Smooth muscle cells are characterised by the specific expres

Smooth muscle cells are characterised by the specific expression of several proteins such as α-smooth muscle Silmitasertib (α-SMA), transgelin (SM22α), calponin and smooth muscle myosin heavy chain (MYH11) (Owens, 1995). α-SMA, encoded by the Acta2 gene, is one of the six actin genes expressed in mammalian cells and is the first known marker of differentiated smooth muscle cells during vasculogenesis (Owens, 1995). During murine embryogenesis, vascular smooth muscle cells express high levels of α-SMA, (Mack and Owens, 1999; McHugh, 1995), initially within the embryonic heart rudiment around E8.5, then in the yolk sac and the aorta vasculature by E9.5–10.5 (Armstrong et al., 2010). The embryonic origin of smooth muscle remains poorly characterised and lineage-tracing studies suggest that, in distinct vessels, vascular smooth muscles or pericytes have different embryonic origins (Cheung et al., 2012; Majesky, 2007). The hemangioblast, or its in vitro equivalent — the blast colony forming cell (BL-CFC), is defined as a mesodermal progenitor cell with endothelial and hematopoietic potentials (Choi et al., 1998). A vascular smooth muscle cell developmental potential was also attributed to the hemangioblast, as both embryo-derived hemangioblast (Huber et al., 2004) and embryonic stem (ES) cell-derived BL-CFCs (Ema et al., 2003; Lu et al., 2009) were demonstrated to contain vascular smooth muscle potential. The generation of blood precursors from the hemangioblast was recently shown to occur through a transient cell population of specialised endothelium, a hemogenic endothelium (Lancrin et al., 2009). To date, the lineage relationship between this specific endothelial cell population and smooth muscle cell progenitors has not been investigated. Several previous studies suggest a close developmental relationship between endothelial and smooth muscle progenitors. For instance, mesodermal FLK1+ cells generated from differentiated ES cells were shown to produce both smooth muscle and endothelial cells that organise into vessel-like structures (Yamashita et al., 2000). Furthermore, smooth muscle actin expression has been detected in CD34+ cord blood endothelium and in endothelial cells at the luminal surface of adult aorta (Azuma et al., 2009; Lu et al., 2004). The generation of cardiomyocytes was also shown to be associated with common endothelial and smooth muscle development. In headfold stage murine embryos, or in differentiated ES cells, Kattman et al. identified a progenitor for cardiomyocytes with additional endothelial and vascular smooth muscle potential (Kattman et al., 2006; Yang et al., 2008). In this study, we generated a mouse reporter ES cell line in which the expression of the fluorescent protein, H2B-VENUS, is driven from α-SMA regulatory sequences. We demonstrated that this reporter cell line allows to efficiently track smooth muscle development during murine ES cell differentiation. Although we observed the presence of rare H2B-VENUS+ cells in enriched hemogenic endothelial cell populations, our findings established that smooth muscle cells were mostly generated independently from the specialised functional hemogenic endothelium.
Materials and methods
Results
Discussion In this study, we investigated the generation of smooth muscle cells upon in vitro differentiation of ES cells. For this we engineered a reporter ES cell line in which expression of the fluorescent protein H2B-VENUS is driven from α-Sma regulatory sequences. We demonstrated that the expression of H2B-VENUS is strongly correlated with α-Sma expression during in vitro differentiation of this reporter ES cell line. In addition, the enrichment for expression of a panel of smooth muscle markers indicates that H2B-VENUS+ cells truthfully represent a smooth muscle cell lineage. It has been shown that vascular smooth muscle cells express the long isoform of Smoothelin (Smoothelin-B) whereas visceral smooth muscle cells express the short isoform (Smoothelin-A) (Kramer et al., 2001). We detected in the H2B-VENUS sorted cells mostly transcripts of Smoothelin-B (Supplemental Fig. 5), suggesting that our ES differentiation conditions preferentially generate vascular rather than visceral smooth muscle cells. Altogether these results indicate that H2B-VENUS detection allows directly quantifying or isolating smooth muscle cells. With this reporter ES cell line, we also confirmed that clonal BL-CFCs generate smooth muscle cells. It has been recently established that BL-CFC generates blood cells through a hemogenic endothelium intermediate (Lancrin et al., 2009). To determine if the generation of smooth muscle cells is associated, or independent, from the emergence of this hemogenic endothelium, we examined the presence of H2B-VENUS+ cells in cell subpopulations containing this precursor. We indeed observed the presence of few H2B-VENUS+ cells in these hemogenic endothelium populations. However these cells lacked hematopoietic potential and therefore did not correspond to functional hemogenic endothelium, indicating that smooth muscle cells are largely generated independently form the hemogenic endothelium.