Myocardin overexpression is sufficient for promoting the development of a mature smooth muscle cell-like phenotype from human embryonic stem cells

Background

Myocardin is thought to have a key role in smooth muscle cell (SMC) development by acting on CArG-dependent genes. However, it is unclear whether myocardin-induced SMC maturation and increases in agonist-induced calcium signalling are also associated with increases in the expression of non-CArG-dependent SMC-specific genes. Moreover, it is unknown whether myocardin promotes SMC development from human embryonic stem cells.

Methodology/Principal

Findings The effects of adenoviral-mediated myocardin overexpression on SMC development in human ESC-derived embryoid bodies were investigated using immunofluorescence, flow cytometry and real time RT-PCR. Myocardin overexpression from day 10 to day 28 of embryoid body differentiation increased the number of smooth muscle α-actin⁺ and smooth muscle myosin heavy chain⁺ SMC-like cells and increased carbachol-induced contractile function. However, myocardin was found to selectively regulate only CArG-dependent SMC-specific genes. Nevertheless, myocardin expression appeared to be sufficient to specify the SMC lineage.

Conclusions/Significance

Myocardin increases the development and maturation of SMC-like cells from human embryonic stem cells despite not activating the full repertoire of SMC genes. These findings have implications for vascular tissue engineering and other applications requiring large numbers of functional SMCs.     

Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.     

Angiotensin II promotes differentiation of mouse embryonic stem cells to smooth muscle cells through PI3-kinase signaling pathway and NF-κB

Embryonic stem cells (ES cells), the pluripotent derivatives of the inner cell mass from blastocysts, have the capacity for unlimited growth, self-renewal and differentiation toward all types of somatic cells. Angiotensin II (Ang II), the most important effector peptide of the renin–angiotensin system, is also an angiogenesis factor. However, the potential impact of Ang II on ES cell differentiation is still unknown. In the present study, we have successfully induced the differentiation of ES cells into smooth muscle cells (SMCs) on collagen IV. Interestingly, incubation of ES cells with Ang II further promoted SMC differentiation from ES cells, which was abolished by prior treatment with Ang II type 1 (AT1) receptor antagonist losartan, but not Ang II type 2 (AT2) receptor antagonist PD123319. Moreover, we found that, in parallel with SMC specific-marker induction, the expression levels of phosphoAkt and NF-Kappa B (NF-κB) p50 were up-regulated by Ang II. Importantly, addition of phosphoinositide-3 kinase (PI3K) inhibitor LY294002 led to a marked inhibition of Ang II induced SMC specific markers, phosphoAkt and NF-κB p50 expression. Furthermore, NF-κB inhibitor BAY11-7082 can inhibit Ang II induced expression of SMC specific markers. Thus, we demonstrate for the first time that Ang II plays a promotive role in the stage of ES cell differentiation to SMCs through AT1 receptor. We further confirmed that PI3K/Akt signaling pathway and NF-κB play key roles in this process.     

Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α–actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10⁺, Sox17⁺) and a glia marker (S100β⁺). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-β1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.     

Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, including vascular smooth muscle cells (SMCs), and have tremendous potential as a cell source for cardiovascular regeneration. We postulate that specific vascular environmental factors will promote MSC differentiation into SMCs. However, the effects of the vascular mechanical environment on MSCs have not been characterized. Here we show that mechanical strain regulated the expression of SMC markers in MSCs. Cyclic equiaxial strain downregulated SM alpha-actin and SM-22alpha in MSCs on collagen- or elastin-coated membranes after 1 day, and decreased alpha-actin in stress fibers. In contrast, cyclic uniaxial strain transiently increased the expression of SM alpha-actin and SM-22alpha after 1 day, which subsequently returned to basal levels after the cells aligned in the direction perpendicular to the strain direction. In addition, uniaxial but not equiaxial strain induced a transient increase of collagen I expression. DNA microarray experiments showed that uniaxial strain increased SMC markers and regulated the expression of matrix molecules without significantly changing the expression of the differentiation markers (e.g., alkaline phosphatase and collagen II) of other cell types. Our results suggest that uniaxial strain, which better mimics the type of mechanical strain experienced by SMCs, may promote MSC differentiation into SMCs if cell orientation can be controlled. This study demonstrates the differential effects of equiaxial and uniaxial strain, advances our understanding of the mechanical regulation of stem cells, and provides a rational basis for engineering MSCs for vascular tissue engineering and regeneration.