N004 Isolation of cardiac precursor cells from the human fetal heart

Abstract

To identify a suitable source of human cardiac precursor cells (CPC) for replacement cell therapies, we established the conditions to isolate precursors from the human fetal heart. Ventricles were collected from donation following voluntary pregnancy termination at 12 weeks of gestation. Non-myocyte cells were isolated, and expanded in vitro. CPC were identified using immunostaining for Nkx2.5, Flk1 and Isl1, and by measuring the expression levels of these markers by real-time PCR. Proliferating cells demonstrated Nkx2.5 staining and no α-actinin expression. A significant fraction of Nkx2.5+ cells expressed Flk1. A few Nkx2.5+ cells were also Isl1+ whereas no Flk1+ cells expressed this marker. The capacity of expanded cells to produce differentiated cardiomyocytes, smooth muscle cells and endothelial cells was then tested by switching the culture to a differentiation serum-free medium. Differentiation into cardiomyocytes, smooth muscle and endothelial cells was evaluated by immunostaining for α-actinin, sm-MHC and CD31 respectively. In addition, expression of cardiac-specific (Nkx2.5, α-MHC, β-MHC), smooth muscle-specific (sm-MHC) and endothelial-specific (CD31) genes was measured using real-time PCR. Evidence for cardiogenic differentiation in the culture is supported by the significant increase in α-actinin+ cells (1 % and 20 % under expansion and differentiation conditions respectively) as well as by the upregulation of cardiac marker expression. Differentiation into smooth muscle cell type was also dramatically stimulated (0 % and 4 % respectively). Accordingly, sm-MHC expression was 25-fold increased. In contrast, no differentiation into endothelial cells was detected under these conditions. The engraftment and differentiation capacity of GFP-labeled CPC were tested in vivo by transfer into the heart of SCID mice after myocardial infarction. Seven days thereafter, engrafted cells were readily detected and demonstrated cardiac-commitment as shown by Nkx2.5 staining. At this stage, no differentiation into cardiomyocytes could be observed.