The role of Wnt regulation in heart development, cardiac repair and disease: A tissue engineering perspective.
ABSTRACT: Wingless-related integration site (Wnt) signaling has proven to be a fundamental mechanism in cardiovascular development as well as disease. Understanding its particular role in heart formation has helped to develop pluripotent stem cell differentiation protocols that produce relatively pure cardiomyocyte populations. The resultant cardiomyocytes have been used to generate heart tissue for pharmaceutical testing, and to study physiological and disease states. Such protocols in combination with induced pluripotent stem cell technology have yielded patient-derived cardiomyocytes that exhibit some of the hallmarks of cardiovascular disease and are therefore being used to model disease states. While FDA approval of new treatments typically requires animal experiments, the burgeoning field of tissue engineering could act as a replacement. This would necessitate the generation of reproducible three-dimensional cardiac tissues in a well-controlled environment, which exhibit native heart properties, such as cellular density, composition, extracellular matrix composition, and structure-function. Such tissues could also enable the further study of Wnt signaling. Furthermore, as Wnt signaling has been found to have a mechanistic role in cardiac pathophysiology, e.g. heart attack, hypertrophy, atherosclerosis, and aortic stenosis, its strategic manipulation could provide a means of generating reproducible and specific, physiological and pathological cardiac models.
Project description:The protocol described here efficiently directs human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of a glycogen synthase kinase 3 (GSK3) inhibitor combined with the expression of ?-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8-1.3 million cardiomyocytes per cm(2)) of virtually pure (80-98%) functional cardiomyocytes in 14 d from multiple hPSC lines without cell sorting or selection. Qualitative (immunostaining) and quantitative (flow cytometry) characterization of differentiated cells is described to assess the expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjunction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening and cell-based therapeutic applications.
Project description:Stem cell-derived cardiomyocytes and vascular cells can be used for a variety of applications such as studying human heart development and modelling human disease in culture. In particular, protocols based on modulation of Wnt signaling were able to produce high quality of cardiomyocytes or vascular cells from human pluripotent stem cells (hPSCs). However, the mechanism behind the development of 3D cardiovascular spheroids into either vascular or cardiac cells has not been well explored. Hippo/Yes-associated protein (YAP) signaling plays important roles in the regulation of organogenesis, but its impact on cardiovascular differentiation has been less evaluated. In this study, the effects of seeding density and a change in YAP signaling on 3D cardiovascular spheroids patterning from hPSCs were evaluated. Compared to 2D culture, 3D cardiovascular spheroids exhibited higher levels of sarcomeric striations and higher length-to-width ratios of α-actinin+ cells. The spheroids with high seeding density exhibited more α-actinin+ cells and less nuclear YAP expression. The 3D cardiovascular spheroids were also treated with different small molecules, including Rho kinase inhibitor (Y27632), Cytochalasin D, Dasatinib, and Lysophosphatidic acid to modulate YAP localization. Nuclear YAP inhibition resulted in lower expression of active β-catenin, vascular marker, and MRTF, the transcription factor mediated by RhoGTPases. Y27632 also promoted the gene expression of MMP-2/-3 (matrix remodeling) and Notch-1 (Notch signaling). These results should help our understanding of the underlying effects for the efficient patterning of cardiovascular spheroids after mesoderm formation from hPSCs.
Project description:Early heart development takes place through a complex series of steps, including the induction of cardiac mesoderm, formation of the cardiovascular progenitor cells and the commitment of cardiovascular lineage cells, such as cardiomyocytes (CMs), smooth muscle cells (SMCs) and endothelial cells (ECs). Recently, microRNAs, a family of endogenous, small non-coding RNAs, have been identified as critical regulators in cardiogenesis and cardiovascular disease. Previous studies demonstrated that microRNA-1 (miR-1) could promote cardiac differentiation from mouse and human embryonic stem (ES) cells. However, the underlying mechanism remained largely unclear. We performed microRNA deep sequencing among human ES cells, ES cell derived-multipotent cardiovascular progenitors (MCPs), and MCP-specified CMs, ECs, and SMCs. A specific enrichment of miR-1 was found in CMs, not in SMCs or ECs, implying a key role of miR-1 in determining cardiovascular commitment from MCPs. When overexpressed in human induced pluripotent stem cells, miR-1 enhanced the expression of key cardiac transcriptional factors and sarcomeric genes. Importantly, we found miR-1 promoted CM differentiation and suppressed EC commitment from MCPs by modulating the activities of WNT and FGF signaling pathways. FZD7 and FRS2 were confirmed as miR-1 targets using luciferase reporter assay and western blot. Overall, this study reveals a fate-switching role of miR-1 at early human cardiovascular commitment stage via suppressing both WNT and FGF signaling pathways.
Project description: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 transforming growth factor ? superfamily ligands to generate cardiomyocytes. However, insulin did not repress cardiomyocyte differentiation in a defined protocol that used 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/?-catenin signaling and that the cardiomyocyte differentiation defect resulting from insulin exposure was rescued by inhibition of Wnt/?-catenin during the cardiac mesoderm (Nkx2.5+) stage. Thus, insulin and Wnt/?-catenin signaling pathways, as a network, coordinate to influence hPSC differentiation to cardiomyocytes, with the Wnt/?-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.
Project description:The generation of human ventricular cardiomyocytes from human embryonic stem cells and/or induced pluripotent stem cells could fulfill the demand for therapeutic applications and in vitro pharmacological research; however, the production of a homogeneous population of ventricular cardiomyocytes remains a major limitation. By combining small molecules and growth factors, we developed a fully chemically defined, directed differentiation system to generate ventricular-like cardiomyocytes (VCMs) from human embryonic stem cells and induced pluripotent stem cells with high efficiency and reproducibility. Molecular characterization revealed that the differentiation recapitulated the developmental steps of cardiovascular fate specification. Electrophysiological analyses further illustrated the generation of a highly enriched population of VCMs. These chemically induced VCMs exhibited the expected cardiac electrophysiological and calcium handling properties as well as the appropriate chronotropic responses to cardioactive compounds. In addition, using an integrated computational and experimental systems biology approach, we demonstrated that the modulation of the canonical Wnt pathway by the small molecule IWR-1 plays a key role in cardiomyocyte subtype specification. In summary, we developed a reproducible and efficient experimental platform that facilitates a chemical genetics-based interrogation of signaling pathways during cardiogenesis that bypasses the limitations of genetic approaches and provides a valuable source of ventricular cardiomyocytes for pharmacological screenings as well as cell replacement therapies.
Project description:Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.
Project description:Understanding pathways controlling cardiac development may offer insights that are useful for stem cell-based cardiac repair. Developmental studies indicate that the Wnt/beta-catenin pathway negatively regulates cardiac differentiation, whereas studies with pluripotent embryonal carcinoma cells suggest that this pathway promotes cardiogenesis. This apparent contradiction led us to hypothesize that Wnt/beta-catenin signaling acts biphasically, either promoting or inhibiting cardiogenesis depending on timing. We used inducible promoters to activate or repress Wnt/beta-catenin signaling in zebrafish embryos at different times of development. We found that Wnt/beta-catenin signaling before gastrulation promotes cardiac differentiation, whereas signaling during gastrulation inhibits heart formation. Early treatment of differentiating mouse embryonic stem (ES) cells with Wnt-3A stimulates mesoderm induction, activates a feedback loop that subsequently represses the Wnt pathway, and increases cardiac differentiation. Conversely, late activation of beta-catenin signaling reduces cardiac differentiation in ES cells. Finally, constitutive overexpression of the beta-catenin-independent ligand Wnt-11 increases cardiogenesis in differentiating mouse ES cells. Thus, Wnt/beta-catenin signaling promotes cardiac differentiation at early developmental stages and inhibits it later. Control of this pathway may promote derivation of cardiomyocytes for basic research and cell therapy applications.
Project description:Differentiation of cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) is critically dependent upon the regulation of the Wnt signaling pathway. The mechanisms remain unclear with regard to the dose and timing of each differentiation inducer, and the interaction of the inducers that regulate the Wnt in mesendoderm specification to cardiac mesoderm. Consequently, it remains far from optimal in differentiation efficiency and consistency from hiPSC lines to CMs. Here, we have carefully deciphered the role of Wnt signaling pathway manipulation on mesoderm specification in a dosage and time dependent manner. To examine the hypothesis of that fate specification of hiPSC-CMs differentiation is dictated by temporal and spatial factors that regulate Wnt, we evaluate hiPSC-CM differentiation with: (1) two-phase modulation of Wnt, (2) dosage variant of GSK3? inhibitors, (3) treatment with insulin, and (4) 3-dimentional suspension culture environment on iPSC-CM differentiation. The results highlight the importance of mesendoderm specification to cardiac mesoderm, which needs precisely regulation of Wnt in a dosage dependent and temporal on/off manner. This temporal regulation dictates the final efficiency and purity of derived cardiomyocytes. After the initial activation of Wnt signaling pathway to generate mesendoderm, the maintenance of Wnt signaling at an appropriate dose is critical to direct the cell fate into cardiac mesoderm. Otherwise, lower Wnt signals lead to definitive endoderm and higher Wnt signals induce presomitic mesoderm differentiation. The precisely specification of cardiac mesoderm results in not only greater than 90% of cTnT+ cardiomyocytes but also high cardiomyocytes yield under both monolayer and suspension culture conditions. Thus, the current findings provide critical insights to decipher the temporal mechanism of Wnt activation in regulation of hiPSC-CMs differentiation, and more importantly provide the guidelines for the consistent and high-yield and high-quality hiPSC-CMs production in cardiovascular research.
Project description:Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-? and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (?90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays.Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (?90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.
Project description:Cardiac fibroblasts (CFs) play critical roles in heart development, homeostasis, and disease. The limited availability of human CFs from native heart impedes investigations of CF biology and their role in disease. Human pluripotent stem cells (hPSCs) provide a highly renewable and genetically defined cell source, but efficient methods to generate CFs from hPSCs have not been described. Here, we show differentiation of hPSCs using sequential modulation of Wnt and FGF signaling to generate second heart field progenitors that efficiently give rise to hPSC-CFs. The hPSC-CFs resemble native heart CFs in cell morphology, proliferation, gene expression, fibroblast marker expression, production of extracellular matrix and myofibroblast transformation induced by TGF?1 and angiotensin II. Furthermore, hPSC-CFs exhibit a more embryonic phenotype when compared to fetal and adult primary human CFs. Co-culture of hPSC-CFs with hPSC-derived cardiomyocytes distinctly alters the electrophysiological properties of the cardiomyocytes compared to co-culture with dermal fibroblasts. The hPSC-CFs provide a powerful cell source for research, drug discovery, precision medicine, and therapeutic applications in cardiac regeneration.