Identifying the best source of autologous progenitors for cardiac regeneration

Abstract

Preliminary efficacy data indicates that stem cell transplantation has the potential to enhance myocardial perfusion and/or contractile performance in patients with ischemic heart disease. A significant challenge in cardiovascular regenerative medicine is the identification and selection of the best suited stem/progenitor cell type. Cardiac progenitor cells (CPCs) and cardiac stem cells (CSCs) have been reviewed as the most promising cell types for autologous transplantation. The other extracardiac cell types that have been considered include bone marrow derived stem cells (BMCs), adipose tissue derived stem cells (ADSCs), peripheral blood derived endothelial progenitor cells (EPCs) and skeletal myoblasts.

CPCs have not been directly compared with extra-cardiac cell types isolated from same patients for properties associated with cardiac repair. Interestingly, even within the heart the best location to isolate CPCs remains undecided. Moreover, atrial and ventricular CPCs may have phenotypic/functional differences owing to the differences in the atrio-ventricular microenvironment. While comparing different progenitor cell types, it is essential to isolate these cells from the same patients because the donor’s physiological and pathological characteristics may have a profound effect on the quality and quantity of progenitor cells isolated. This project, therefore, is aimed at comparing the functional characteristics of resident CPCs from the right atrial appendage (RAA), the left ventricle (LV) and an extra-cardiac source: EPCs from the peripheral blood – all isolated from the same patients, for the properties associated with cardiac repair.

CPCs were isolated and characterised from the RAA and LV tissues from nineteen patients undergoing coronary artery bypass surgery (CABG). Early EPCs were isolated from the same patients from the peripheral blood and characterised for the expression of progenitor cell associated markers.

To examine whether RAA and LV CPCs from the same hearts show different functional characteristics, the stem cell surface marker expression, cardiomyocyte differentiation capacity, expansion potential, senescence in culture and migration properties of these cells were examined. The RAA CPCs showed a higher expression of CD90 (a surface protein involved in cell migration and adhesion). In consonance with this, a scratch assay revealed a higher migration potential of RAA CPCs compared to LV CPCs isolated from the same hearts. The ex vivo expansion potential of RAA CPCs was greater than LV CPCs with higher cumulative population doublings after six weeks in culture. In contrast, RAA CPCs underwent greater senescence in culture at the same time point with a higher expression of cyclin dependent kinase inhibitor 2A (CDKN2A), a gene involved in the transcription of the senescence protein P16inka.

To determine the differences in response to ischemia, the three progenitor cell types were exposed to hypoxia and serum starvation in vitro and their apoptotic cell death and gene expression was quantified. RAA and LV CPCs exhibited a similar pattern of resistance to apoptotic cell death under ischemia. Interestingly, EPCs exhibited the highest resistance to apoptotic cell death, however, they also showed the lowest proliferation under ‘hypoxia only’ treatment. The RT2 profiler gene array showed comparable gene expression pattern in RAA and LV CPCs, however, EPCs differentially expressed certain genes associated with paracrine properties and cell survival.

To compare the paracrine properties of the progenitor cells exposed to ischemia, the secretion of cytokines was quantified in the conditioned medium (CM) from all three cell types. EPCs showed a significantly lower secretion of cytokines compared to CPCs. When human umbilical vein endothelial cells (HUVECs) were treated with the CM from the three cell types, LV CPC CM induced the highest angiogenesis in a tube formation assay. Conversely, ischemic mouse cardiomyocytes (HL-1 cells) treated with CM from the RAA CPCs showed the greatest survival. Further, it was identified that RAA CPC CM mediated its effect via both the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) and protein kinase C epsilon (PKCε) pathways, whereas LV CPC CM and EPC CM mediated their effect by PKCε and PI3K-Akt pathways respectively.

In conclusion, this study provides the first evidence that progenitor cells from the atria, ventricle and peripheral blood of the same patients show differences in functional properties associated with cardiac repair. The selection of the best cell type could, therefore, be influenced by the nature of the injury and the type of effect desired.