246. Characterization of Peripheral Cardiac Progenitors
Authors: José R. Navarro-Betancourt, Salomón Hernández, Felipe Masso, Luis A. Lara-Martínez, Alejandra Contreras-Ramos, Andrés R. Martinez-Calderón, Victor M. Arenas, José L. Aceves-Chimal
Journal: Molecular Therapy (2016)

Introduction We propose that cells expressing cardiomyocyte-specific structural proteins have a specific cardiac differentiation commitment and that an immunophenotype distinguished by the presence of cardiac stem cell (CSC) related surface markers, namely CD117, CD34 and Sca-1, identifies peripheral cardiac progenitors, therefore we explored the presence of these attributes in bone marrow-derived cells, aiming to establish an association between cardiac differentiation potential and a particular cellular profile. Methods Whole bone marrow was extracted from Wistar rat femur, cells were isolated through enzymatic desegregation and plated for incubation. Twenty-four hours later non-adherent supernatant cells were recovered and re-seeded, seventy-two hours later adherent cells were harvested and underwent dilution cloning. Flow cytometry was used to characterize each clone searching particularly for the surface markers CD117, CD34 and Sca-1. The subpopulation expressing the highest levels of these markers was isolated and labeled bone marrow supernatant (BMSN). Subsequently, a complete immunophenotypic profile of BMSN was performed, their capacity to differentiate into typical mesenchymal tissues was assessed using a commercial kit. BMSN were evaluated through rt-PCR and q-PCR for the expression of cardiac-related genes. The presence of proteins coded by these genes was evaluated through western blot and immunofluorescence. Co-cultures of green fluorescent protein (GFP) labeled BMSN with mature cardiomyocytes were performed, the expression of prototypal cardiac proteins was verified through immunofluorescence. In a rat model of myocardial infarction, 5×106 GFP-labeled BMSN were injected directly into the infarcted tissue. Cardiac function was tracked with left-ventricle ejection fraction (LVEF), measured though transthoracic echocardiography, and myocardial perfusion measured through single-photon emission computed tomography (SPE-CT). Myocardium histological analysis was carried out to evaluate engraftment. Results In addition to CD117, CD34 and Sca-1, BMSN express CD29, CD44, CD90, CD105 and CD106. BMSN are able to differentiate into osteocytes, but not into adipocytes or chondrocytes. BMSN express the following genes: GATA4, MEF2, Nkx2.5, a-MHC, connexin-43 and troponin-T, connexin-43 reached a 60% higher expression than mature cardiomyocytes. Through western-blot analysis the presence of GATA4, MEF2, Nkx2.5, a-MHC, connexin-43 and troponin-T in CMSC-like is unequivocal. By means of immunofluorescence protein detection only a meager proportion was positive for GATA4, a-MHC and troponin-T, compared to the quantity presumed by western-blot. BMSN phenotypically differentiate into cardiomyocytes when co-cultured with mature cardiomyocytes, cells that expressed Troponin-T before co-culture acquired spontaneous rhythmic contractile movement at the seventh day of follow-up. Sixty days after BMSN injection LVEF increased from an average value of 29% to a mean of 56% and myocardial perfusion increased significantly. BMSN co-expressing GFP and Troponin T were detected inside and around the infarcted area of treated rats. Conclusions The available data allows to infer an association between CSC-related surface marker expression, cardiomyocyte-specific gene transcription, prototypal cardiac protein presence and a differentiation potential restricted to cardiac tissue.