Isolation and characterization of pluripotent cells from the subendocardial layer of human hearts from chronic heart failure.

Abstract

Purpose: Several literature reports indicated the existence of pluripotent cells residing into the heart wall, even if in vivo heart regeneration is still a poorly comprised phenomenon. Post-infarct chronic heart failure is a pathology for which heart transplant remains a key option of clinical intervention. The amount of data collected in recent years forces researchers to better characterize the properties of adult stem cells, especially for those isolated from heart. Methods: We developed a new protocol to obtain, from the subendocardial layer of post-infarct hearts explanted from chronic heart failure patients undergoing heart transplantation, populations of mesenchymal stem cells. The isolation protocol allowed obtaining homogeneous fibroblast-like cellular populations from the subendocardial layer of left ventricle. A first step comprised a brief collagenase digestion in order to remove the endocardial endothelial cells. Subsequently, the cells were allowed to migrate out spontaneously from the heart wall slices, and when confluent were sub-cultured using standard techniques for stem cells maintenance. The phenotypical characterization of these cellular populations was carried out by immunocytochemistry and RT-PCR, and allowed to establish their adherence to the “mesenchymal stem cells” phenotype. Results: These cells did not express CD31, CD34, CD45, CD4, CD8 or CD20, while they did express c-kit (CD117), CD44, CD73, CD90, CD10, CD13, CD68, CD105, CD166. Moreover, not only these key markers of the MSC type were present, but also GATA4, a key transcription factor for myocardiocyte differentiation, Connexin-43, expressed in myocytes, and Nestin, were expressed, indicating the potentiality for these cells to differentiate towards different lineages. To assess the differentiative properties of such cardiac populations, we successfully performed differentiation experiments towards the adipogenic and osteoblastic lineage. The acquisition of the differentiated phenotype was demonstrated by histochemical stainings and RT-PCR analyses of adipocyte-specific and osteoblast-specific molecules. In addition, such cells were able to undergo several passages in culture, without significant phenotypical alterations. Moreover, the self-renewal, which is a key property for stem cells, was demonstrated by clonogenicity experiments. Conclusions: The data obtained allowed to characterize a novel population of mesenchymal stem cells resident in the subendocardial layer of diseased human hearts. Further experiments are in progress in order to establish the feasibility of in vitro differentiation towards myocardiocytes.