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

Abstract

Rationale Human embryonic stem cells (hESCs) can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that enrich for cardiac lineages are poorly understood and limit the yield of cardiomyocytes. Objective Our aim was to develop a hESC-based high content screening (HCS) assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology. 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 About 550 known pathway modulators were screened in a HCS 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, demonstrating that Wnt inhibition alone is sufficient for deriving cardiomyocytes from hESC originating mesoderm cells. Transcriptional profiling of inhibitor-treated compared to vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors are very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, confirming Wnt inhibition as the relevant biological activity. Conclusions Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes, yielding novel tools for the benefit of pharmaceutical and clinical applications.

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

“…This model system provides unique opportunities to study the molecular pathways that control cardiac lineage commitment and cell fate specification. In recent years, the ability to efficiently generate cardiomyogenic cells from hPSCs has greatly improved [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] . However, among protocols there is cell line variation with respect to the efficiency in generating cardiomyogenic cells and timing at which the cells express chamber-specific markers (e.g., ventricle and atria).…”

“…In contrast to previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or Bone morphogenetic protein 4(BMP4) and robustly generates cultures highly positive for TNNI3, TNNT2, IRX4, MLC2v, and MLC2a by day 10 cells across all hESC and hiPSC lines tested to date. The strategy is technically simple to implement, especially compared to three-dimensional cultures, mass culture, or embryoid body based strategies [4][5][6][7][8][9] , and was recently defined in a study that describes a small molecule with selective toxicity to hPSCs (Boheler et al) Flow cytometry is a powerful analytical tool for assessing the quality of cells in culture and determining subpopulation homogeneity, and with proper experimental design, can provide quantitative measurements. As with all antibody-based strategies, accurate interpretation of experimental results requires that elements of the assay design including antibody concentration and fixation and permeabilization conditions (when targeting intracellular antigens) are carefully tested for each antibody as sub-optimal conditions significantly affect efficiency of antibody binding, and therefore, interpretation of results.…”

We heart cultured hearts. A comparative review of methodologies for targeted differentiation and maintenance of cardiomyocytes derived from pluripotent and multipotent stem cells

“…Undoubtedly, this was related, on the one hand, to the relatively common occurrence of spontaneous differentiation along the cardiogenic lineage, and, on the other hand, to the characteristic phenotypic feature of rhythmical pulsation that facilitated the acknowledgement of the presence of cardiomyocytes in embryoid bodies and cells grown in monolayers. The protocols for targeted differentiation of embryonic stem cells to cardiomyocyte-like cells by sequential induction (that is, directing the cells to the mesodermal lineage first and then into the cardiogenic pathway) developed over the next decade years [14][15][16][17]. The yield of cardiomyocytes was, however, quite low, especially with embryoid body-based protocols that patently provided no more that several per cent of robustly beating cells.…”

“…The inhibition may be implemented by small-molecule agents such as IWR-1, IWP-2, IWP-3, XAV939, etc., or small hairpin RNAs (shRNAs) targeting the expression of β-catenin; provided that the cells have already reached mesodermal stage. Inhibition of the Wnt-associated signalling in hESC that have been partially differentiated to mesodermal fate results in about 30 % of beating cardiomyocytes [17]. The more recently established protocol of sequential two-phase modulation of the Wntassociated pathways has been reported to increase the yield of cardiomyocytes further [68,102].…”

Potential for pharmacological manipulation of human embryonic stem cells

“…WNT Cardiomyocytes (Hudson et al, 2011) IWR-1 WNT Cardiomyocytes (Ren et al, 2011) IWR-1, IWP-3 WNT Cardiomyocytes (Willems et al, 2011) LY294002 PI3K PI3K/AKT/mTOR Endodermal (Touboul et al, 2010) Myocyte progenitors (Mahmood et al, 2010) hESC-derived hemogenic epithelial cells into HPCs (Wang et al, 2011) Cardiomyocytes (Graichen et al, 2008;Xu et al, 2008), Endothelial cells (James et al, 2010).…”