA-dependent myocardin expression and SMC differentiation in mesenchymal stem cells. 5 Thrombin generation occurs at sites of injury and could conceivably contribute to smooth muscle differentiation of circulating progenitor cells based on several lines of evidence. Under various in vitro conditions in mature SMCs, thrombin stimulates upregulation of the SMC marker genes smooth muscle myosin heavy chain (SM-MHC) 6 and calponin. 7 Thrombin also enhances the angiogenic potential of endothelial progenitor cells 8 and may influence vascular progenitor phenotype and function. 9 In the context of circulating progenitors, no study to date has fully investigated the downstream effects of thrombin on circulating mononuclear cells. Here, we show that thrombin induces robust sequential upregulation of the master smooth muscle cell transcription factor myocardin, as well as smooth muscle specific protein expression in peripheral blood mono...