Small molecule Wnt inhibitors enhance the efficiency of BMP-4-directed cardiac differentiation of human pluripotent stem cells

Abstract

Human induced pluripotent stem (iPS) cells potentially provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms and treatments. However, existing approaches to cardiomyocyte production from human iPS cells are inefficient, limiting the application of iPS cells in basic and translational cardiac research. Furthermore, strategies to accurately record changes in iPS cell-derived cardiomyocyte action potential duration (APD) are needed to monitor APD-related cardiac disease and for rapid drug screening. We examined whether modulation of the bone morphogenetic protein 4 (BMP-4) and Wnt/β-catenin signaling pathways could induce efficient cardiac differentiation of human iPS cells. We found that early treatment of human iPS cells with BMP-4 followed by late treatment with small molecule Wnt inhibitors led to a marked increase in production of cardiomyocytes compared to existing differentiation strategies. Using immunocytochemical staining and real-time intracellular calcium imaging, we showed that these induced cardiomyocytes expressed typical sarcomeric markers, exhibited normal rhythmic Ca 2+ transients, and responded to both β-adrenergic and electric stimulation. Furthermore, human iPS cell-derived cardiomyocytes demonstrated characteristic changes in action potential duration in response to cardioactive drugs procainamide and verapamil using voltage-sensitive dye-based optical recording. Thus, modulation of the BMP-4 and Wnt signaling pathways in human iPS cells leads to highly efficient production of cardiomyocytes with typical electrophysiological function and pharmacologic responsiveness. The use of human iPS cell-derived cardiomyocytes and the application of calcium-and voltage-sensitive dyes for the direct, rapid measurement of iPS cell-derived cardiomyocyte activity promise to offer attractive platforms for studying cardiac disease mechanisms and therapeutics.

Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

“…Our results and prior studies (21)(22)(23)(24) indicate that early induction of canonical Wnt signaling and suppression of canonical Wnt signaling at later stages of differentiation synergistically enhance yield of cardiomyocytes with other growth factors and/or serum. We next sought to determine whether modulating regulatory elements of Wnt signaling alone, in the absence of serum and exogenous growth factors, was sufficient to induce cardiogenesis.…”

Disruption of Wnt/β-Catenin Signaling and Telomeric Shortening Are Inextricable Consequences of Tankyrase Inhibition in Human Cells

“…Tnks and Porcn inhibitors, including IWR-1 and IWP-2, are now widely used for the in vitro engineering of various tissues, including cardiomyocytes, retinal pigmented epithelial cells, pneumocytes, and dopaminergic neurons (40)(41)(42)(43)(44)(45)(46)(47). Given the transient nature of the chemical exposure in these protocols (typically 2 to 3 days) and similar efficacies of Tnks and Porcn inhibitors for directing cell fate in a direct comparison (46), we assume the chemical induction of these cell types is due to suppression of Wnt/␤-catenin transcriptional responses and not telomeric stress or shortening.…”

Potential for pharmacological manipulation of human embryonic stem cells

“…IWP-4 and IWR-1 act by inhibiting the palmitylation of WNT proteins by porcupine (PORCN), a membrane-bound O-acyltransferase, thereby blocking WNT secretion and activity (Chen et al, 2009a). An additional study demonstrated that following BMP4-treatment of hESCs and hiPSCs, IWR-1 significantly improved cardiomyocyte differentiation resulting in cells with typical electrophysiological functions and pharmacological responsiveness (Ren et al, 2011). An interesting recent study demonstrated that successive, mutually exclusive waves of non-canonical and canonical WNT signalling precede mesoderm differentiation, and blocking these two waves leads to differential differentiation (Rai et al, 2011).…”

“…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).…”