ETV2 Upregulation Marks the Specification of Early Cardiomyocytes and Endothelial Cells During Co-differentiation.

The ability to differentiate human-induced pluripotent stem cells (hiPSCs) efficiently into defined cardiac lineages, such as cardiomyocytes and cardiac endothelial cells, is crucial to study human heart development and model cardiovascular diseases in vitro. The mechanisms underlying the specification of these cell types during human development are not well understood which limits fine-tuning and broader application of cardiac model systems. Here, we used the expression of ETV2, a master regulator of hematoendothelial specification in mice, to identify functionally distinct subpopulations during the co-differentiation of endothelial cells and cardiomyocytes from hiPSCs. Targeted analysis of single-cell RNA-sequencing data revealed differential ETV2 dynamics in the 2 lineages. A newly created fluorescent reporter line allowed us to identify early lineage-predisposed states and show that a transient ETV2-high-state initiates the specification of endothelial cells. We further demonstrated, unexpectedly, that functional cardiomyocytes can originate from progenitors expressing ETV2 at a low level. Our study thus sheds light on the in vitro differentiation dynamics of 2 important cardiac lineages.

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.

Homozygous Missense Variants in NTNG2, Encoding a Presynaptic Netrin-G2 Adhesion Protein, Lead to a Distinct Neurodevelopmental Disorder.

NTNG2 encodes netrin-G2, a membrane-anchored protein implicated in the molecular organization of neuronal circuitry and synaptic organization and diversification in vertebrates. In this study, through a combination of exome sequencing and autozygosity mapping, we have identified 16 individuals (from seven unrelated families) with ultra-rare homozygous missense variants in NTNG2; these individuals present with shared features of a neurodevelopmental disorder consisting of global developmental delay, severe to profound intellectual disability, muscle weakness and abnormal tone, autistic features, behavioral abnormalities, and variable dysmorphisms. The variants disrupt highly conserved residues across the protein. Functional experiments, including in silico analysis of the protein structure, in vitro assessment of cell surface expression, and in vitro knockdown, revealed potential mechanisms of pathogenicity of the variants, including loss of protein function and decreased neurite outgrowth. Our data indicate that appropriate expression of NTNG2 plays an important role in neurotypical development.

Cartilage from human-induced pluripotent stem cells: comparison with neo-cartilage from chondrocytes and bone marrow mesenchymal stromal cells.

Cartilage has little intrinsic capacity for repair, so transplantation of exogenous cartilage cells is considered a realistic option for cartilage regeneration. We explored whether human-induced pluripotent stem cells (hiPSCs) could represent such unlimited cell sources for neo-cartilage comparable to human primary articular chondrocytes (hPACs) or human bone marrow-derived mesenchymal stromal cells (hBMSCs). For this, chondroprogenitor cells (hiCPCs) and hiPSC-derived mesenchymal stromal cells (hiMSCs) were generated from two independent hiPSC lines and characterized by morphology, flow cytometry, and differentiation potential. Chondrogenesis was compared to hBMSCs and hPACs by histology, immunohistochemistry, and RT-qPCR, while similarities were estimated based on Pearson correlations using a panel of 20 relevant genes. Our data show successful differentiations of hiPSC into hiMSCs and hiCPCs. Characteristic hBMSC markers were shared between hBMSCs and hiMSCs, with the exception of CD146 and CD45. However, neo-cartilage generated from hiMSCs showed low resemblances when compared to hBMSCs (53%) and hPACs (39%) characterized by lower collagen type 2 and higher collagen type 1 expression. Contrarily, hiCPC neo-cartilage generated neo-cartilage more similar to hPACs (65%), with stronger expression of matrix deposition markers. Our study shows that taking a stepwise approach to generate neo-cartilage from hiPSCs via chondroprogenitor cells results in strong similarities to neo-cartilage of hPACs within 3 weeks following chondrogenesis, making them a potential candidate for regenerative therapies. Contrarily, neo-cartilage deposited by hiMSCs seems more prone to hypertrophic characteristics compared to hPACs. We therefore compared chondrocytes derived from hiMSCs and hiCPCs with hPACs and hBMSCs to outline similarities and differences between their neo-cartilage and establish their potential suitability for regenerative medicine and disease modelling.

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.

Quantifying Ca2+ signaling and contraction in vascular pericytes and smooth muscle cells.

Vascular pericytes and smooth muscle cells surround many blood vessels of the body. Their primary roles include vessel stabilization and regulation of the blood flow. The high degree of heterogeneity among these cells is dictated by (1) differences in their developmental origin and (2) their location in the vascular bed. Phenotype switching contributes to this heterogeneity especially following in vitro culture. In the absence of distinguishing molecular markers, functional assays that capture their heterogeneity in vitro are needed. Spatiotemporal changes in intracellular Ca2+ levels and contraction in response to vasoconstrictors reflect the differences between vascular pericyte and smooth muscle cell. In order to capture this heterogeneity in vitro, large ensembles of cells need to be analyzed. Here we developed an automated image processing method to measure intracellular Ca2+ and contraction in large cell groups which in combination with a computational approach for integrative analysis allowed vascular pericytes and smooth muscle cells to be distinguished without knowledge of their anatomical origin.

Squaramide-Based Supramolecular Materials for Three-Dimensional Cell Culture of Human Induced Pluripotent Stem Cells and Their Derivatives.

Synthetic hydrogel materials can recapitulate the natural cell microenvironment; however, it is equally necessary that the gels maintain cell viability and phenotype while permitting reisolation without stress, especially for use in the stem cell field. Here, we describe a family of synthetically accessible, squaramide-based tripodal supramolecular monomers consisting of a flexible tris(2-aminoethyl)amine (TREN) core that self-assemble into supramolecular polymers and eventually into self-recovering hydrogels. Spectroscopic measurements revealed that monomer aggregation is mainly driven by a combination of hydrogen bonding and hydrophobicity. The self-recovering hydrogels were used to encapsulate NIH 3T3 fibroblasts as well as human-induced pluripotent stem cells (hiPSCs) and their derivatives in 3D. The materials reported here proved cytocompatible for these cell types with maintenance of hiPSCs in their undifferentiated state essential for their subsequent expansion or differentiation into a given cell type and potential for facile release by dilution due to their supramolecular nature.

In Brief: Endothelial-to-mesenchymal transition.

Recent evidence has highlighted the role of endothelial-to-mesenchymal transition (EndoMT) in the onset and progression of a number of human pathologies. EndoMT involves the loss of an endothelial signature to generate unspecialized mesenchymal-like cells, susceptible to being re-differentiated into mesodermal cell types, including osteoblasts, chondrocytes, and adipocytes. Therefore, modulation of the molecular pathways controlling EndoMT can be considered as a therapeutic approach for particular human diseases. In addition, EndoMT may be harnessed for tissue engineering by producing multipotent mesenchymal cells able to differentiate into mutiple cell types.

Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts.

OBJECTIVE: Endothelial cells (ECs), pericytes, and vascular smooth muscle cells (vSMCs) are essential for vascular development, and their dysfunction causes multiple cardiovascular diseases. Primary vascular cells for research are, however, difficult to obtain. Human-induced pluripotent stem cells (hiPSCs) derived from somatic tissue are a renewable source of ECs and vSMCs; however, their use as disease models has been limited by low and inconsistent efficiencies of differentiation and the lack of phenotypic bioassays. APPROACH AND RESULTS: Here, we developed defined conditions for simultaneous derivation of ECs and pericytes with high efficiency from hiPSCs of different tissue origin. The protocol was equally efficient for all lines and human embryonic stem cells (hESCs). The ECs could undergo sequential passage and were phenotypically indistinguishable, exhibiting features of arterial-like embryonic ECs. Moreover, hiPSC-derived ECs formed an authentic vascular plexus when cocultured with hiPSC-derived pericytes. The coculture system recapitulated (1) major steps of vascular development including EC proliferation and primary plexus remodeling, and (2) EC-mediated maturation and acquisition of contractile vSMC phenotype by pericytes. In addition, hiPSC-derived ECs integrated into developing vasculature as xenografts in zebrafish. This contrasts with more widely used ECs from human umbilical vein, which form only unstable vasculature and were completely unable to integrate into zebrafish blood vessels. CONCLUSIONS: We demonstrate that vascular derivatives of hiPSC, such as ECs and pericytes, are fully functional and can be used to study defective endothelia-pericyte interactions in vitro for disease modeling and studies on tumor angiogenesis.

Self-assembling 3D vessel-on-chip model with hiPSC-derived astrocytes.

Functionality of the blood-brain barrier (BBB) relies on the interaction between endothelial cells (ECs), pericytes, and astrocytes to regulate molecule transport within the central nervous system. Most experimental models for the BBB rely on freshly isolated primary brain cells. Here, we explored human induced pluripotent stem cells (hiPSCs) as a cellular source for astrocytes in a 3D vessel-on-chip (VoC) model. Self-organized microvascular networks were formed by combining hiPSC-derived ECs, human brain vascular pericytes, and hiPSC-derived astrocytes within a fibrin hydrogel. The hiPSC-ECs and pericytes showed close interactions, but, somewhat unexpectedly, addition of astrocytes disrupted microvascular network formation. However, continuous fluid perfusion or activation of cyclic AMP (cAMP) signaling rescued the vascular organization and decreased vascular permeability. Nevertheless, astrocytes did not affect the expression of proteins related to junction formation, transport, or extracellular matrix, indicating that, despite other claims, hiPSC-derived ECs do not entirely acquire a BBB-like identity in the 3D VoC model.

Breast cancer-on-chip for patient-specific efficacy and safety testing of CAR-T cells.

Physiologically relevant human models that recapitulate the challenges of solid tumors and the tumor microenvironment (TME) are highly desired in the chimeric antigen receptor (CAR)-T cell field. We developed a breast cancer-on-chip model with an integrated endothelial barrier that enables the transmigration of perfused immune cells, their infiltration into the tumor, and concomitant monitoring of cytokine release during perfused culture over a period of up to 8 days. Here, we exemplified its use for investigating CAR-T cell efficacy and the ability to control the immune reaction with a pharmacological on/off switch. Additionally, we integrated primary breast cancer organoids to study patient-specific CAR-T cell efficacy. The modular architecture of our tumor-on-chip paves the way for studying the role of other cell types in the TME and thus provides the potential for broad application in bench-to-bedside translation as well as acceleration of the preclinical development of CAR-T cell products.

Targeted JAM-C deletion in germ cells by Spo11-controlled Cre recombinase.

Meiosis is a crucial process for the production of functional gametes. However, the biological significance of many genes expressed during the meiotic phase remains poorly understood, mainly because of the lethal phenotypes of the knockout mice. Functional analysis of such genes using the conditional knockout approach is hindered by the lack of suitable Cre transgenic lines. We describe here the generation of transgenic mice expressing Cre recombinase under the control of the meiotic Spo11 gene. Using LacZ-R26(loxP) and EYFP-R26(loxP) reporter mice, we show the specific expression and activity of Cre during meiosis in males and females. Spo11(Cre) mice were then crossed with floxed Nbs1 and JAM-C mice to produce conditional knockouts. A strong reduction of Nbs1 and JAM-C protein levels was found in the testis. Although Nbs1-deleted mice developed minor gonadal abnormalities, JAM-C-knockout mice showed a spermiogenetic arrest, as previously described for the null mice. These results provide strong evidence that Spo11(Cre) transgenic mice represent a powerful tool for deleting genes of interest specifically in meiotic and/or in postmeiotic germ cells.

Genetic repair of a human induced pluripotent cell line from patient with Dutch-type cerebral amyloid angiopathy.

Dutch-type cerebral amyloid angiopathy (D-CAA), also known as hereditary cerebral haemorrhage with amyloidosis-Dutch type (HCHWA-D), is an autosomal dominant disorder caused by a G to C transversion in codon 693 of the amyloid precursor protein (APP) that results in a Gln-to-Glu amino acid substitution. CRISPR-Cas9 editing was used for genetic correction of the mutation in a human induced pluripotent stem cell (hiPSC-) line established previously. The isogenic hiPSCs generated showed typical pluripotent stem cell morphology, expressed all markers of undifferentiated state, displayed a normal karyotype and had the capacity to differentiate into the three germ layers.

Vascularized hiPSC-derived 3D cardiac microtissue on chip.

Functional vasculature is essential for delivering nutrients, oxygen, and cells to the heart and removing waste products. Here, we developed an in vitro vascularized human cardiac microtissue (MT) model based on human induced pluripotent stem cells (hiPSCs) in a microfluidic organ-on-chip by coculturing hiPSC-derived, pre-vascularized, cardiac MTs with vascular cells within a fibrin hydrogel. We showed that vascular networks spontaneously formed in and around these MTs and were lumenized and interconnected through anastomosis. Anastomosis was fluid flow dependent: continuous perfusion increased vessel density and thus enhanced the formation of the hybrid vessels. Vascularization further improved endothelial cell (EC)-cardiomyocyte communication via EC-derived paracrine factors, such as nitric oxide, and resulted in an enhanced inflammatory response. The platform sets the stage for studies on how organ-specific EC barriers respond to drugs or inflammatory stimuli.

Maturation of hiPSC-derived cardiomyocytes promotes adult alternative splicing of SCN5A and reveals changes in sodium current associated with cardiac arrhythmia.

AIMS: Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs. METHODS AND RESULTS: We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch. CONCLUSIONS: Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B. TRANSLATIONAL PERSPECTIVE: The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications.

Junctional adhesion molecule-C regulates vascular endothelial permeability by modulating VE-cadherin-mediated cell-cell contacts.

We recently reported that junctional adhesion molecule (JAM)-C plays a role in leukocyte transendothelial migration. Here, the role of JAM-C in vascular permeability was investigated in vitro and in vivo. As opposed to macrovascular endothelial cells that constitutively expressed JAM-C in cell-cell contacts, in quiescent microvascular endothelial cells, JAM-C localized mainly intracellularly, and was recruited to junctions upon short-term stimulation with vascular endothelial growth factor (VEGF) or histamine. Strikingly, disruption of JAM-C function decreased basal permeability and prevented the VEGF- and histamine-induced increases in human dermal microvascular endothelial cell permeability in vitro and skin permeability in mice. Permeability increases are essential in angiogenesis, and JAM-C blockade reduced hyperpermeability and neovascularization in hypoxia-induced retinal angiogenesis in mice. The underlying mechanisms of the JAM-C-mediated increase in endothelial permeability were studied. JAM-C was essential for the regulation of endothelial actomyosin, as revealed by decreased F-actin, reduced myosin light chain phosphorylation, and actin stress fiber formation due to JAM-C knockdown. Moreover, the loss of JAM-C expression resulted in stabilization of VE-cadherin-mediated interendothelial adhesion in a manner dependent on the small GTPase Rap1. Together, through modulation of endothelial contractility and VE-cadherin-mediated adhesion, JAM-C helps to regulate vascular permeability and pathologic angiogenesis.

Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis.

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.

Perspectives for Future Use of Cardiac Microtissues from Human Pluripotent Stem Cells.

Cardiovascular disorders remain a critical health issue worldwide. While animals have been used extensively as experimental models to investigate heart disease mechanisms and develop drugs, their inherent drawbacks have shifted focus to more human-relevant alternatives. Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs, collectively called hPSCs) have been identified as a source of different cardiac cells, but to date, they have rarely offered functional and structural maturity of the adult human heart. However, the combination of patient derived hPSCs with microphysiological tissue engineering approaches has presented new opportunities to study heart development and disease and identify drug targets. These models often closely mimic specific aspects of the native heart tissue including intercellular crosstalk and microenvironmental cues such that maturation occurs and relevant disease phenotypes are revealed. Most recently, organ-on-chip technology based on microfluidic devices has been combined with stem cell derived organoids and microtissues to create vascularized structures that can be subjected to fluidic flow and to which immune cells can be added to mimic inflammation of tissue postinjury. Similarly, the integration of nerve cells in these models can provide insight into how the cardiac nervous system affects heart pathology, for example, after myocardial infarction. Here, we consider these models and approaches in the context of cardiovascular disease together with their applications and readouts. We reflect on perspectives for their future implementation in understanding disease mechanisms and the drug discovery pipeline.

Microphysiological stem cell models of the human heart.

Models of heart disease and drug responses are increasingly based on human pluripotent stem cells (hPSCs) since their ability to capture human heart (dys-)function is often better than animal models. Simple monolayer cultures of hPSC-derived cardiomyocytes, however, have shortcomings. Some of these can be overcome using more complex, multi cell-type models in 3D. Here we review modalities that address this, describe efforts to tailor readouts and sensors for monitoring tissue- and cell physiology (exogenously and in situ) and discuss perspectives for implementation in industry and academia.