Cardiac Progenitor Cell Therapy Research Articles

Therapeutic effects of CXCR4+ subpopulation of transgene‐free induced cardiosphere‐derived cells on experimental myocardial infarction

ObjectivesMyocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor‐like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus‐based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene‐free approaches.Materials and MethodsFour transgene‐free approaches for somatic reprogramming, including episome, minicircle, self‐replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations.ResultsThe self‐replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C‐X‐C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach.ConclusionThus, the optimized strategy for iCS generation is safer and has more therapeutic potentials.

Abstract 16769: Intravenous Injection of Neonatal Cardiac Mesenchymal Stem Cells is a Viable Alternative Route to Treat Acute Myocardial Infarction

Introduction: Cardiovascular disease is major cause of morbidity and mortality around the world and major health care burden indeed. Ischemic heart disease (IHD) and myocardial ischemia (MI) are most devastating cardiovascular disease. Multiple stem cell/ cardiac progenitor cell therapy has been reported previously to treat cardiovascular disease safely. However randomized clinical trials with adult cardiac progenitor cells or cardiosphere-derived cells unable to show long-term efficacy. We have our unique source of human neonatal cardiac tissue derived neonatal cardiac mesenchymal stem cells (nMSCs). Systemic administration is preferred route of stem cell delivery in order to consecutive dosage for most of the clinical trials. We hypothesized that nMSCs have unique proteome profile, which supports their survival, migration and homing. They home to the injured myocardium when administered intravenously (IV) to a wild type male rat subjected to MI. Methods: This model was created in 6-week-old Brown-Norway Rats. Rats were subjected to an anterior myocardial Infraction (MI) by permanent LAD ligation. The rats were treated with nMSCs (1^10 6 cells/Kg, 5^10 6 cells/Kg and 10^10 6 cells/Kg) along with placebo and sham, which are delivered intravenously by tail vein injection. Rats are once again treated with nMSCs/placebo 4 days after MI. Baseline echocardiography is performed 24 hours after MI. Results: LVEF was significantly higher in the nMSC-treated group than in the placebo group. Other parameters, including fractional shortening (FS) and decreased end-systolic volume (ESV), were also significantly improved when compared with the placebo group, and other LV functional parameter, including cardiac output/body weight and posterior wall thickness tended towards improvement of remodeled heart. Conclusions: Twice intravenous administration of nMSCs for MI attenuate the progressive deterioration of left ventricle and adverse remodeling of rat heart.

The Biological Mechanisms of Action of Cardiac Progenitor Cell Therapy.

Cell therapy for cardiovascular diseases is regarded as a rapidly growing field within regenerative medicine. Different cellular populations enriched for cardiac progenitor cells (CPCs), or derivate a-cellular products, are currently under preclinical and clinical evaluation. Here, we have reviewed the described mechanisms whereby resident post-natal CPCs, isolated in different ways, act as a therapeutic product on the damaged myocardium. Several biological mechanisms of action have been described which can explain the multiple therapeutic effects of CPC treatment observed on cardiac function and remodelling. These mechanisms span from direct cardiovascular differentiation, through induction of resident progenitor proliferation, to paracrine effects on cardiac and non-cardiac cells mediated by exosomes and non-coding RNAs. All the reported mechanisms of action support an integrated view including cardiomyogenesis, cardioprotection, and anti-fibrotic effects. Moreover, future developments of CPC therapy approaches may support cell-free strategies, exploiting effective pleiotropic cell-derived products, such as exosomes.

The current status and future of cardiac stem/progenitor cell therapy for congenital heart defects from diabetic pregnancy.

Pregestational maternal diabetes induces congenital heart defects (CHDs). Cardiac dysfunction after palliative surgical procedures contributes to the high mortality of CHD patients. Autologous or allogeneic stem cell therapies are effective for improving cardiac function in animal models and clinical trials. c-kit+ cardiac progenitor cells (CPCs), the most recognized CPCs, have the following basic properties of stem cells: self-renewal, multicellular clone formation, and differentiation into multiple cardiac lineages. However, there is ongoing debate regarding whether c-kit+ CPCs can give rise to sufficient cardiomyocytes. A new hypothesis to address the beneficial effect of c-kit+ CPCs is that these cells stimulate endogenous cardiac cells through a paracrine function in producing a robust secretome and exosomes. The values of other cardiac CPCs, including Sca1+ CPCs and cardiosphere-derived cells, are beginning to be revealed. These cells may be better choices than c-kit+ CPCs for generating cardiomyocytes. Adult mesenchymal stem cells are considered immune-incompetent and effective for improving cardiac function. Autologous CPC therapy may be limited by the observation that maternal diabetes adversely affects the biological function of embryonic stem cells and CPCs. Future studies should focus on determining the mechanistic action of these cells, identifying new CPC markers, selecting highly effective CPCs, and engineering cell-free products.

Empowering human cardiac progenitor cells by P2Y14 nucleotide receptor overexpression.

Autologous cardiac progenitor cell (CPC) therapy is a promising approach for treatment of heart failure (HF). There is an unmet need to identify inherent deficits in aged/diseased human CPCs (hCPCs) derived from HF patients in the attempts to augment their regenerative capacity prior to use in the clinical setting. Here we report significant functional correlations between phenotypic properties of hCPCs isolated from cardiac biopsies of HF patients, clinical parameters of patients and expression of the P2Y14 purinergic receptor (P2Y14 R), a crucial detector for extracellular UDP-sugars released during injury/stress. P2Y14 R is downregulated in hCPCs derived from HF patients with lower ejection fraction or diagnosed with diabetes. Augmenting P2Y14 R expression levels in aged/diseased hCPCs antagonizes senescence and improves functional responses. This study introduces purinergic signalling modulation as a potential strategy to rejuvenate and improve phenotypic characteristics of aged/functionally compromised hCPCs prior to transplantation in HF patients. Autologous cardiac progenitor cell therapy is a promising alternative approach to current inefficient therapies for heart failure (HF). However, ex vivo expansion and pharmacological/genetic modification of human cardiac progenitor cells (hCPCs) are necessary interventions to rejuvenate aged/diseased cells and improve their regenerative capacities. This study was designed to assess the potential of improving hCPC functional capacity by targeting the P2Y14 purinergic receptor (P2Y14 R), which has been previously reported to induce regenerative and anti-senescence responses in a variety of experimental models. c-Kit+ hCPCs were isolated from cardiac biopsies of multiple HF patients undergoing left ventricular assist device implantation surgery. Significant correlations existed between the expression of P2Y14 R in hCPCs and clinical parameters of HF patients. P2Y14 R was downregulated in hCPCs derived from patients with a relatively lower ejection fraction and patients diagnosed with diabetes. hCPC lines with lower P2Y14 R expression did not respond to P2Y14 R agonist UDP-glucose (UDP-Glu) while hCPCs with higher P2Y14 R expression showed enhanced proliferation in response to UDP-Glu stimulation. Mechanistically, UDP-Glu stimulation enhanced the activation of canonical growth signalling pathways ERK1/2 and AKT. Restoring P2Y14 R expression levels in functionally compromised hCPCs via lentiviral-mediated overexpression improved proliferation, migration and survival under stress stimuli. Additionally, P2Y14 R overexpression reversed senescence-associated morphology and reduced levels of molecular markers of senescence p16INK4a , p53, p21 and mitochondrial reactive oxygen species. Findings from this study unveil novel biological roles of the UDP-sugar receptor P2Y14 in hCPCs and suggest purinergic signalling modulation as a promising strategy to improve phenotypic properties of functionally impaired hCPCs.

High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker

Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes.

In This Issue

HomeCirculation ResearchVol. 118, No. 7In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published1 Apr 2016https://doi.org/10.1161/RES.0000000000000102Circulation Research. 2016;118:1041Glut1, Myelopoiesis, and Atherosclerosis (p 1062)Download figureDownload PowerPointInhibiting extramedullary hematopoietic activity may halt progression of atherosclerosis, say Sarrazy et al.To proliferate and differentiate hematopoietic stem and progenitor cells (HSPCs) in the bone marrow increase their glucose uptake to meet the rising metabolic demand. Indeed PET-CT imaging can identify increased metabolic activity in HSPCs by the uptake of glucose (or rather its radiolabeled analogue) during bone marrow and extramedullary hematopoietic activity. Cell proliferation dependent glucose uptake has been observed in atherosclerotic plaques as well as the spleen. Overproduction of hematopoietic cells, including neutrophils and monocytes, could both contribute to atherosclerosis onset and exacerbate plaque growth. To study extramedullary hematopoiesis more closely, Sarrazy and colleagues examined atherosclerosis-prone mice. They found that glucose uptake was increased in the aortic arch, spleens and bone marrow of these animals, which correlated with increased expression of the glucose transporter Glut1 in their HSPCs. Furthermore, suppression of Glut1 activity reduced not only HSPC proliferation and differentiation—specifically myelopoiesis—but also the progression of atherosclerosis. The authors propose that preventing glucose uptake by HSPCs with an appropriately targeted Glut1 inhibitor could be a potential treatment strategy for atherosclerosis.Long-Term Effects of Cardiac Progenitor Cells (p 1091)Download figureDownload PowerPointThe benefits of cardiac progenitor cell therapy lasts at least a year in rats, report Tang et al.Several studies show that the therapeutic use of cardiac progenitor cells (CPCs) can promote repair and improve function of damaged hearts in animals after myocardial infarction. However, long-term effects of CPC therapy remain unknown as most studies have examined the potential benefits for only four-to-six weeks. Therefore, Tang and colleagues studied the outcome of CPC therapy for myocardial infarction in rats, one year after CPC transplant. They found that while the overall survival rate was no different between control and CPC-treated animals, the treated group exhibited less hypertrophy, smaller scars, increased left ventricle wall thickness, and evidence of less fibrosis. Significantly, CPC treatment did not result in tumor formation. The team used male CPCs and female recipients to enable identification of the cells—by the Y chromosome—and this approach revealed that while a small number of the cells were still present even after 1 year, the transplanted cells had not differentiated into mature cardiomyocytes, although increased proliferation of endogenous CPCs was observed. Together these results indicate that CPC therapy is safe, and that it provides long-term benefits, and induces endogenous CPCs to proliferate and promote repair. The study paves the way for long-term studies evaluating the clinical efficacy of stem cells, say the authors.GRK2 and Prognosis in Heart Failure (p 1116)Download figureDownload PowerPointRengo et al assess the usefulness of a possible new biomarker for heart failure.Heart failure (HF) is a leading cause of death worldwide, and about five million people in the United States suffer from the condition. A major characteristic of HF is chronic sympathetic nervous system (SNS) hyperactivity, which while compensatory in the initial stages of disease becomes deleterious in the long run. Indeed, chronic SNS hyperactivity boosts cardiac levels of G-protein coupled receptor (GPCR) kinase 2 (GRK2), which in turn leads to dysfunctional β-adrenergic signaling—a key factor contributing to the progress of HF pathology. Importantly, the levels of GRK2 in peripheral lymphocytes correlate with those in the failing heart. Rengo and colleagues studied levels of lymphocyte GRK2 in 257 HF patients and found that they accurately reflect disease severity. Patients at low risk of death has lower levels of GRK2, while high levels of protein were identified in high risk individuals. Currently, left ventricle ejection fraction and levels of NT-proBNP are used as biomarkers for HF; however, the team suggests that GRK2 could offer independent and additional prognostic value over and above current approaches. The authors therefore suggest that further larger-scale clinical investigations are warranted to assess the value of the prognostic value of GRK2 in heart failure patients. Previous Back to top Next FiguresReferencesRelatedDetails April 1, 2016Vol 118, Issue 7 Advertisement Article InformationMetrics © 2016 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000102 Originally publishedApril 1, 2016 PDF download Advertisement

Long-Term Outcome of Administration of c-kit(POS) Cardiac Progenitor Cells After Acute Myocardial Infarction: Transplanted Cells Do not Become Cardiomyocytes, but Structural and Functional Improvement and Proliferation of Endogenous Cells Persist for at Least One Year.

Cardiac progenitor cells (CPCs) improve left ventricular remodeling and function after acute or chronic myocardial infarction. However, the long-term (>5 weeks) effects, potential tumorigenicity, and fate of transplanted CPCs are unknown. To assess the outcome of CPC therapy at 1 year. Female rats underwent a 90-minute coronary occlusion; 4 hours after reperfusion, they received intracoronarily vehicle or 1 million male, syngeneic CPCs. One year later, CPC-treated rats exhibited smaller scars and more viable myocardium in the risk region, along with improved left ventricular remodeling and regional and global left ventricular function. No tumors were observed. Some transplanted (Y-chromosome(POS)) CPCs (or their progeny) persisted and continued to proliferate, but they failed to acquire a mature cardiomyocyte phenotype and were too few (4-8% of nuclei) to account for the benefits of CPC therapy. Surprisingly, CPC transplantation triggered a prolonged proliferative response of endogenous cells, resulting in increased formation of endothelial cells and Y-chromosome(NEG) CPCs for 12 months and increased formation, for at least 7 months, of small cells that expressed cardiomyocytic proteins (α-sarcomeric actin) but did not have a mature cardiomyocyte phenotype. The beneficial effects of CPCs on left ventricular remodeling and dysfunction are sustained for at least 1 year and thus are likely to be permanent. Because transplanted CPCs do not differentiate into mature myocytes, their major mechanism of action must involve paracrine actions. These paracrine mechanisms could be very prolonged because some CPCs engraft, proliferate, and persist at 1 year. This is the first report that transplantation of any cell type in the heart induces a proliferative response that lasts at least 1 year. The results strongly support the safety and clinical utility of CPC therapy.

Fibronectin and Cyclic Strain Improve Cardiac Progenitor Cell Regenerative Potential In Vitro.

Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2–4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment.

An in vitro model for the assessment of stem cell fate following implantation within the infarct microenvironment identifies ISL-1 expression as the strongest predictor of c-Kit+ cardiac progenitor cells' therapeutic potential

Cell therapy has the potential to drastically improve clinical outcomes for the 1.45 million patients suffering from a myocardial infarction (MI) each year in the U.S. However, the limitations associated with this treatment – including poor engraftment, significant cell death and poor differentiation potential – have prevented its widespread application clinically. To optimize functional improvements provided by transplanted cells, there is a need to develop methods that increase cellular retention and viability, while supporting differentiation and promoting paracrine signaling. Current in vivo models are expensive, difficult to access and manipulate and are time consuming. We have developed an in vitro model of MI which allows for a straightforward, consistent and relatively accurate prediction of cell fate following injection in vivo. The model demonstrated how the infarct environment impairs cellular engraftment and differentiation, but identified an implantation strategy which enhanced cell fate in vitro. Multivariate linear regression identified variables within the model that regulated vascular differentiation potential including oxygen tension, stiffness and cytokine presence, while cardiac differentiation was more accurately predicted by Isl-1 expression in the original cell isolate than any other variable present within the model system. The model highlighted how the cells' sensitivity to the infarct variables varied from line to line, which emphasizes the importance of the model system for the prediction of cell fate on a patient specific basis. Further development of this model system could help predict the clinical efficacy of cardiac progenitor cell therapy at the patient level as well as identify the optimal strategy for cell delivery.

Glutamine Regulates Cardiac Progenitor Cell Metabolism and Proliferation.

Autologous transplantation of cardiac progenitor cells (CPCs) alleviates myocardial dysfunction in the damaged heart; however, the mechanisms that contribute to their reparative qualities remain poorly understood. In this study, we examined CPC metabolism to elucidate the metabolic pathways that regulate their proliferative capacity. In complete growth medium, undifferentiated CPCs isolated from adult mouse heart proliferated rapidly (Td = 13.8 hours). CPCs expressed the Glut1 transporter and their glycolytic rate was increased by high extracellular glucose (Glc) concentration, in the absence of insulin. Although high Glc concentrations did not stimulate proliferation, glutamine (Gln) increased CPC doubling time and promoted survival under conditions of oxidative stress. In comparison with Glc, pyruvate (Pyr) or BSA-palmitate, Gln, when provided as the sole metabolic substrate, increased ATP-linked and uncoupled respiration. Although fatty acids were not used as respiratory substrates when present as a sole carbon source, Gln-induced respiration was doubled in the presence of BSA-palmitate, suggesting that Gln stimulates fatty acid oxidation. Additionally, Gln promoted rapid phosphorylation of the mTORC1 substrate, p70S6k, as well as retinoblastoma protein, followed by induction of cyclin D1 and cdk4. Inhibition of either mTORC1 or glutaminolysis was sufficient to diminish CPC proliferation, and provision of cell permeable α-ketoglutarate in the absence of Gln increased both respiration and cell proliferation, indicating a key role of Gln anaplerosis in cell growth. These findings suggest that Gln, by enhancing mitochondrial function and stimulating mTORC1, increases CPC proliferation, and that interventions to increase Gln uptake or oxidation may improve CPC therapy.

Lenti‐Angiotensin‐(1–7) transduction of Islet+ cardiac progenitor cells improves the reparative capacity in Doxorubicin induced Cardiomyopathy

Doxorubicin (DOX), a highly effective antineoplastic agent, can have devastating effects on the heart. Recent studies indicate that cardiac progenitor cell (CPC) loss plays a major role in the pathophysiology of DOX‐induced cardiomyopathy. We have recently demonstrated the potential for Angiotensin‐(1–7) (Ang‐(1–7)) to improve the beneficial effects of CPCs when given as therapy in a model of myocardial infarction. We hypothesize that giving CPCs transduced with lenti‐Ang‐(1–7) will further improve the regenerative capabilities of CPC therapy in DOX‐induced cardiomyopathy. Male SD rats were injected via tail vein with 2.5mg/kg/week DOX for 5 weeks, reaching a cumulative dose of 12.5mg/kg. 4 × 10^8 Islet+ CPCs, or lenti‐Ang‐(1–7) CPCs, were injected via jugular vein 3 days post Dox injection at weeks 3 and 5. Hemodynamic parameters were measured 2 weeks later. DOX significantly reduced dP/dtmax (3769 ± 322 vs 6880 ± 345 for control, mmHg/sec) and ventricular mass (VM, 1.75 ± 0.09 vs 3.07 ± 0.2 for control, (g/cm)). In comparison to DOX, CPCs alone restored dP/dtmax by 7.5% and increased VM by 5.3%. Ang‐(1–7) expressing CPCs further improved dP/dtmax by 13.8%, and increased VM by 8.3%. Ang‐(1–7) transduced CPCs seems to further improve the reparative capacity of CPC therapy and may prove to be a significantly beneficial therapy in preventing DOX‐induced cardiomyopathy.

Abstract 344: Naturally Derived Cardiac Extracellular Matrix for Cardiac Progenitor Cell Therapy

Cardiovascular disease is the leading cause of death in the United States. Following acute myocardial infarction, there is dramatic myocyte loss, and the cells are replaced with a collagen scar. Cell therapy directed toward regenerating infarcted myocardium suffers from poor retention of cells, possibly because the conditions are not ideal to support injected cells. Many studies show the local microenvironment is a potent regulator of cell fate. Therefore, we hypothesize that culturing cardiac progenitor cells (CPCs) on a naturally derived, cardiac extracellular matrix (cECM) will increase their function. CPCs were isolated from rat cardiac tissue-homogenate with magnetic beads conjugated with c-kit antibodies. Porcine, cardiac extracellular matrix (cECM) was decellularized, digested, and lyophillized to be used as a coating material. CPCs were cultured on cECM and examined for cardiogenic lineage markers, survival, proliferation and adherence. Our results show that CPCs cultured on cECM proliferate better (2.9-fold) as compared to COL (2.3-fold; p<0.05). Additionally, CPCs demonstrate improved resistance to serum-starvation-induced apoptosis when cultured on cECM (40% ± 14%) as compared to COL (53% ± 14%; p<0.001). Furthermore, CPCs adhered more strongly to cECM than COL as measured using microfluidics. CPCs showed enhanced progression toward the cardiomyoctye lineage when cultured on cECM (nkx2.5: 2.3 ± 0.4-fold; α-myosin heavy chain: 14.6 ± 4.4-fold; troponinC: 2.4 ± 0.2-fold; p-values <0.05) as compared to COL. This was confirmed at the protein level with Gata-4 (1.8 ± 0.1-fold; p<0.001) and Nkx2.5 (1.6 ± 0.2-fold; p<0.05). A reduction in fibroblast specific protein-1 (0.5 ± 0.1-fold) was also seen on cECM as compared to COL and suggests the CPCs are less likely to contribute to scar formation. While these conditioned cells have not yet been examined in an in vivo setting, our comparison to COL mimics both the naked injection of these cells into an infarct zone, and comparison to a commonly used cell delivery vehicle. Our data suggest that CPCs delivered with cECM are more likely to survive and differentiate in the infarct zone than cells delivered by themselves or in collagen.

Abstract 9306: Site-Specific Reporter Gene Imaging of Stem Cell Transplantation: Early Engraftment Predicts Late Improvement in Cardiac Function

Stable genetic modification of cardiac progenitor cells (CPCs) enables quantitative assessment of their engraftment and proliferation using reporter gene imaging. However, random lentiviral integration techniques carry the concomitant risk of introducing inadvertent mutations, including the unacceptable risk of proto-oncogene activation. Here, we demonstrate a safe, clinically applicable method for site-specific PET reporter gene imaging of transplanted CPCs. A triple fusion reporter gene was integrated specifically into chromosome 19 of Sca-1+ human CPCs using the ΦC31 integrase. Targeted integration into this genomic locus was confirmed using nested PCR and Southern blotting. Stably-integrated CPCs (1×10 6 ) were then transplanted into ischemic murine myocardium (n=40), and monitored noninvasively using PET, bioluminescence, cardiac MR, and echocardiography for four weeks. hCPC-treated hearts demonstrated a statistically significant improvement in fractional shortening (27.2±2.3% vs. 21.5±1.4%; P=0.04) as well as ejection fraction (48.3±2.6% vs. 42.2±1.7%; P=0.03) at day 14 post-transplant compared to PBS controls, as assessed by echocardiography and cardiac MRI, respectively. Initial PET signal intensity, as a surrogate marker of cellular engraftment, predicted the degree of functional improvement at week 2 (R 2 =0.73) in a “dose-effect” relationship. To investigate the mechanism of hCPC benefit on myocardial function, we isolated the transplanted cells from recipient myocardium by laser capture microdissection and found direct evidence for upregulated expression of the VEGF-α and CTGF pro-angiogenic growth factors in vivo. Here, we demonstrate the first use of site-specific reporter gene imaging to enable prognostic prediction of the ultimate success of CPC therapy. Given the encouraging but variable results of recent cell therapy trials, our results provide valuable insight into the determinants of successful cell therapy.

Cardiac Progenitor Cells and Biotinylated Insulin-Like Growth Factor-1 Nanofibers Improve Endogenous and Exogenous Myocardial Regeneration After Infarction

Cardiac progenitor cells (CPCs) possess the insulin-like growth factor-1 (IGF-1)-IGF-1 receptor system, and IGF-1 can be tethered to self-assembling peptide nanofibers (NF-IGF-1), leading to prolonged release of this growth factor to the myocardium. Therefore, we tested whether local injection of clonogenic CPCs and NF-IGF-1 potentiates the activation and differentiation of delivered and resident CPCs enhancing cardiac repair after infarction. Myocardial infarction was induced in rats, and untreated infarcts and infarcts treated with CPCs or NF-IGF-1 only and CPCs and NF-IGF-1 together were analyzed. With respect to infarcts exposed to CPCs or NF-IGF-1 alone, combination therapy resulted in a greater increase in the ratio of left ventricular mass to chamber volume and a better preservation of +dP/dt, -dP/dt, ejection fraction, and diastolic wall stress. Myocardial regeneration was detected in all treated infarcts, but the number of newly formed myocytes with combination therapy was 32% and 230% higher than with CPCs and NF-IGF-1, respectively. Corresponding differences in the volume of regenerated myocytes were 48% and 115%. Similarly, the length density of newly formed coronary arterioles with both CPCs and NF-IGF-1 was 73% and 83% greater than with CPCs and NF-IGF-1 alone, respectively. Importantly, activation of resident CPCs by paracrine effects contributed to cardiomyogenesis and vasculogenesis. Collectively, CPCs and NF-IGF-1 therapy reduced infarct size more than CPCs and NF-IGF-1 alone. The addition of nanofiber-mediated IGF-1 delivery to CPC therapy improved in part the recovery of myocardial structure and function after infarction.

Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4 Expression

Myocardial infarction rapidly depletes the endogenous cardiac progenitor cell pool, and the inefficient recruitment of exogenously administered progenitor cells limits the effectiveness of cardiac cell therapy. Recent reports indicate that interactions between the CXC chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone marrow-derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Here, we studied the influence of hypoxia on CXCR4 expression in cardiac progenitor cells, on the recruitment of intravenously administered cells to ischemic heart tissue, and on the preservation of heart function in a murine myocardial infarction model. We found that hypoxic preconditioning increased CXCR4 expression in CLK (cardiosphere-derived, Lin(-)c-kit(+) progenitor) cells and markedly augmented CLK cell migration (in vitro) and recruitment (in vivo) to the ischemic myocardium. Four weeks after surgically induced myocardial infarction, infarct size and heart function were significantly better in mice administered hypoxia-preconditioned CLK cells than in mice treated with cells cultured under normoxic conditions. Furthermore, these effects were largely abolished by the addition of a CXCR4 inhibitor, indicating that the benefits of hypoxic preconditioning are mediated by the stromal cell-derived factor 1/CXCR4 axis, and that therapies targeting this axis may enhance cardiac-progenitor cell-based regenerative therapy.

Cardiac Progenitor Cells

See related article, pages 1090–1092 In the last few years we have witnessed one of most extraordinary revolutions in cardiovascular medicine, namely, an explosion of basic and clinical studies that support the notion that the diseased heart can be repaired by administration of stem cells, resulting in formation of functional new myocytes and vessels. Although the mechanism by which cell therapy improves cardiac function and anatomy remains uncertain, translation of basic findings to the clinical setting is proceeding at a feverish pace.1 A multitude of small, mostly nonrandomized clinical studies have reported improvement in cardiac perfusion and function after therapy with various cell types in patients with acute myocardial infarction or chronic ischemic cardiomyopathy.1 Larger, randomized, double-blinded studies will be reported soon; if they confirm the salubrious effects of cell-based therapies observed in the initial trials, our management of acute myocardial infarction and heart failure will change dramatically. One of the most important and unresolved issues in this scenario is the identity of the ideal cell for myocardial reconstitution. Although most of the clinical studies reported to date have used bone marrow– or skeletal muscle–derived cells, a host of other cells are being investigated in the experimental laboratory. Among these, resident cardiac stem cells (CSCs), discovered by Anversa’s group in 2003,2 hold great promise. In their initial report,2 Beltrami et al identified a lin−/c-kit+ population of primitive cells that can be clonally expanded, differentiates into cardiac myocytes, smooth muscle cells, and endothelial cells in vitro, and is able to reconstitute infarcted myocardium in vivo. It is of translational interest that these cells can effect cardiac repair when delivered via the intravascular route.3 CSCs have also been shown to be present in the human heart, where they give rise to new myocytes in patients with …