0129 : Adult human mononuclear clones isolated from peripheral blood can differentiate into immature cardiomyocytes

Breakthroughs in stem cell biology and demonstrations of the heart’s endogenous regenerative capacities have incited an intense race towards cardiac regeneration, i.e. the replacement of lost myocardium after myocardial infarction. In preclinical trials, cell transplantation therapies, using adult human multipotent stem cells (e. g, hematopoietic stem cells) into infarcted myocardium has shown enhanced cardiogenesis in animal models. Nevertheless, results in clinical trials remain unsatisfactory and determining a suitable cell population that is easily harvested and improves cardiac repair is challenging. In previous research, our lab isolated human peripheral blood mononuclear clones (PBMCs) bearing cardiac mesodermal markers, e.g. c-kit, Islet-1 or Flk-1. Potentially, these cells can differentiate within the cardiac lineage to mature cardiomyocytes and partMyosin heavy chainicipate in heart repair. We sought to establish an in vitro cardioinstructive differentiation protocol to derive cardiomyocytes from our PBMC population. For this, we screened three differentiation protocols, i.e. Keller, Smith and He protocols. To follow cell differentiation RT-qPCR and immunostainings were performed for relevant genes, e.g. Nkx2.5, GATA4, Troponin T (cTNT), (β-Myosin heavy chain ((β-MHC) and (-actinin. Results for mRNA expression showed that at least 75% of PBMCs can derive to a cardiac precursor population (Nkx2.5 + /GATA4 + ) following Keller and Smith protocol. In Smith protocol, 75% of PBMCs differentiated to immature cardiomyocytes (cTNT + ) of which 50% expressed both cTNT and (- MHC when co-cultured with neonatal mice ventricular myocytes. Immunofluoresence assay showed that PBMCs in both Keller and Smith protocol are Nkx2.5 + /(α-actinin + demonstrating differentiation at the protein level. Next we evaluated the safety, survival and integration of injected PBMCs in neonatal Gt(ROSA) 26 -Tomato mice tissues in vivo. By staining PBMC with membrane marker PKH2GL, we followed cell homing within the animal tissues visualized by fluorescent microscopy on tissue sections. In conclusion, we demonstrated that human PBMCs can differentiate, under certain conditions, into cardiac precursor cells or immature cardiomyocytes in vitro . These results make them an interesting and promising cell source for stem cell therapies in cardiac repair.