397 Molecular signature of EX vivo proliferated resident cardiac progenitor cells

Abstract

A number of laboratories have documented small reservoirs of resident cardiac progenitor cells (CPCs) within the post-natal heart. Because these cells reside within the heart and have the ability to reconstitute all types of cardiac cells, they represent a promising therapy to regenerate the heart. In that view, we have developed techniques to proliferate CPCs directly from a patient's own heart biopsy with a view towards transplanting these cells back into damaged myocardium. Despite the start of clinical trials using these cells, remarkably little is known about the molecular mechanisms that underlie their multipotency. We sectioned, enzymatically digested and plated myocardial tissue from 8-9 week old c-KitBAC-GFP transgenic mice. This mouse model genetically labels CPCs with green fluorescent protein (GFP) as transgene expression is controlled by the stem cell kit locus within a bacterial artificial chromosome (BAC). After several days in culture, a layer of stromal-like cells arose from the adherent plated tissue with clusters of small GFP+ CPCs (Figure panel A). As compared to non-transgenic mice, the overall GFP fluorescence of the c-KitBAC-GFP cultures progressively increased (Figure panel B and C; Day 14: 77 ± 45% vs. 12 ± 18%, P < 0.01). Given that CPCs represented 0.1 ± 0.1% of all cells in the native heart (aggregate data from 6 random fields, n = 6 mice), flow cytometry of collected cells confirmed the ability of explant culture to markedly increased the overall CPC yields (9.2 ± 2.3%, 1.3×106 ± 1.7×105 cells collected). To identify commonalities between the transcriptional profile of explant cultured CPCs and unrestricted pluripotent cells, we compared the expression profiles of mouse embryonic stem cells (C2 mESCs), CPCs and somatic cells (dermal fibroblasts; DF) using a gene expression micro-array (Affymetrix). The gene expression patterns of CPC lines showed many similarities with the mESCs cell line and more distantly with the somatic DF cell lines. We identified 587 genes that were expressed at greater levels in mouse CPCs than in somatic control cell lines. As shown in the Figure panel D, quantitative PCR of key pluripotent markers (Oct3, nanog and sox 2) were upregulated in both CPCs and C2 mESCs with a tendency to greater expression in the more pluripotent C2 mESCs. Explant culture of myocardial biopsies from c-KitBAC-GFP transgenic mice increased the proportion of CPCs above that found in the native heart. These CPCs express genetic markers similar to mESCs and not somatic cells; hinting these genes are involved in the multipotent phenotype of CPCs.