Harnessing developmental cues for cardiomyocyte production

Abstract

Developmental research has attempted to untangle the exact signals that control heart growth and size, with knockout studies in mice identifying pivotal roles for Wnt and Hippo signaling during embryonic and fetal heart growth. Despite this improved understanding, no clinically relevant therapies are yet available to compensate for the loss of functional adult myocardium and the absence of mature cardiomyocyte renewal that underlies cardiomyopathies of multiple origins. It remains of great interest to understand which mechanisms are responsible for the decline in proliferation in adult hearts and to elucidate new strategies for the stimulation of cardiac regeneration. Multiple signaling pathways have been identified that regulate the proliferation of cardiomyocytes in the embryonic heart and appear to be upregulated in postnatal injured hearts. In this Review, we highlight the interaction of signaling pathways in heart development and discuss how this knowledge has been translated into current technologies for cardiomyocyte production.

Keywords: Cardiomyocyte production; Cardiomyocyte proliferation; Cardiomyocyte self-renewal; Embryonic growth pathways; Fetal gene program; Heart regeneration; Hippo signaling; Wnt signaling; hiPSC-CM.

Fig. 1.

There is interplay between Wnt, Hippo and insulin signaling during cardiomyocyte proliferation. Activation of the Hippo, canonical Wnt or IGF1/PI3K/AKT signaling pathways causes YAP/TAZ and β-catenin to enter the nucleus and cluster with their DNA-binding partners TEAD and TCF/LEF, which activates transcription of target genes and induces cell cycle activation. Conversely, when Dsh is inactivated due to lack of Wnt proteins, activated GSK-3β can phosphorylate β-catenin or YAP/TAZ, ultimately resulting in their degradation by the proteasome. Hippo signaling induced by, for example, N-cadherin junction-mediated cell-cell contact leads to the degradation of the YAP/TAZ complex through the MST1/2-SAV1-LATS1/2-MOB1-YAP/TAZ cascade. IGF1/PI3K/AKT signaling can facilitate the entry of β-catenin into the nucleus via AKT kinase, which phosphorylates GSK-3β to inhibit its activation. AKT, RACα serine/threonine-protein kinase; APC, adenomatous polyposis coli protein; CK1α, casein kinase 1α; Dsh, disheveled; GSK-3β, glycogen synthase kinase-3; IGF1, insulin-like growth factor 1; IGF1R, IGF1 receptor; LATS1/2, large tumor suppressor homologue 1/2; LRP5/6, low-density-lipoprotein-related protein 5/6; MOB1, MOB kinase activator 1; MST1/2, mammalian STE20-like protein kinase 1/2; PI3 K, phosphoinositide 3-kinase; SAV1, protein Salvador homologue 1; TAZ, transcriptional coactivator with PDZ-binding motif; TCF/LEF, T-cell factor/lymphoid enhancer-binding factor; TEAD, TEA domain transcription factor family members; YAP, yes-associated protein; +p, phosphorylation; +u, ubiquitination.

Fig. 2.

Heart size at selected stages of normal cardiac development. (A) Brightfield images capturing murine hyperplasia over embryonic day (E)9.5 (whole embryo; dashed white lines indicate the location of the heart), E12.5, E18.5 and postnatal day (P)2. (B) Brightfield images capturing murine hypertrophy from P13 to P60. (C) Immunofluorescent images showing pHH3⁺ cells in E12.5 and Ki67⁺ in LV cells of P2 and P13 hearts. pHH3 and Ki67 are proliferation markers and TnT is a cardiomyocyte marker. Ao, aorta; H, heart; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; TnT, troponin T; pHH3, phosphohistone H3. Figure is adapted from Buikema et al. (2013, 2020) and is available to view on Figshare alongside detailed Materials and Methods: 10.6084/m9.figshare.23607306. Scale bars: ∼1 mm (A,C); 5 mm (B).

Fig. 3.

Summary of recent directed cardiac differentiation and expansion protocols for human pluripotent stem cell-derived cardiomyocytes. Schematics of pluripotent stem cell monolayer-based cardiomyocyte differentiation protocols. The days of each protocol are indicated by the vertical lines. Corresponding articles are shown on the left. Initial stem cell conditions, before the start of the differentiation, are shown to the left of day 0 on each timeline. Differentiation efficiency is indicated on the right side, where D indicates the day on which the differentiation efficiency was measured. The media used for differentiation are indicated by different colors below the time axis, with any additional components listed above the time axis. The red and grey boxes depicted above the time axis indicate activation or inhibition of the Wnt pathway, respectively. Key components responsible for modulating Wnt activity are indicated in bold for Wnt activation or italics for Wnt inhibition. Abbreviations: AA, L-ascorbic acid 2-phosphate; bFGF/FGF2, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; BSA, bovine serum albumin; CHIR, CHIR99021; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; hESCs, human embryonic stem cells; hiPSC, human induced pluripotent stem cells; IWP, inhibitor of Wnt production; KO-DMEM, knockout DMEM; LPA, lysophosphatidic acid; MEFs, mouse embryonic fibroblasts; RPMI, Roswell Park Memorial Institute medium; S1P, sphingosine-1-phosphate; VNT, vitronectin. E8, FDTA and mTeSR1 are stem cell media. C59 and IWR1 are inhibitors of the Wnt pathway. B27 is a commercially available cell culture supplement. Created with BioRender.com.

Fig. 4.

Overview of Wnt modulation during hiPSC-CM differentiation and expansion. (A) Schematic overview of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation. Created with BioRender.com. (B) Brightfield images of hiPSC-CMs over multiple passages including expansion illustrating the process of cell-cell contact removal via sparse passaging (∼2.5×10⁴ cells/cm²) with concomitant CHIR99021 administration in B27+insulin/RPMI media to facilitate massive expansion. (C) Brightfield images of hiPSC-CMs in expansion from days 15-20. (D) Immunofluorescence images of hiPSC-CMs stained for α-actinin (green), proliferation marker Ki67 (red) and nuclei (blue) as indicated. (E) Quantification of Ki67⁺ cells indicates that a 37% increase in hiPSC-CM proliferation can be promoted by administering 2 μM CHIR99021. (F) Quantification of hiPSC-CM number from P1 to P5 by sequentially expanding the hiPSC-CMs using CHIR99021 (CHIR). D, differentiation day; P, passage; RPMI, Roswell Park Memorial Institute medium. Figure is adapted from Maas et al. (2021) and is available to view on Figshare alongside detailed Materials and Methods: 10.6084/m9.figshare.23607282. Scale bars: 200 µM (B,C); 100 µM (D).