We have explored the possibility of using the cardiac MSCs of the human fetal heart for expansion of angiotensin receptor blocker with characteristics similar to those of mesenchymal progenitors that contribute to mammalian cardiogenesis. In this study cardiac MSCs were reproducibly recovered from the adherent cell fraction from first trimester human fetal hearts by using defined culturing conditions, including the use of specific laminin (LN) isoforms, in combination with canonical Wnt/β-catenin stimulation. The MSCs expressed ISL1, PDGFR-α, and NKX2.5, where subpopulations also expressed the stemness and potential cardiovascular progenitor markers: kinase insert domain protein receptor (KDR), the human orthologue of FLK1; c-KIT, the receptor for the stem cell factor (Beltrami et al., 2003; (Bearzi et al., 2007, 2009), stage-specific embryonic antigen 1 (SSEA-1) (Blin et al., 2010), and TBX18 (Christoffels et al., 2006; Mommersteeg et al., 2010). The fetal cardiac MSCs were multipotent and could be differentiated into elongated, striated, and spontaneously beating cardiomyocytes, endothelial cells, and smooth muscle cell-like cells, also when using defined laminins as substrata. This implies that the unique culturing conditions of human fetal cardiac MSCs using a combination of canonical Wnt/β-catenin stimulation and LN-based matrices facilitate the in vitro propagation of cells with cardiac progenitor characteristics.
Discussion The origin of the different subpopulations of cardiac MSCs has not been examined in this study. It has previously been reported that interstitial cells of both the atria and ventricles, display a strong expression of PDGFR-α (Chong et al., 2011, 2013). Chong et al. (2013) showed that PDGFR-α+ cells isolated from human fetal hearts of the second trimester were multipotent and could be differentiated into smooth muscle as well as endothelial cells, but they did not differentiate into cardiomyocytes. In the present study, the majority of the cultured cardiac MSCs stained positive for PDGFR-α. Whether these cells possibly originate from interstitial cells of the atria and ventricles remains to be elucidated, but they displayed a similar capacity to differentiate into endothelial cells and also cells with polarized α-SMA expression akin to that of mature smooth muscle cells. In contrast to the PDGFR-α+ cells previously derived from second trimester hearts (Chong et al., 2013), the fetal cardiac MSCs derived from a 9-week human heart in the present study could be differentiated into spontaneously beating, striated TnT+ cardiomyocytes, although it was only a small proportion of the cardiac MSCs that demonstrated this ability. This restricted differentiation capacity might be related to the age of the fetal heart from which the cardiac MSCs are derived, although in the present study it could not be linked to age-related differences in expression of markers known to characterize cardiovascular progenitors like KDR, NKX2.5, c-KIT, and SSEA-1 (Urbanek et al., 2003; Beltrami et al., 2003; Bearzi et al., 2007; Bruneau, 2002; Kwon et al., 2008; Durocher et al., 1996; Jho et al., 2002; Millauer et al., 1993; Motoike et al., 2003). c-KIT is a tyrosine kinase receptor that is expressed on purported cardiovascular progenitors in the heart (Urbanek et al., 2003; Beltrami et al., 2003; Bearzi et al., 2007; Bearzi et al., 2009). The origin of these cells is still unknown. Chong et al. (2013) have reported that a limited number of PDGFR-α+ cells in the atria and ventricles of the fetal hearts also express c-KIT, which is similar to our cardiac MSCs, where approximately 2% expressed c-KIT. Although subpopulations of the cardiac MSCs expressed c-KIT, we do not know if these cells are similar to the previously described cardiac progenitors in the adult heart (Urbanek et al., 2003; Beltrami et al., 2003; Bearzi et al., 2007, 2009). A small population of the fetal cardiac MSCs also expresses SSEA-1, which has been reported to represent an early marker of cardiovascular progenitors derived from pluripotent stem cells (Blin et al., 2010). The existence of a corresponding progenitor in the human fetal heart has yet to be identified, and it remains to be further investigated whether the SSEA-1-expressing cells identified within the cardiac MSC population specifically have the potential to develop into the different cell types of the heart.