Uncoupling DNA damage from chromatin damage to detoxify doxorubicin.

The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.

Cardiac microtissues from human pluripotent stem cells recapitulate the phenotype of long-QT syndrome.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) and their derivative cardiomyocytes (hiPSC-CMs) have been successfully used to study the electrical phenotype of cardiac ion channel diseases. However, strategies to mature CMs and more comprehensive systems recapitulating the heart complexity are required to advance our ability to capture adult phenotypes. METHODS: We differentiated wild-type (WT) and long QT syndrome type 1 (LQT1) hiPSCs into CMs, endothelial cells and cardiac fibroblasts. The three cell types were combined to form three-dimensional (3D) spheroids, termed "cardiac microtissues" (cMTs) and the electrophysiological properties were measured using 96-well multi-electrode arrays. RESULTS: LQT1 cMTs displayed prolonged field potential duration compared to WT controls, thus recapitulating the typical feature of LQTS. Isoprenaline caused a positive chronotropic effect on both LQT1 and WT cMTs. The 96-well multi-electrode array format proved suitable to detect electrical changes directly in the 3D tissues. CONCLUSIONS: 3D hiPSC cMTs are a scalable tool that can be used to identify LQT electrical hallmarks and drug responses. We anticipate this tool can be adopted by pharmaceutical companies to screen cardio-active compounds.

Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells.

Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we have generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers that were also present in primary cardiac microvasculature, suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducible in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

RATIONALE: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. OBJECTIVE: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. METHODS AND RESULTS: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. CONCLUSIONS: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.

Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs.

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development.

Generation of human cerebral organoids with a structured outer subventricular zone.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.

Generation of human cerebral organoids with a structured outer subventricular zone.

Mammalian outer radial glia (oRG) emerge as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX , which closely matches human oRG in vivo . In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF- producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell-types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.

Human stem cell models of neurodegeneration: From basic science of amyotrophic lateral sclerosis to clinical translation.

Neurodegenerative diseases are characterized by progressive cell loss leading to disruption of the structure and function of the central nervous system. Amyotrophic lateral sclerosis (ALS) was among the first of these disorders modeled in patient-specific iPSCs, and recent findings have translated into some of the earliest iPSC-inspired clinical trials. Focusing on ALS as an example, we evaluate the status of modeling neurodegenerative diseases using iPSCs, including methods for deriving and using disease-relevant neuronal and glial lineages. We further highlight the remaining challenges in exploiting the full potential of iPSC technology for understanding and potentially treating neurodegenerative diseases such as ALS.

Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells.

Tissue-like structures from human pluripotent stem cells containing multiple cell types are transforming our ability to model and understand human development and disease. Here we describe a protocol to generate cardiomyocytes (CMs), cardiac fibroblasts (CFs) and cardiac endothelial cells (ECs), the three principal cell types in the heart, from human induced pluripotent stem cells (hiPSCs) and combine them in three-dimensional (3D) cardiac microtissues (MTs). We include details of how to differentiate, isolate, cryopreserve and thaw the component cells and how to construct and analyze the MTs. The protocol supports hiPSC-CM maturation and allows replacement of one or more of the three heart cell types in the MTs with isogenic variants bearing disease mutations. Differentiation of each cell type takes ~30 d, while MT formation and maturation requires another 20 d. No specialist equipment is needed and the method is inexpensive, requiring just 5,000 cells per MT.

Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease.

Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.

Co-Differentiation of Human Pluripotent Stem Cells-Derived Cardiomyocytes and Endothelial Cells from Cardiac Mesoderm Provides a Three-Dimensional Model of Cardiac Microtissue.

The formation of cardiac mesodermal subtypes is highly regulated in time and space during heart development. In vitro models based on human pluripotent stem cells (hPS cells) provide opportunities to study mechanisms underlying fate choices governing lineage specification from common cardiovascular progenitors in human embryos. The generation of cardiac endothelial cells in particular allows the creation of complex models of cardiovascular disorders in which either cardiomyocytes or endothelial cells are affected. Here, a protocol for co-differentiation of cardiomyocytes and endothelial cells from cardiac mesoderm using hPS cells is described. Precise details for the enrichment of each cell population from heterogeneous-differentiated cultures, a description of how to maintain and dissociate enriched cardiomyocytes, and the expansion and cryopreservation of enriched endothelial cells are all provided. The generation and culture of three-dimensional cardiac microtissues from these cell populations is described and guidelines for the characterization of microtissues by immunofluorescent staining and re-plating for downstream applications are provided. © 2017 by John Wiley & Sons, Inc.

Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells.

Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (pro)epicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (pro)epicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (pro)epicardial-like cells displaying functional properties (adhesion and spreading over the myocardium) of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (pro)epicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration.