Kristin M French

Identification of Therapeutic Covariant MicroRNA Clusters in Hypoxia-Treated Cardiac Progenitor Cell Exosomes Using Systems Biology

Rationale: Myocardial infarction is a leading cause of death in developed nations, and there remains a need for cardiac therapeutic systems that mitigate tissue damage. Cardiac progenitor cells (CPCs) and other stem cell types are attractive candidates for treatment of myocardial infarction; however, the benefit of these cells may be as a result of paracrine effects. Objective: We tested the hypothesis that CPCs secrete proregenerative exosomes in response to hypoxic conditions. Methods and Results: The angiogenic and antifibrotic potential of secreted exosomes on cardiac endothelial cells and cardiac fibroblasts were assessed. We found that CPC exosomes secreted in response to hypoxia enhanced tube formation of endothelial cells and decreased profibrotic gene expression in TGF-&bgr;–stimulated fibroblasts, indicating that these exosomes possess therapeutic potential. Microarray analysis of exosomes secreted by hypoxic CPCs identified 11 miRNAs that were upregulated compared with exosomes secreted by CPCs grown under normoxic conditions. Principle component analysis was performed to identify miRNAs that were coregulated in response to distinct exosome-generating conditions. To investigate the cue–signal–response relationships of these miRNA clusters with a physiological outcome of tube formation or fibrotic gene expression, partial least squares regression analysis was applied. The importance of each up- or downregulated miRNA on physiological outcomes was determined. Finally, to validate the model, we delivered exosomes after ischemia–reperfusion injury. Exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis. Conclusions: These data provide a foundation for subsequent research of the use of exosomal miRNA and systems biology as therapeutic strategies for the damaged heart.

A naturally derived cardiac extracellular matrix enhances cardiac progenitor cell behavior in vitro.

Myocardial infarction (MI) produces a collagen scar, altering the local microenvironment and impeding cardiac function. Cell therapy is a promising therapeutic option to replace the billions of myocytes lost following MI. Despite early successes, chronic function remains impaired and is likely a result of poor cellular retention, proliferation, and differentiation/maturation. While some efforts to deliver cells with scaffolds have attempted to address these shortcomings, they lack the natural cues required for optimal cell function. The goal of this study was to determine whether a naturally derived cardiac extracellular matrix (cECM) could enhance cardiac progenitor cell (CPC) function in vitro. CPCs were isolated via magnetic sorting of c-kit(+) cells and were grown on plates coated with either cECM or collagen I (Col). Our results show an increase in early cardiomyocyte markers on cECM compared with Col, as well as corresponding protein expression at a later time. CPCs show stronger serum-induced proliferation on cECM compared with Col, as well as increased resistance to apoptosis following serum starvation. Finally, a microfluidic adhesion assay demonstrated stronger adhesion of CPCs to cECM compared with Col. These data suggest that cECM may be optimal for CPC therapeutic delivery, as well as providing potential mechanisms to overcome the shortcomings of naked cell therapy.

Self-assembling peptide-based delivery of therapeutics for myocardial infarction.

Cardiovascular disease, including myocardial infarction, is the number one cause of death. Current treatments are palliative and slow the progression toward heart failure, but to not regenerate healthy tissue. Self-assembling peptides are biomimietic, readily produced, non-immunogenic and non-cytotoxic. They do not assemble into hydrogels until triggered, allowing them to be injected into the myocardium and providing opportunities for minimally invasive therapies. The ability to tune the mechanical and bioactive properties of self-assembling peptides will continue to make them readily adaptable for mimicking natural microenvironments. To date, a variety of growth factors and signaling moieties have been incorporated into self-assembling peptide hydrogels, enhancing cell behavior and tissue function. Furthermore, the hydrogels serve as delivery vehicles for cells in vivo and platforms for improved cell culture. In addition to a brief review of self-assembling peptides, we will discuss a variety of their approaches for myocardial infarction therapy. Moreover, we will assess approaches taken in other tissue and discuss how these could benefit therapies for myocardial infarction.

Age-Dependent Effect of Pediatric Cardiac Progenitor Cells After Juvenile Heart Failure

Children with congenital heart diseases have increased morbidity and mortality, despite various surgical treatments, therefore warranting better treatment strategies. Here we investigate the role of age of human pediatric cardiac progenitor cells (hCPCs) on ventricular remodeling in a model of juvenile heart failure. hCPCs isolated from children undergoing reconstructive surgeries were divided into 3 groups based on age: neonate (1 day to 1 month), infant (1 month to 1 year), and child (1 to 5 years). Adolescent athymic rats were subjected to sham or pulmonary artery banding surgery to generate a model of right ventricular (RV) heart failure. Two weeks after surgery, hCPCs were injected in RV musculature noninvasively. Analysis of cardiac function 4 weeks post‐transplantation demonstrated significantly increased tricuspid annular plane systolic excursion and RV ejection fraction and significantly decreased wall thickness and fibrosis in rats transplanted with neonatal hCPCs compared with saline‐injected rats. Computational modeling and systems biology analysis were performed on arrays and gave insights into potential mechanisms at the microRNA and gene level. Mechanisms including migration and proliferation assays, as suggested by computational modeling, showed improved chemotactic and proliferative capacity of neonatal hCPCs compared with infant/child hCPCs. In vivo immunostaining further suggested increased recruitment of stem cell antigen 1‐positive cells in the right ventricle. This is the first study to assess the role of hCPC age in juvenile RV heart failure. Interestingly, the reparative potential of hCPCs is age‐dependent, with neonatal hCPCs exerting the maximum beneficial effect compared with infant and child hCPCs.

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.

Isolation and Expansion of C-Kit-Positive Cardiac Progenitor Cells by Magnetic Cell Sorting

Cell therapy techniques are a promising option for tissue regeneration; especially in cases such as heart failure where transplantation is limited by donor availability. Multiple cell types have been examined for myocardial regeneration, including mesenchymal stem cells (and other bone marrow-derived cells), induced pluripotent stem cells, embryonic stem cells, cardiosphere-derived cells, and cardiac progenitor cells (CPCs). CPCs are multipotent and clonogenic, can be harvested from mature tissue, and have the distinct advantages of autologous transplant and lack of tumor formation in a clinical setting. Here we focus on the isolation, expansion, and myocardial differentiation of rat CPCs. Brief adaptations of the protocol for isolation from mouse and human tissue are also provided.