Abstract 15972: Fibronectin is Essential for Mesendoderm Induction During Cardiac Differentiation of Human Pluripotent Stem Cells

Abstract

Extracellular matrix (ECM) plays important roles in development, and we have previously demonstrated a matrix sandwich protocol for efficient cardiac differentiation of human pluripotent stem cells (hPSCs). The purpose of the present study was to identify the key ECM proteins required to initiate cardiogenic differentiation of hPSCs and define their mechanism of action. We tested purified ECM proteins including human laminin (LN111, LN521), fibronectin (FN) and collagen IV in the matrix sandwich protocol, and found FN promoted efficient cardiac differentiation. To determine if FN is essential for the early stages of cardiogenesis we tested three FN blockers: FUD peptide that binds the N-terminal 70-kDa fragment of FN, anti-human FN antibody clone10 which recognizes FN domain III2, and anti-human FN antibody clone 3E3 that binds on the cell attachment domain. All three blockers showed concentration-dependent inhibition of cardiac differentiation of hPSCs when added at day 0 for 24 hours, measured by flow cytometry for cTnT+ cells in the Matrigel/Matrigel (bottom/overlay) and FN/FN sandwich culture as well as in the small molecule GiWi protocol. Furthermore, clone10 showed significant inhibition of cardiac differentiation even in the absence of adding exogenous FN due to the robust endogenous expression of FN by the hPSCs. Gene expression analysis with qRT-PCR demonstrated that blocking FN by clone10 caused a significant suppression of gene expression related to: 1) epithelial to mesenchymal transition, e.g. SNAIL1, SNAIL2, GSC, VIM, CDH2 and FN1; 2) Mesendoderm e.g. T, MIXL1, SOX17 and precardiac mesoderm of MESP1; 3) Cardiac transcription factors ISL1 and NKX2.5. To investigate the cellular signaling activated by FN, we tested a blocking antibody (clone P5D2) to the major FN receptor, integrin β1, and found that P5D2 produced a concentration-dependent inhibition of cardiogenesis in multiple hPSC lines. Furthermore, the integrin-linked kinase (ILK) inhibitor, Cpd22, inhibited cardiac differentiation in a concentration-dependent fashion as well when added on day 0. We conclude that FN, acting via integrin β1-activated ILK signaling, is essential to mesendoderm formation enabling cardiogenesis of hPSCs.