Insulin Inhibits Cardiac Mesoderm, Not Mesendoderm, Formation During Cardiac Differentiation of Human Pluripotent Stem Cells and Modulation of Canonical Wnt Signaling Can Rescue This Inhibition

Abstract

The study of the regulatory signaling hierarchies of human heart development is limited by a lack of model systems that can reproduce the precise developmental events that occur during human embryogenesis. The advent of human pluripotent stem cell (hPSC) technology and robust cardiac differentiation methods affords a unique opportunity to monitor the full course of cardiac induction in vitro. Here we show that stage-specific activation of insulin signaling strongly inhibited cardiac differentiation during a monolayer-based differentiation protocol that used TGFβ superfamily ligands to generate cardiomyocytes. However, insulin did not repress cardiomyocyte differentiation in a defined protocol that employed small molecule regulators of canonical Wnt signaling. By examining the context of insulin inhibition of cardiomyocyte differentiation, we determined that the inhibitory effects by insulin required Wnt/β-catenin signaling and that the cardiomyocyte differentiation defect resulting from insulin exposure was rescued by inhibition of Wnt/β-catenin during the cardiac mesoderm (Nkx2.5+) stage. Thus, insulin and Wnt/β-catenin signaling pathways, as a network, coordinate to influence hPSC differentiation to cardiomyocytes, with the Wnt/β-catenin pathway dominant to the insulin pathway. Our study contributes to the understanding of the regulatory hierarchies of human cardiomyocyte differentiation and has implications for modeling human heart development.

MSX2 mediates entry of human pluripotent stem cells into mesendoderm by simultaneously suppressing SOX2 and activating NODAL signaling

“…The Wnt/β-catenin and BMP signaling pathways are critical for hPSC differentiation into the early embryonic lineages. Activation of Wnt signaling results in a loss of pluripotency and drives hPSC differentiation towards endoderm and mesoderm [12][13][14], and activation of BMP signaling leads to mesendoderm or trophoectoderm differentiation, depending on the dose and duration of stimulation [15,16]. Interestingly, Nodal/Activin signaling cooperates with bFGF to maintain pluripotency [17,18], but promotes mesendoderm differentiation of hESCs in the absence of bFGF [19][20][21].…”

Isolation of Contractile Cardiomyocytes from Human Pluripotent Stem-Cell-Derived Cardiomyogenic Cultures Using a HumanNCX1-EGFPReporter

“…Following that samples were blocked/ permeabilized with 10% goat serum and 0.5% Triton-X100 in PBS for 30 min at room temperature. Samples were incubated with primary antibodies against myosin heavy chain/MYH (clone [3,42]; Abcam), CTNT (RV-C2, DSHB), or a-actinin (A7811; Sigma-Aldrich) for 30 min at 4°C. Secondary antibody labeling was performed at room temperature for 30 min.…”

“…We next subjected the H9, HES3, C32, and C11 NCX1cp-EGFP-transgenic hESC/iPSC lines to two different established cardiomyocyte differentiation protocols, using methods based on BMP/activin [4] and small-molecule modulators of Wnt signaling [3,41]. Initial NCX1cp-EGFP expression was observed from day 7 of differentiation.…”

“…H uman cardiomyocytes can be efficiently generated from pluripotent stem cells in vitro through temporal exposure of either monolayer cultures or embryoid bodies to combinations of growth/differentiation factors or small molecules and tailoring of the extracellular matrix and culture medium composition [1][2][3][4][5][6]. Generation of cardiomyocytes from human pluripotent stem cells (hPSCs) represents a reliable source of cardiac cells for modeling of heart disease [7][8][9][10][11][12], identification of molecular pathways involved in cardiac cell type specification [13,14], drug screening in vitro and in vivo [15][16][17][18], and, perhaps, even for regenerative medicine applications [19][20][21][22][23].…”

“…Despite the development of increasingly efficient differentiation protocols, the final population inevitably contains a mixture of cardiomyocytes, smooth muscle cells, stromal cells, and endothelial cells [1,3,5,6,13,[24][25][26][27]. Isolation of pure functional cardiomyocytes and their committed precursors from heterogeneous populations is therefore of significant interest for further in vitro or in vivo applications and cardiac regeneration [28][29][30].…”