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.
Highlights
Heart diseases including coronary artery disease and heart attack, result in cardiomyocyte death and damage to heart function[1]
To examine the simultaneous influence of auto/paracrine factors and the cell–cell contact signals, condition 4 was designed to contain the same number of cells as the sum of the low- and high-density cells; the cells were cultured on the lower layer
ELISA data showed that the high-density cells secreted DKK1, DKK4 and Cerberus during the early stages of differentiation; this secretion commenced on day 1 and peaked at day 3 to 5 after induction (Supplementary Fig. S4). These results suggest that a high cell density exhibits a sufficient Wnt signal inhibition effect, which might be induced through auto/paracrine factors such as the secretion of DKK1, DKK4 and Cerberus to suppress anti-cardiac mesoderm genes and facilitate cardiomyocyte differentiation
Summary
Heart diseases including coronary artery disease and heart attack, result in cardiomyocyte death and damage to heart function[1]. The cell density effects might be Wnt-modulating factors secreted by the cells themselves (auto/paracrine factors). We tested our hypothesis that auto/paracrine factors and Wnt-modulating signals are the primary effects of high cell density during cardiomyocyte differentiation from hiPSCs. To clarify the effects of cell. Density, we have previously established a method to induce cardiomyocytes from hiPSCs at an initial low cell density (1% confluence)[14]. We hypothesized that a low cell density would lack certain essential factors required for cardiac differentiation, as compared to high cell densities. We examined whether employing a co-culture with high-density cells could improve the differentiation efficiency in low-density cultures. This study enables an understanding of the key role of seeding density and the importance of time in modulating Wnt signaling for determining the cardiac differentiation efficiency
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