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, 109 (4), 360-4

Small-molecule Inhibitors of the Wnt Pathway Potently Promote Cardiomyocytes From Human Embryonic Stem Cell-Derived Mesoderm


Small-molecule Inhibitors of the Wnt Pathway Potently Promote Cardiomyocytes From Human Embryonic Stem Cell-Derived Mesoderm

Erik Willems et al. Circ Res.


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.


Figure 1
Figure 1. A high content screen for cardiogenesis in hESC identifies a Wnt inhibitor
(A) In EB, after treatment with the indicated growth factors, mesoderm is induced efficiently from days 1-4 (assessed by mRNA, upper half, blue full line) and cardiomyocytes occur spontaneously from day 6 onward (assessed by mRNA, upper half, red dashed line). For the HCS assay, day 4 EBs were dissociated, plated, and treated with molecules and/or growth factors as depicted in the lower half. (B) Day 4 cells have reached maximum mesoderm levels and were further characterized for early cardiac markers by RT-qPCR for MESP1 and for PDGFRα by flow cytometry (average ± standard deviation, n = 3). (C,D) The HCS assay was screened against a collection of 244 kinase inhibitors (InhibitorSelect) (C) and a collection of 305 pathway modulators (StemSelect) (D) in three concentrations (0.3, 1 and 3μM). All compounds with a Z score larger than 2 are shown for the StemSelect library (D). (E) Fluorescence image of cells expressing the MYH6-mCherry reporter under vehicle or IWR treated cultures.
Figure 2
Figure 2. IWR specifically induces cardiomyocytes
(A) Dose response curves for cardiogenesis by IWR with exposure at different time points show IWR is most active when added at day 4. (B) In the optimal window, IWR has an EC50 for cardiogenesis of about 2241 nM. (C) Flow cytometry analysis for the MYH6-mCherry reporter of vehicle or IWR treated cells, gated against a negative control. Numbers reflect the percentage of MYH6 positive cells. (D) Time course RT-qPCR analysis of IWR treated monolayer samples (Red, squares) versus DMSO treated samples (blue, diamonds) normalized to the DMSO day 5 sample. Early markers for cardiogenic mesoderm include T (BRACHYURY), MESP1 and KDR, cardiac progenitor markers tested were MEF2C and NKX2.5, and cardiomyocyte markers studied were MYH6, TNNT2 and ACTN2. Other mesoderm lineages tested were endothelium (CD31) and smooth muscle (ACTA2 and TAGLN). Error bars represent SEM of 3 biological replicates. Asterisk (*) indicates p<0.05 compared to the corresponding DMSO control. (E) Schematic overview of the lineage diversification of a cardiovascular mesoderm progenitor. Wnt inhibition specifically drives mesoderm progenitors to cardiomyocytes.
Figure 3
Figure 3. Inhibition of Wnt at different levels of the pathway promotes cardiogenesis
(A) Chemical structures of the Wnt inhibitors tested in the cardiogenesis assay. (B) Dose response curves and EC50 values for cardiogenesis. (C) Maximum cardiac induction concentrations of Wnt inhibitors tested to indicate efficacy of each molecule, the natural inhibitor DKK1 is included to demonstrate superior efficacy by small molecules. Asterisk (*) indicates p<0.05 relative to the untreated condition. (D) Schematic overview of the canonical Wnt pathway, and active Wnt inhibitors (green). Green lines indicate general function of the inhibitors on their targets.

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