Small-Molecule Inhibitors of the Wnt Pathway Potently Promote Cardiomyocytes From Human Embryonic Stem Cell–Derived MesodermNovelty and Significance
Rationale: Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes.
Objective: Our aim was to develop a human embryonic stem cell–based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis.
Methods and Results: Approximately 550 known pathway modulators were screened in a high-content screening assay, with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell, which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell–derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors were very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, which confirmed that Wnt inhibition was the relevant biological activity.
Conclusions: Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications.
Heart disease often leads to cardiomyocyte death and pathological remodeling, and the only replacement option is heart transplantation, but its clinical complexity and the limited number of donors have prompted research into stem cell–based alternatives. Stem cell–based approaches include both cell transplantation and mobilization of an endogenous stem and progenitor pool, which show promise for therapeutic regeneration.1 Development of stem cell–based replacement therapies, however, is limited by incomplete understanding of the factors that drive differentiation of cardiomyocytes from either human embryonic stem cells (hESCs) or endogenous cardiac stem and progenitor cells. Therefore, we sought to develop high-throughput screens for simultaneous testing of small molecules for cardiogenic potential in order to identify cellular signals that are required or need to be inhibited at different stages of cardiac development.2 Moreover, such a chemical biology approach would generate small-molecule tools to improve the yield in hESC differentiation protocols and could even result in drug leads for cardiac regeneration should they target hESC-derived progenitors that resemble adult cardiac progenitor cells. Here, we describe the development and implementation of an hESC-based assay that probes the signals that drive the conversion of platelet-derived growth factor receptor-α (PDGFR-α)+–mesoderm posterior 1 homolog (MESP1)+ cardiogenic cells to cardiomyocytes, as well as one of the hits that arose from screening pathway modulator libraries.
MYH6-mCherry hESC were propagated as described previously3 and were differentiated with growth factors as outlined in Figure 1A. For high-content screening (HCS), day 4 embryoid bodies (EBs) were dissociated and plated in the presence of small molecules. For analysis, red fluorescence from the cardiac-specific MYH6 reporter was imaged with a high-throughput microscope and was quantified with an image analysis software package.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
To develop an HCS assay for small molecules that drive mesoderm to differentiate into cardiomyocytes in hESCs, we first profiled cardiac marker expression during differentiation in EBs (Figure 1A, upper half), when cardiac-inducing signals are provided to mesodermal cells by closely juxtaposed endodermal and potentially other cell types present in early embryos4 (Online Figure I). Mesoderm was induced in EBs by addition of activin A and bone morphogenetic protein 4, which resulted in mesoderm induction between days 1 and 4 (Figure 1A, blue full line), and cardiomyocytes appeared spontaneously from day 6 onward (Figure 1A, red dashed line). Because mesoderm induction appeared to be maximal at day 4, we further characterized the day 4 population for the early cardiac markers MESP1 and PDGFR-α (Figure 1B).5 Because this population appeared to be highly enriched for both markers, we developed an HCS assay that would probe these cells and determine what signals would drive differentiation toward cardiomyocytes (Figure 1A, lower half). HCS assays are image-based and have better dynamic range and sensitivity than traditional plate-reader–based assays. Day 4 EBs were collected and dispersed into 384-well plates and subsequently exposed to 3 concentrations (0.3, 1, and 3 μmol/L) of 2 small collections of well-characterized pathway modulators, namely, approximately 244 protein kinase inhibitors (InhibitorSelect; EMD Millipore, Billerica, MA) and 305 pathway agonists and antagonists (StemSelect; EMD Chemicals). In the InhibitorSelect collection, no compound induced cardiogenesis (Figure 1C), whereas in the StemSelect collection, 1 extremely potent hit was identified (Figure 1D). The hit IWR-1, a recently published inhibitor of the canonical Wnt signaling response (IWR),6 induced beating foci from monolayer cultures, whereas none were observed in dimethyl sulfoxide conditions (Figure 1E; Online Movies I and II). Inhibitors of endothelial growth factor, vascular epidermal growth factor, transforming growth factor-β, insulin, and Sonic hedgehog signaling did not result in increased cardiogenesis, which suggests that Wnt inhibition alone is sufficient for cardiac induction (Online Table I). Moreover, agonists of the Wnt, vascular endothelial growth factor, and Sonic hedgehog pathways failed to promote cardiac fate (Online Table I).
Further characterization revealed that maximal cardiac induction by IWR occurs when IWR is added from day 4 to day 5, with cardiac induction levels decreasing as the treatment window is shifted up toward day 10 of differentiation (Figure 2A). The EC50 to induce cardiogenesis at the day 4 time window was 2241 nmol/L (Figure 2B). Flow cytometry analysis demonstrated that IWR yielded up to 30% cardiomyocytes, a 200-fold increase over vehicle-treated cultures (Figure 2C). Reverse transcription quantitative polymerase chain reaction analysis was performed to study the effect of IWR on mesoderm, cardiac progenitors, and mesoderm derivatives such as cardiomyocytes, endothelial cells, and smooth muscle (Figure 2D). The mesoderm-specific gene T/BRACHYURY had a downward tendency as soon as 24 hours after IWR exposure, although this was not statistically significant on most days (Figure 2D). The earliest markers of cardiac fate, MESP1 and kinase insert domain receptor, were not affected in the first few days after IWR treatment, which suggests that IWR drives MESP1+, kinase insert domain receptor+ cardiac mesoderm cells toward cardiomyocytes (Figure 2D). MEF2C and NKX2.5, 2 cardiac transcription factors, as well as the cardiomyocyte structural genes MYH6, ACTN2, and TNNT2, were increased dramatically by IWR (Figure 2D). Further characterization of other mesodermal lineages revealed smooth muscle lineages were not increased (ACTA2 and TGLN) and endothelial cell markers (CD31 and kinase insert domain receptor) even decreased. Flow cytometry confirmed a >50% reduction in CD31+ cells (Online Figure II).
We next questioned whether other inhibitors of the Wnt pathway were also able to induce cardiogenesis in this assay (Figure 3A). We tested 3 more small-molecule inhibitors of Wnt signaling: 1 the PORCN inhibitor IWP-3 (IWP)6; 2 the more potent IWR-1 analog 53AH; and 3 the tankyrase inhibitor XAV939 (XAV), which is cardiogenic in mouse embryonic stem cells.7 All 3 compounds promoted cardiogenesis, with 53AH having an EC50 below the micromolar range (Figure 3B). Because EC50 measurements are not indicative of the efficacy of each compound, we ran maximal effect doses of each compound, as well as the biological Wnt inhibitor DKK1 (Dickkopf-related protein 1), per comparison to evaluate their maximum efficacy in terms of cardiogenesis. IWR and its analog 53AH were the most efficacious, followed by IWP and XAV, and any of the small-molecule inhibitors were at least 40 times more effective in inducing cardiogenesis than DKK1, a purified recombinant Wnt inhibitor protein (Figure 3C).
Definition of the pathways that control stem cell cardiogenesis is important to efficiently derive cardiomyocytes from human pluripotent stem cells and might be useful to mobilize endogenous cardiac stem cells. To discover new molecules and pathways that would direct cardiac differentiation and/or regeneration, we developed an hESC-based HCS assay that allows small-molecule screens in serum-free conditions, on a scale that has not been reported for any hESC-based assay. We prepared cardiogenic mesoderm (platelet-derived growth factor receptorα+MESP1+) cells in bulk EB culture (Figure 1B)5 and dispersed and plated the cells in a monolayer with the intent of identifying small molecules that are able to replace the natural signals provided in the 3-dimensional EB, thus yielding insight into signals that direct mesodermal progenitors to form cardiomyocytes.
By screening approximately 550 pathway modulators, we identified a small-molecule inhibitor of the β-catenin–dependent canonical Wnt pathway. Although it was not entirely unexpected that a Wnt inhibitor would be identified as a cardiac inducer, given that the natural Wnt inhibitor DKK1 is capable of directing cardiogenesis in Xenopus(8 and hESCs,5 the present study showed that Wnt inhibition was sufficient to drive the hESC-derived mesoderm to a cardiac fate in the absence of other signaling modulators, and no other inhibitors had comparable activity (Online Table I). Moreover, Wnt inhibition drove cardiomyocytes specifically and did not increase other mesoderm lineages, which suggests that Wnt inhibition specifically drives a mesoderm progenitor toward cardiomyocytes (Figures 2D and 2E).
To further explore inhibition throughout the Wnt pathway, we evaluated structurally diverse Wnt inhibitors that target different cellular components of the pathway (Figure 3D). Importantly, all of the small molecules were much more cardiogenic than DKK1 (Figure 3C). The most interesting finding, however, was that IWP, an inhibitor that prevents cells from producing Wnt, was also potent, which revealed that endogenous Wnt activity must be blocked. This is an important finding, because it suggests not only that exogenously added factors are important for efficient differentiation but also that endogenous cellular signals may have to be modulated to control differentiation. Because these cultures are typically heterogeneous, further experimentation may unravel which cell type is, in this case, secreting inhibitory Wnts.
In summary, HCS of an hESC-based assay identified Wnt inhibition as critical for cardiogenesis. Active compounds function by blocking Wnt secretion and stabilize Axin to destabilize β-catenin. The present data further suggest that endogenous Wnt signals need to be inhibited to direct the formation of cardiomyocytes from a mesoderm progenitor.
Sources of Funding
E.W. is supported by California Institute for Regenerative Medicine (CIRM) training grant T2-00004 and an American Heart Association postdoctoral grant, M.M. by CIRM RC00132 and NIH HL059502, and J.C. by CIRM RS1-00169 and the T Foundation.
E.W. and M.L. are employees of ChemRegen Inc. M.M. and J.C. are officers of ChemRegen Inc. M.M. is on the advisory board of Vala Sciences Inc. The remaining authors report no conflicts.
The authors would like to thank Dennis Schade for functional confirmation of hits in a Wnt assay and critical reading of the manuscript.
In May 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.
Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, BURCs present seminal findings that have the potential to open up new avenues of research. A decision on BURCs is rendered within 7 days of submission.
Non-standard Abbreviations and Acronyms
- structural analog of IWR-1
- embryoid body
- high-content screen
- human embryonic stem cell
- small-molecule collection of 244 kinase inhibitors
- inhibitor of Wnt response, IWR-1
- inhibitor of Wnt production, IWP-3
- small-molecule collection of 305 pathway modulators
- inhibitor of tankyrase, XAV939
- Received May 27, 2011.
- Revision received June 22, 2011.
- Accepted June 24, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Human embryonic stem cells (hESCs) show great promise as a source for generating myocardial cells for use in cell transplantation therapies.
hESCs form cardiac myocytes if they are treated appropriately; however, the yield is typically low, because the mechanisms that drive hESC toward a cardiac myocyte phenotype are poorly understood.
What New Information Does This Article Contribute?
We developed an hESC-based high-throughput small-molecule screen assay using a cardiac myocyte–specific fluorescent reporter to identify molecules that drive hESCs to cardiac myocytes.
Small-molecule inhibitors of the Wnt signaling pathways were identified as potent inducers of cardiac myocytes. They increased cardiac myocyte yield dramatically over recombinant protein inhibitors. Many other pathway modulators were inactive. Thus, Wnt inhibition is uniquely important for directed cardiogenesis.
The development of a high-throughput assay for hESC cardiogenesis was deemed essential because it allowed simultaneous probing of the many signals that may drive hESCs to cardiac myocytes. Screening of more than 500 pathway modulators demonstrated that inhibition of the Wnt pathway alone was sufficient to drive cardiac cell formation specifically and did not induce other mesodermal derivatives, such as endothelial or smooth muscle cells. Small-molecule inhibitors of the Wnt pathway are thus useful tools for increasing cardiac myocyte yield. These molecules have great potential benefits for clinical and pharmaceutical applications.