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In spite of all the efforts for generating efficient pharmacological treatment options for cancer patients, the unwanted side effect of these substances on the cardiovascular system is becoming a major issue for cancer survivors. The fast pacing oncology field necessitate the quest for more accurate and reliable preclinical screenings. hiPSCs derived cardiomyocytes, endothelial and vascular smooth muscle cells provide unlimited source of physiologically relevant cells that could be used in the screening platforms. Cells derived from hiPSCs can measure drug induced alterations to different aspect of the heart including electrophysiology, contractility and structure. In this review, we will give an overview of the different in vivo and in vitro preclinical drug safety screenings. In following sections, we will focus on hiPSCs derived cardiomyocytes, endothelial and vascular smooth muscle cells and present the current knowledge of the application of these cells in unicellular cardiotoxicity assays. In the final part, we will focus on cardiac organoids as multi cell type platform and their role in cardiotoxicity screening of the chemotherapeutic drugs.

A scalable, robust, and integrated differentiation platform for large‐scale production of human pluripotent stem cell‐cardiomyocyte (hPSC‐CM) in a stirred suspension bioreactor as a single‐unit operation was developed. This platform could become a valuable tool for 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), 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‐β 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. Significance 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.

Summary Pluripotent stem cells hold enormous potential for regenerative therapies, however their ability to provide insight into early human development and the origins of disease could arguably provide an even greater outcome. This is primarily due to their contribution to the establishment of a powerful knowledge base of human development, something which all researchers and clinicians can potentially benefit from. Modeling human heart development and disease using pluripotent stem cells has already provided many important insights into cardiogenesis and cardiovascular disease mechanisms however, it is important to be aware of the complexities of this model system. Thorough contemplation of experimental models and specialized techniques is required to provide high‐quality evidence of the intricacies of both normal early development, and when this process goes awry in disease states. Stem Cells Translational Medicine 2017;6:1452–1457

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of \"off-targets\", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease. Many genes working within large gene networks influence the development of heart muscle cells in humans and other animals. The activity of these genes is controlled in part by proteins called transcription factors, which bind to DNA and act as molecular switches. One transcription factor that is particularly important for the development of heart muscle cells is called NKX2-5. Mice lacking NKX2-5 have abnormal hearts and many humans who are born with congenital heart disease carry mutations in the gene that encodes this protein. Many of these mutations alter a section of the protein called the homeodomain, and therefore interfere with the ability of NKX2-5 to bind to DNA or associate with other important cardiac proteins called cofactors. However, it is not clear how such mutations alter the behaviour of NKX2-5 across all of its targets. Bouveret et al. have now used a technique called ‘DNA adenine methyltransferase identification’ to study how NKX2-5 interacts with other proteins and DNA. The experiments found that, as expected, the mutant NKX2-5 proteins were unable to associate with many of the usual gene and protein targets of normal NKX2-5. However, the mutant proteins were still able to bind to some of their usual targets, plus many other targets that the normal NKX2-5 protein was not able to bind to. A particular NKX2-5 mutant protein that the experiments analysed was missing the entire homeodomain, yet it was still able to associate with the normal NKX2-5 protein and bind to cofactors that help NKX2-5 to find its usual targets. This finding led Bouveret et al. to discover the role of a section of the NKX2-5 protein called the tyrosine-rich domain, which in the absence of the homeodomain can direct interactions of NKX2-5 with itself and its cofactors. Bouveret et al.'s findings suggest that protein cofactors of NKX2-5 help mutant NKX2-5 proteins retain some of their normal activities, but also direct the mutant proteins to abnormal gene targets, which could contribute to congenital heart disease. The next steps are to carry out experiments in animals to confirm these findings, and to understand the activities of mutant NKX2-5 and other mutant transcription factors across the whole genome. This could lead to new therapeutic approaches to treat congenital heart disease and other conditions.

Human embryonic stem cells (hESCs) have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes. We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1) recombinant protein into the cells. We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 µg/ml recombinant ISL1 protein during days 1-8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4) doubled. This protocol was also reproducible for another hESC line. This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.

Derivation of cardiomyocytes directly from patients’ own fibroblasts could offer a new therapeutic approach for those with ischemic heart disease. An essential step toward clinical application is to establish safe conversion of human fibroblasts into a cardiac fate. Here we aimed to efficiently and safely generate cardiomyocytes from human fibroblasts by direct delivery of reprogramming recombinant cell permeant form of reprogramming proteins followed by cardio-inductive signals. Human fetal and adult fibroblasts were transiently exposed to transactivator of transcription-fused recombinant OCT4, SOX2, KLF4 and c-MYC for 2 weeks and then were directly differentiated toward protein-induced cardiomyocyte-like cells (p-iCLCs) in a cardiac fate niche, carried out by treatment with a set of cardiogenic small molecules (sequential treatment of Chir, and IWP-2, SB431542 and purmorphamine). The cells showed cardiac phenotype over a period of 3 weeks without first undergoing reprogramming into or through a pluripotent intermediate, shown by lack of expression of key pluripotency markers. p-iCLCs exhibited cardiac features at both the gene and protein levels. Our study provides an alternative method for the generation of p-iCLCs which shortcut reprogramming toward allogeneic cardiomyocytes in a safe and efficient manner and could facilitate generation of genetic material-free cardiomyocytes.

Derivation of cardiomyocytes directly from patients' own fibroblasts could offer a new therapeutic approach for those with ischemic heart disease. An essential step toward clinical application is to establish safe conversion of human fibroblasts into a cardiac fate. Here we aimed to efficiently and safely generate cardiomyocytes from human fibroblasts by direct delivery of reprogramming recombinant cell permeant form of reprogramming proteins followed by cardio-inductive signals. Human fetal and adult fibroblasts were transiently exposed to transactivator of transcription-fused recombinant OCT4, SOX2, KLF4 and c-MYC for 2 weeks and then were directly differentiated toward protein-induced cardiomyocyte-like cells (p-iCLCs) in a cardiac fate niche, carried out by treatment with a set of cardiogenic small molecules (sequential treatment of Chir, and IWP-2, SB431542 and purmorphamine). The cells showed cardiac phenotype over a period of 3 weeks without first undergoing reprogramming into or through a pluripotent intermediate, shown by lack of expression of key pluripotency markers. p-iCLCs exhibited cardiac features at both the gene and protein levels. Our study provides an alternative method for the generation of p-iCLCs which shortcut reprogramming toward allogeneic cardiomyocytes in a safe and efficient manner and could facilitate generation of genetic material-free cardiomyocytes.

Human embryonic stem cells (hESCs) have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes. We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1) recombinant protein into the cells. We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 [micro]g/ml recombinant ISL1 protein during days 1-8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4) doubled. This protocol was also reproducible for another hESC line. This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.

Human embryonic stem cells (hESCs) have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes. We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1) recombinant protein into the cells. We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 [micro]g/ml recombinant ISL1 protein during days 1-8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4) doubled. This protocol was also reproducible for another hESC line. This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.