Abstract 17837: Fibronectin is Sufficient for Robust Matrix Sandwich-based Cardiac Differentiation of Human Pluripotent Stem Cells in Fully Defined Conditions
Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research including disease modeling and hold promise for clinical applications. To enable this technology, efficient protocols for cardiac differentiation that are broadly applicable across cell lines are essential. Based on the role of extracellular matrix (ECM) in cardiac development, we recently described a matrix sandwich protocol using GFR-Matrigel for efficient cardiogenesis in combination with growth factors Activin A, BMP4, and bFGF (Figure 1). However, Matrigel is a mixture of ECM proteins and is not fully defined. The purpose of the present study was to determine if purified individual ECM proteins can effectively support cardiogenesis in this protocol. Human PSCs were cultured as monolayers on Matrigel in mTeSR1 medium and subsequently overlayed with Matrigel or purified ECM proteins (Figure 1). Cardiogenesis efficiency was measured by flow cytometry for cTnT-labeled CMs. ECM overlays using laminin (5 μg/cm2) or collagenIV (5 μg/cm2) resulted in 42.4±16.0% CMs and 49.1±17.6% CMs, respectively, which were significantly less than the Matrigel (8.7 μg/cm2) overlay (77.4±8.4%, p < 0.05). In contrast, fibronectin (0.3 μg/cm2) overlay potently resulted efficient cardiogenesis with 80.7±4.6% CMs. To test differentiation in fully defined conditions, human PSCs were grown on a synthetic culture substrate, Synthemax (Corning), instead of Matrigel, followed by differentiation using the matrix sandwich protocol. The Synthemax surface combined with the fibronectin overlay resulted in a high purity of CMs (98%). These results showed that fibronectin is sufficient to promote highly efficient cardiogenesis of human PSCs in fully defined conditions which will enable robust and reproducible production of CMs. Figure 1. Schematic of the matrix sandwich protocol
- © 2012 by American Heart Association, Inc.