Auto/paracrine factors and early Wnt inhibition promote cardiomyocyte differentiation from human induced pluripotent stem cells at initial low cell density

Abstract

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) have received increasing attention for their clinical use. Many protocols induce cardiomyocytes at an initial high cell density (confluence) to utilize cell density effects as hidden factors for cardiomyocyte differentiation. Previously, we established a protocol to induce hiPSC differentiation into cardiomyocytes using a defined culture medium and an initial low cell density (1% confluence) to minimize the hidden factors. Here, we investigated the key factors promoting cardiomyocyte differentiation at an initial low cell density to clarify the effects of cell density. Co-culture of hiPSCs at an initial low cell density with those at an initial high cell density showed that signals secreted from cells (auto/paracrine factors) and not cell–cell contact signals, played an important role in cardiomyocyte differentiation. Moreover, although cultures with initial low cell density showed higher expression of anti-cardiac mesoderm genes, earlier treatment with a Wnt production inhibitor efficiently suppressed the anti-cardiac mesoderm gene expression and promoted cardiomyocyte differentiation by up to 80% at an initial low cell density. These results suggest that the main effect of cell density on cardiomyocyte differentiation is inhibition of Wnt signaling at the early stage of induction, through auto/paracrine factors.

Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo

“…Several studies reported that Wnt and Smad signaling pathways play an important role in the fate determination of stem cells and their potential for cardiovascular differentiation. Several studies reported that early application of Wnt/β-catenin signaling pathway inhibitor, IWR1 promotes cardiomyocyte differentiation[ 12 ]. Similarly, IWP-4 is a potent small molecule that has been reported to differentiate embryonic stem cells into cardiomyocyte like cells[ 7 ].…”

Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells

“…CD49f is a marker of stem/progenitor cell populations [ 37 ], including cardiac stem cells, and was expressed here only in PRRX1 low Day 4 cells. High cell density inhibits WNT signaling to promote cardiomyocyte differentiation from hiPSCs [ 38 ] and the expression level of PRRX1 in cardiac mesoderm is lower than that of forelimb mesoderm [ 18 , 39 ], indicating that high cell density may have promoted the cardiac mesoderm-directed differentiation of LPM cells.…”

Electric‐Field‐Driven Printed 3D Highly Ordered Microstructure with Cell Feature Size Promotes the Maturation of Engineered Cardiac Tissues

“…This allows us to replace dysfunctional cardiomyocytes and build cardiovascular disease models for drug screening and also cardiotoxicity testing. [6][7][8] However, the predictive powers of these models are limited by the immature state of the ECTs. Many studies have been conducted to improve the maturity of hiPSC-CMs and ECTs to overcome this limitation.…”