Smooth Muscle Cells

Generation of smooth muscle cells by haemangioblast
There is a worldwide shortage of matched donors for blood stem cell transfer of leukaemia or lymphoma patients. The generation of blood cells upon the in vitro differentiation of embryonic stem (ES) cells or induced pluripotent stem (iPS) cells could represent a powerful approach to generate the autologous cell populations required for these transplantations.  In this context, it is important to further understand the development of blood cells.

The earliest site of blood cell development in the mouse embryo is the yolk sac where blood islands, consisting of haematopoietic cells surrounded by a layer of angioblasts, develop at approximately day 7.5 of gestation.  The parallel development of these two lineages in close association provided the basis for the hypothesis that they arise from a common precursor, a cell called the haemangioblast. A conflicting theory however, associates the first haematopoietic cells to a phenotypically differentiated endothelial cell with haematopoietic potential, i.e. a haemogenic endothelium.  Support for the haemangioblast concept was initially provided by the identification during embryonic stem (ES) cells differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise after four days of culture to blast colonies with both endothelial, smooth muscle and haematopoietic potential. Recent studies have now provided further evidence for the presence of this tri-potential precursor in vivo.  We have recently established a new model of haematopoietic development based on the in vitro differentiation of embryonic stem cells. We demonstrated that haematopoietic cells are generated from the haemangioblast through the formation of a haemogenic endothelium intermediate. During this process, the haemogenic endothelial cells lose their endothelial identity, altering their flat, adherent appearance into the characteristic round shape of mobile haematopoietic precursor cells. 

The haemangioblast generates blast colonies containing both haematopoietic, endothelial and smooth muscle cells. These vascular smooth muscle cells represent a major component of the cardiovascular system.  During vasculogenesis, newly formed endothelial vessels become rapidly associated with mural cells of the smooth muscle cell lineage. These cells are either referred to as vascular smooth muscle cells if they encircle larger vessels, or as pericytes if they reside within the wall of small vessels such as capillaries and post-capillary venules. These cells regulate blood flow through contraction and have been proposed to control endothelial cell proliferation and differentiation. Although the role of smooth muscle cell in the pathophysiology of cancer is still unclear, evidence suggests that aberrations in pericyte-endothelial cell signalling networks could contribute to tumour angiogenesis and metastasis.


Figure 2: Smooth Muscle cells develop independently from haemogenic endothelium A. Immmuno-staining of smooth muscle cells generated upon ES cell differentiation (red: SMA Smooth muscle actin, green; H2B:Venus).  B. Model of generation of smooth muscle cells by Flk1+ cells and haemangioblast.

To further investigate the development of smooth muscle cells, we generated a mouse reporter ES cell line in which the expression of the fluorescent protein, H2B-VENUS, is driven from the α-SMA (Smooth Muscle Actin) regulatory sequences. We demonstrated that this reporter cell line allows us to efficiently track smooth muscle development during murine ES cell differentiation. The expression of H2B-VENUS was strongly correlated with α-Sma expression during the in vitro differentiation of this reporter ES cell line (Figure 2A). In addition, the enrichment for expression of a panel of smooth muscle markers indicated that H2B-VENUS+ cells accurately represent a smooth muscle cell lineage.  Furthermore, we detected, mostly in the H2B-VENUS sorted cells, transcripts of the long isoform of Smoothelin-B, suggesting that our ES differentiation conditions preferentially generate vascular rather than visceral smooth muscle cells.  Altogether, these results indicate that H2B-VENUS detection allows the direct quantification or isolation of smooth muscle cells.  With this reporter ES cell line, we then confirmed that clonal BL-CFCs generate smooth muscle cells. To determine if the generation of smooth muscle cell is associated with, or independent from, the emergence of the haemogenic endothelium intermediate cell population, we examined the presence of H2B-VENUS+ cells in subpopulations containing this precursor. We indeed observed the presence of some H2B-VENUS+ cells in these haemogenic endothelium populations. However, these cells lacked haematopoietic potential and therefore did not correspond to functional haemogenic endothelium, indicating that smooth muscle cells are largely generated independently from the haemogenic endothelium. Altogether, these findings are consistent with an early separation between haemogenic endothelium and smooth muscle lineages during development (Figure 2B). Whether signalling by smooth muscle contributes to some extent to the generation of blood cells from haemogenic endothelium is still unclear. This study provides new and important insights into haematopoietic and vascular development, which may help drive further progress towards the development of bioengineered vascular grafts, or help discover new opportunities to control tumour angiogenesis and metastasis.