¶ These authors are joint first authors on this work.
The authors have declared that no competing interests exist.
Conceived and designed the experiments: SS AT CC. Performed the experiments: LR SS AT CC. Analyzed the data: LR SS AT CC. Contributed reagents/materials/analysis tools: SS AT CC. Wrote the paper: SS AT CC.
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.
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.
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 was originally identified as a serum response factor (SRF) co-factor expressed only in cardiomyocytes and smooth muscle cells (SMCs)
Other studies have also investigated the role of myocardin in SMC development and whether overexpression of this transcription factor led to SMC lineage commitment. Yoshida and colleagues
Although SMC development has been studied in a wide range of systems such as
We examined whether overexpression of myocardin was able to promote the formation of SMCs in differentiating human embryoid bodies, whether contractile function in response to a vaso-active agonist was affected and whether increased gene expression was confined to the subset of CArG-dependent genes.
Human ESC clumps were induced to undergo serum-induced differentiation in embryoid bodies initially in suspension then plated onto gelatin coated plates as described in the materials and methods. This resulted in a dense outgrowth of differentiating cells (marked by arrowheads in
(A) Low power dark field image of day 28 human embryoid body grown in 20% FBS showing outgrowth of cells (white arrowheads) from the central embryoid body mass. (B and C) Expression of SMC specific genes in embryoid bodies by real time RT-PCR at days 0, 15 and 28 is normalised by three housekeeping genes (GAPDH, UBC, 18S) and then presented relative to undifferentiated human ESCs. RT-PCR data represent means from three independent experiments. Bars represent s.e.m. Cells that stain for SMαA (D) and SMMHC (E) clearly seen at day 28 within embryoid bodies by immunofluorescence. Nuclei counterstained (blue) with DAPI. Bar in A = 1000 µm, bars in D and E = 20 µm.
Immunocytochemistry for SMαA and SMMHC at day 28 revealed the presence of groups of cells that stained for SMC markers within the embryoid body outgrowth (
To investigate whether exogenous myocardin would promote SMC development, differentiating embryoid bodies were transduced with adenoviruses expressing myocardin (Ad-Myo) or a negative control, β-galactosidase (Ad-LacZ) at days 10, 14, 18 and 23, as described in the materials and methods. Since the embryoid body is a multi-layered structure, viral transduction efficiency was optimised using varying concentrations of Ad-LacZ; the optimal dose of virus was chosen as 0.5×107 pfu/ml (
Immunoblotting of embryoid bodies transduced with Ad-LacZ or Ad-Myo confirm myocardin overexpression at a variety of developmental times (A). Immunofluorescent assessment of day 28 embryoid bodies reveals an increase in SMαA, calponin and SMMHC (B) staining following treatment with Ad-Myo from day 10 onwards, in comparison to no treatment or Ad-LacZ negative controls. Photomicrograph exposure times were kept constant across all three treatment groups and deliberately low enough to clearly show the large increases in fluorescent intensity in the Ad-Myo group. Nuclei counterstained (blue) with DAPI. Bar = 100 µm.
Nevertheless, treatment with Ad-Myo resulted in a characteristic change in morphology of some outgrowth regions in the embryoid bodies, such that the cells appeared more spindle shaped or ‘SMC-like’ (
In order to quantify SMC numbers, we carried out flow cytometric analysis of SMαA and SMMHC expression on enzyme dispersed embryoid bodies at day 17 and day 28 (
Embryoid bodies were enzymatically dispersed into single cells at day 17 or day 28 and flow cytometric assessment for SMC markers was carried out. Groups that had been treated with no virus, Ad-LacZ or Ad-Myo from day 10 onwards were used to quantify the proportion of SMαA+ cells (A & B) and SMMHC+ cells (C & D). Both FL1 and FL2 channels were measured for all samples to distinguish specific signal for SMαA (FL1 in A) and SMMHC (FL2 in B) due to the high levels of autofluorescence in embryoid body-derived cells. In the no virus group, SMαA staining was quantified as median SMαA+ signal/median SMαA− signal at both day 17 and day 28 (E). Data presented in A and C are representative flow cytometric plots from a single study with the means from three independent experiments specified in the gated regions and as bar charts ± s.e.m. (B, D & E). **p<0.01.
An interesting observation is that although there was a significant increase in SMαA mRNA during normal embryoid body differentiation (
To examine the specific genes regulated by myocardin in differentiating human embryoid bodies, we carried out quantitative RT-PCR of both CArG-dependent and CArG-independent SMC genes (
Embryoid bodies were treated with no virus, Ad-LacZ or Ad-Myo from day 10 to day 28 and then harvested for RNA. SMC marker expression for a range of CArG-dependent and non-dependent genes was measured using real time RT-PCR and is normalised by three housekeeping genes and then presented relative to no virus controls. RT-PCR data represent means from at least three independent experiments. Error bars represent s.e.m. SMαA and SMMHC expression levels in response to myocardin were significantly higher than the other genes and thus the precise levels are depicted by numbers above the black bars (± s.e.m.).
We also examined expression of markers of different germ layers including nestin & PAX6 (ectoderm), SOX17 & FOXA2 (endoderm), Nkx2–5 & Isl1 (lateral plate mesoderm) and PAX1 & TCF15 (paraxial mesoderm) (
We then considered how incomplete activation of SMC genes would impact upon SMC function. We initially examined Ca2+ concentration in the embryoid body and the response to a vasoactive agonist, carbachol, as a measure of SMC function and an estimate of contractile potential. Day 28 embryoid bodies were dispersed into single cells and loaded with Fluo-4, a Ca2+-sensitive fluorophore. Ca2+ influx in response to carbachol was quantified by flow cytometry (
Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28 and then dispersed into a single cell suspension. Cells were loaded with Fluo-4, a calcium sensitive fluorophore, and intracellular [Ca2+] was measured by flow cytometry before and after the addition of the muscarinic agonist, carbachol in arbitrary units (AU). Representative data from a single experiment (A) and means of three studies (B) show significant increases in fluorescence following addition of carbachol. Error bars represent s.e.m., ns = not significant, * = p<0.05, C = carbachol.
Next, we carried out direct measurements of SMC contractility to assess the effects of myocardin overexpression. Day 28 embryoid bodies were enzymatically dispersed and the cells reseeded in a collagen gel (
Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28, dispersed into a single cell suspension and seeded into collagen gels in 24 well plates (A). Gel contraction in response to carbachol was significantly increased in the Ad-myo group (B). Data points represent the means (±s.e.m.) of three experiments. SMCs derived from human ESCs using a 2-dimensional culture protocol were individually examined for contraction following transduction with Ad-LacZ or Ad-Myo using time lapse microscopy. Percentage of contractile cells increased from 29% with Ad-LacZ to 53% with Ad-Myo (C). Results represent the mean values (±s.e.m.) from 10 randomly chosen optical fields. **p<0.01.
Together, these data make a compelling case that myocardin overexpression increases hESC-derived SMC contractility. Moreover, myocardin is able to promote a phenotype that displays functional properties consistent with a more mature contractile phenotype without activating the entire repertoire of SMC genes.
An intriguing question raised by our studies is whether myocardin expression promotes the SMC phenotype in all developing cells, ie affects lineage commitment or whether myocardin simply promotes SMC maturation and contractility in cells that are already committed to the SMC phenotype. Our flow cytometry data show that even in the Ad-Myo treated group, only a minority of cells ultimately expressed SMC markers whilst a large proportion remained SMαA and SMMHC negative. To determine whether these SMC marker-negative cells were a result of low Ad-Myo transduction efficiency or the inability of myocardin to promote the SMC lineage in all developing cells, we carried out flow cytometry with co-labelling for the FLAG tag on the myocardin transgene and SMMHC (
Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28 then dispersed and fixed for flow cytometry. (A) The subset of cells transduced with the Ad-Myo virus was identified by flow cytometric detection of the 3′ FLAG tag fused to the myocardin transgene. (B) The effect of Ad-Myo treatment on %SMMHC+ cells was analysed by flow cytometry. The majority (90%) of FLAG+ Ad-Myo transduced cells demonstrated a SMC-like phenotype. Data are representative of three independent experiments.
We also investigated whether timing of adenoviral delivery was important and compared induction of SMC marker positive populations by flow cytometry when embryoid body transduction was carried out early (day 10 or days 10 & 14) or late (days 18 & 23) or throughout differentiation (days 10, 14, 18 & 23) (
Embryoid bodies were transduced with Ad-LacZ or Ad-Myo early (day 10 alone or days 10 & 14), late (days 18 & 23) or throughout differentiation (days 10, 14, 18 &13). Late delivery of Ad-Myo reduced number of SMαA+ cells (A) (*p<0.05 by ANOVA and Tukey HSD) but had no significant effect on SMMHC+ cell numbers (B). Values represent mean cell proportions from three independent experiments (± s.e.m.). *p<0.05.
We have demonstrated that myocardin overexpression in human embryoid bodies upregulates the subset of SMC genes that are CArG-dependent and is not able to activate the full SMC developmental program. These results extend the findings of Yoshida and colleagues
The SMCs that develop using the described embryoid body protocol, in the absence of exogenous myocardin, appear to be relatively immature given the low expression of SMMHC and the lack of spontaneous SMC-like contraction by day 28. Other groups have reported higher efficiencies of SMC induction from human ESCs, for example Huang and colleagues
When assessing SMC development and identity, in addition to a range of SMC markers it is essential to measure SMC function. Thus, a particular strength of this study is that the effects of myocardin on hESC-derived SMC contractile function have been evaluated in a variety of different ways. We chose to examine Ca2+ influx secondary to vasoactive drugs as this is usually a prerequisite for SMC contraction and also directly measured contraction in seeded collagen gels. Moreover, we used a novel chemically-defined in vitro system that we have recently described to visualise the contraction of individual SMCs. These multiple lines of evidence strongly suggested that myocardin overexpression promotes a contractile phenotype in human ESC-derived SMCs. This may be of importance in regenerative medicine applications where mature contractile SMCs are required
It is instructive to examine the pattern of gene regulation associated with myocardin overexpression in developing human embryoid bodies. The most striking feature is a dramatic upregulation of CArG-dependent SMC genes, consistent with myocardin’s known mechanism of action through promoting SRF-CArG box interactions
A key question is whether myocardin promotes commitment to the SMC lineage in uncommitted multipotent cells in the embryoid body or whether it acts only to accelerate/promote SMC maturation once precursor cells have already undergone specification. Previous studies have shown that in A404 cells, the expression of at least a low level of myocardin is associated with the ability of these cells to form SMCs efficiently in contrast to the parental P19 line which does not express myocardin and does not form SMC at high frequency
Further analyses on timing of transgene delivery suggest that early transduction is important for SMC lineage determination, with reduced induction of SMαA+ cells with late transduction only. In keeping with this, adenoviral delivery timing did not influence numbers of SMMHC+ cells, perhaps since this marker reflects maturation better than lineage induction (as immature SMCs may be SMαA+ yet SMMHC−). It is recognised that a limitation of the experiments presented is the sub-optimal adenoviral transduction of embryoid bodies, likely due to the multilayered nature of these structures, which may dilute the experimental effects of myocardin. One possibility for further experiments would be to generate an inducible myocardin system with lentiviral delivery prior to embryoid body formation, which would result in transgene integration into the host genome and may lead to more effective transgene expression. For the purposes of this study, we have focussed exclusively on myocardin gain of function. Studies in the mouse using myocardin-null ESCs
In conclusion, these studies demonstrate that myocardin overexpression was able to promote the formation and maturation of SMC-like cells in differentiating human embryoid bodies in a dominant manner despite increased gene expression being predominantly confined to the subset of CArG-dependent genes. These findings have implications for vascular tissue engineering and other applications requiring large numbers of functional SMCs.
Human H9 ESCs were obtained from Wicell (Madison, Wisconsin) and cultured on irradiated mouse embryonic fibroblasts (MEFs) using KSR medium [advanced DMEM/F12 (Life Sciences), 20% knockout serum replacer (Life Sciences), 2 mM L-glutamine and 0.1 mM β-mercaptoethanol] supplemented with 4 ng/ml FGF-2 (R&D Systems) or on gelatin coated plates using chemically defined media (CDM) [IMDM and F12 (1∶1 mix), 5 mg/ml bovine serum albumin (BSA, Sigma), 1% lipid concentrate (Life Sciences), 450 µM monothioglycerol (Sigma), 7 µg/ml insulin (Roche) and 15 µg/ml transferrin (Roche)] supplemented with 10 ng/ml Actinic A (R&D Systems) and 12 ng/ml FGF-2. ESC colonies were passaged by a brief treatment with 1 mg/ml collagenase IV (dissolved in advanced DMEM/F12, 20% KSR and 2 mM L-glutamine) and then mechanically scraped using a 5 ml pipette. ESC clumps were washed with PBS and replated or cultured in suspension in embryoid body medium [DMEM high glucose (Life Sciences), 20% foetal bovine serum, 0.1 mM non-essential amino acids, 1 mM pyruvate and 1% penicillin-streptomycin-glutamine (Life Sciences)] to generate embryoid bodies. As previously described for mouse ESCs
Two-dimensional human ESC differentiation was carried out as described previously
Replication deficient adenoviruses encoding either β-galactosidase (Ad-LacZ) or myocardin (Ad-Myo) were generated using standard methods by the University of Iowa Gene Transfer Vector Core
RNA was extracted using Trizol (Life Sciences) according to the manufacturer’s instructions and cDNA was synthesised using the Superscript III kit (Life Sciences). Quantitative PCR was carried out using the SYBR Green PCR Master Mix (Applied Biosystems) in a Rotor Gene 6000 (Corbett). A full list of primers can be found in
Gene | Forward primer | Reverse primer | Annealing Temp (°C) |
SMαA |
|
|
65 |
SM22α |
|
|
65 |
SMMHC |
|
|
65 |
Myocardin |
|
|
60 |
Smoothelin-B |
|
|
60 |
ACLP |
|
|
60 |
FRNK |
|
|
60 |
cTnT |
|
|
60 |
UBC |
|
|
60 |
GAPDH |
|
|
60 |
18S |
|
|
60 |
Nestin |
|
|
60 |
NKX2.5 |
|
|
60 |
ISL1 |
|
|
60 |
FoxA2 |
|
|
60 |
Sox17 |
|
|
60 |
PAX1 |
|
|
60 |
TCF15 |
|
|
60 |
MYH2 |
|
|
60 |
MYH4 |
|
|
60 |
Embryoid bodies were fixed in 4% paraformaldehyde and then permeabilised with 0.2% triton in PBS. Specimens were blocked with 3% BSA and then the following primary antibodies were used: anti-SMαA (Sigma, A2547) at 1∶1000 and anti-SMMHC (Sigma, M7786) at 1∶2000. Anti-mouse or anti-rabbit secondaries labelled with Alexa Fluor 568 (Life Sciences) were used at 1∶400 and nuclei were counterstained with 0.5 µg/ml 4′,6-diamidino-2-phenylindole (DAPI). For x-gal staining, paraformaldehyde fixed embryoid bodies were incubated overnight at 37°C with x-gal staining solution (1 mg/ml x-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM magnesium chloride in PBS). Images were taken using a Zeiss Axiovert 200 M microscope.
Fixed single cell suspensions were blocked with 3% BSA and then labelled with primary antibodies in BD Perm/Wash buffer (BD Biosciences) as follows: anti-SMαA-FITC (Sigma, F3777) at 1∶1000, anti-SMMHC (Sigma, M7786) at 1∶500 and anti-FLAG-PE (Abcam, ab72469) at 1∶300. Anti-mouse-PE or anti-mouse Alexa Fluor 647 secondaries (Life Sciences, 1∶400) were used for anti-SMMHC detection. Samples were analysed on a Cyan ADP analyzer (Beckman Coulter). Both FL1 and FL2 channels were measured for all samples to distinguish specific signal for SMαA (FL1 in
Proteins were extracted using lysis buffer (10 mM Tris-HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease and phosphatase inhibitor cocktail (Sigma)), and concentrations determined using a BCA Protein Assay Kit (Thermo Scientific). Samples were separated by SDS-PAGE and proteins transferred on to polyvinylidene difluoride membranes, blocked in 5% milk in Tris-buffered saline and 0.05% Tween 20, incubated with primary antibodies against myocardin (Sigma M8948; 1∶1000 dilution) and β-actin (Sigma A2228; 1∶10,000 dilution) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Dako). Signals were detected using ECL Western Blotting Detection Reagents (GE Healthcare Life Sciences).
The collagen gel was prepared by mixing 8 parts of ice-cold collagen Type-1 solution (Sigma Aldrich, UK) with 1 part of 10× PBS. The pH of the mixture was adjusted to 7.2–7.4. The cells were resuspended at a density of 0.6×106 cells/ml of collagen mixture and 500 µl was pipetted into each well of a 24-well plate. Gels were polymerised at 37°C 45–60 min. The contraction assay was initiated by the addition of 50 µM of carbachol. The gel areas were measured at 0, 6 & 18 hours of carbachol stimulation using Image J software.
For assessment of individual cell contraction, SMCs were generated using the two-dimensional directed differentiation protocol. SMCs were transduced with Ad-LacZ or Ad-Myo and examined for contractile responses by time-lapse microscopy 48 h after viral transduction in response to 50 µM carbachol.
Means between two groups were compared using the student’s t-test. Multiple groups were compared using one way ANOVA and differences between groups confirmed by Tukey HSD test.
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The authors acknowledge the excellent technical assistance of Ms Morgan Alexander. We would also like to thank Dr Helle Jorgensen for critical reading of the manuscript.