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Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Preclinical data have confirmed that human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can remuscularize the injured or diseased heart, with several clinical trials now in planning or recruitment stages. However, because ventricular arrhythmias represent a complication following engraftment of intramyocardially injected PSC-CMs, it is necessary to provide treatment strategies to control or prevent engraftment arrhythmias (EAs). Here, we show in a porcine model of myocardial infarction and PSC-CM transplantation that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. Two unique surface marker signatures, signal regulatory protein α (SIRPA) + CD90 - CD200 + and SIRPA + CD90 - CD200 - , identify arrhythmogenic and non-arrhythmogenic cardiomyocytes, respectively. Our data suggest that modifications to current PSC-CM-production and/or PSC-CM-selection protocols could potentially prevent EAs. We further show that pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias., (© 2024. The Author(s).)

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function., Competing Interests: Declaration of interests E.P., R.J.M., and J.E.H. are co-inventors on patents relating to cardiac organoid maturation and cardiac therapeutics. J.E.H. is co-inventor on licensed patents for engineered heart muscle. E.P., R.J.M., and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics. N.J.P. is an inventor on patents relating to stem cells and heart regeneration and is a co-founder and equity holder in Infensa Bioscience., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor β (PDGFRβ) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models., Competing Interests: Declaration of interests R.J.M., J.E.H., G.A.Q.-R., and E.R.P. are listed as co-inventors on pending patents that relate to cardiac organoid maturation and cardiac regeneration therapeutics. E.R.P. and J.E.H. are listed as co-inventors on pending patents that relate to endothelial cells in 3D cardiac products. J.E.H. is a co-inventor on licensed patents relating to engineered heart muscle. R.J.M., E.R.P., and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The adult mammalian heart displays a cardiomyocyte turnover rate of ≈1%/y throughout postnatal life and after injuries such as myocardial infarction (MI), but the question of which cell types drive this low level of new cardiomyocyte formation remains contentious. Cardiac-resident stem cells marked by stem cell antigen-1 (Sca-1, gene name Ly6a) have been proposed as an important source of cardiomyocyte renewal. However, the in vivo contribution of endogenous Sca-1 + cells to the heart at baseline or after MI has not been investigated., Methods: Here we generated Ly6a gene-targeted mice containing either a constitutive or an inducible Cre recombinase to perform genetic lineage tracing of Sca-1 + cells in vivo., Results: We observed that the contribution of endogenous Sca-1 + cells to the cardiomyocyte population in the heart was <0.005% throughout all of cardiac development, with aging, or after MI. In contrast, Sca-1 + cells abundantly contributed to the cardiac vasculature in mice during physiological growth and in the post-MI heart during cardiac remodeling. Specifically, Sca-1 lineage-traced endothelial cells expanded postnatally in the mouse heart after birth and into adulthood. Moreover, pulse labeling of Sca-1 + cells with an inducible Ly6a-MerCreMer allele also revealed a preferential expansion of Sca-1 lineage-traced endothelial cells after MI injury in the mouse., Conclusions: Cardiac-resident Sca-1 + cells are not significant contributors to cardiomyocyte renewal in vivo. However, cardiac Sca-1 + cells represent a subset of vascular endothelial cells that expand postnatally with enhanced responsiveness to pathological stress in vivo.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During vertebrate development, mesodermal fate choices are regulated by interactions between morphogens such as activin/nodal, BMPs and Wnt/β-catenin that define anterior-posterior patterning and specify downstream derivatives including cardiomyocyte, endothelial and hematopoietic cells. We used human embryonic stem cells to explore how these pathways control mesodermal fate choices in vitro. Varying doses of activin A and BMP4 to mimic cytokine gradient polarization in the anterior-posterior axis of the embryo led to differential activity of Wnt/β-catenin signaling and specified distinct anterior-like (high activin/low BMP) and posterior-like (low activin/high BMP) mesodermal populations. Cardiogenic mesoderm was generated under conditions specifying anterior-like mesoderm, whereas blood-forming endothelium was generated from posterior-like mesoderm, and vessel-forming CD31(+) endothelial cells were generated from all mesoderm origins. Surprisingly, inhibition of β-catenin signaling led to the highly efficient respecification of anterior-like endothelium into beating cardiomyocytes. Cardiac respecification was not observed in posterior-derived endothelial cells. Thus, activin/BMP gradients specify distinct mesodermal subpopulations that generate cell derivatives with unique angiogenic, hemogenic and cardiogenic properties that should be useful for understanding embryogenesis and developing therapeutics., (© 2015. Published by The Company of Biologists Ltd.)

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Cardiomyocytes, the cells of the heart muscle, lose nearly all of their proliferative capacity after birth, limiting the heart's ability to regenerate. Naqvi et al. now identify a transient burst of cardiomyocyte proliferation during preadolescence, driven by a thyroid hormone surge, with therapeutic implications for congenital and acquired heart diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host-graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models.

Genetic regulation of the cell fate transition from lateral plate mesoderm to the specification of cardiomyocytes requires suppression of Wnt/β-catenin signaling, but the mechanism for this is not well understood. By analyzing gene expression and chromatin dynamics during directed differentiation of human embryonic stem cells (hESCs), we identified a suppressor of Wnt/β-catenin signaling, transmembrane protein 88 (TMEM88), as a potential regulator of cardiovascular progenitor cell (CVP) specification. During the transition from mesoderm to the CVP, TMEM88 has a chromatin signature of genes that mediate cell fate decisions, and its expression is highly upregulated in advance of key cardiac transcription factors in vitro and in vivo. In early zebrafish embryos, tmem88a is expressed broadly in the lateral plate mesoderm, including the bilateral heart fields. Short hairpin RNA targeting of TMEM88 during hESC cardiac differentiation increases Wnt/β-catenin signaling, confirming its role as a suppressor of this pathway. TMEM88 knockdown has no effect on NKX2.5 or GATA4 expression, but 80% of genes most highly induced during CVP development have reduced expression, suggesting adoption of a new cell fate. In support of this, analysis of later stage cell differentiation showed that TMEM88 knockdown inhibits cardiomyocyte differentiation and promotes endothelial differentiation. Taken together, TMEM88 is crucial for heart development and acts downstream of GATA factors in the pre-cardiac mesoderm to specify lineage commitment of cardiomyocyte development through inhibition of Wnt/β-catenin signaling.

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

Cardiac troponin I (cTnI) functions as the molecular switch of the thin filament. Studies have shown that a histidine button engineered into cTnI (cTnI A164H) specifically enhances inotropic function in the context of numerous pathophysiological challenges. To gain mechanistic insight into the basis of this finding, we analyzed histidine ionization states in vitro by studying the myofilament biophysics of amino acid substitutions that act as constitutive chemical mimetics of altered histidine ionization. We also assessed the role of histidine-modified cTnI in silico by means of molecular dynamics simulations. A functional in vitro analysis of myocytes at baseline (pH 7.4) indicated similar cellular contractile function and myofilament calcium sensitivity between myocytes expressing wild-type (WT) cTnI and cTnI A164H, whereas the A164R variant showed increased myofilament calcium sensitivity. Under acidic conditions, compared with WT myocytes, the myocytes expressing cTnI A164H maintained a contractile performance similar to that observed for the constitutively protonated cTnI A164R variant. Molecular dynamics simulations showed similar intermolecular atomic contacts between the WT and the deprotonated cTnI A164H variant. In contrast, simulations of protonated cTnI A164H showed various potential structural configurations, one of which included a salt bridge between His-164 of cTnI and Glu-19 of cTnC. This salt bridge was recapitulated in simulations of the cTnI A164R variant. These data suggest that differential histidine ionization may be necessary for cTnI A164H to act as a molecular sensor capable of modulating sarcomere performance in response to changes in the cytosolic milieu., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Stem cell biology has increasingly gained scientific and public interest in recent years. In particular, the use of stem cells for treatment of heart disease has been strongly pursued within the scientific and medical communities. Significant effort has gone into the use of adult tissue-derived stem cells for cardiac repair including bone marrow, blood, and cardiac-derived cell populations. Significant interest in this area has been balanced by the difficulties of understanding stem cells, cardiac injury, and the amalgamation of these areas of investigation in translational medicine. Recent studies have emerged on adipose-derived stem cells which show the potential for cardiac lineage development in vitro and may have application in cell-mediated in vivo therapy for the diseased heart. This review provides a summary of current findings within the field of adipose-derived stem cell biology regarding their cardiac differentiation potential.

Recent reports have described a stem cell population termed stromal vascular cells (SVCs) derived from the stromal vascular fraction of adipose tissue, which are capable of intrinsic differentiation into spontaneously beating cardiomyocytes in vitro. The objective of this study was to further define the cardiac lineage differentiation potential of SVCs in vitro and to establish methods for enriching SVC-derived beating cardiac myocytes. SVCs were isolated from the stromal vascular fraction of murine adipose tissue. Cells were cultured in methylcellulose-based murine stem cell media. Analysis of SVC-derived beating myocytes included Western blot and calcium imaging. Enrichment of acutely isolated SVCs was carried out using antibody-tagged magnetic nanoparticles, and pharmacologic manipulation of Wnt and cytokine signaling. Under initial media conditions, spontaneously beating SVCs expressed both cardiac developmental and adult protein isoforms. Functionally, this specialized population can spontaneously contract and pace under field stimulation and shows the presence of coordinated calcium transients. Importantly, this study provides evidence for two independent mechanisms of enriching the cardiac differentiation of SVCs. First, this study shows that differentiation of SVCs into cardiac myocytes is augmented by non-canonical Wnt agonists, canonical Wnt antagonists, and cytokines. Second, SVCs capable of cardiac lineage differentiation can be enriched by selection for stem cell-specific membrane markers Sca1 and c-kit. Adipose-derived SVCs are a unique population of stem cells that show evidence of cardiac lineage development making them a potential source for stem cell-based cardiac regeneration studies.