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.

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.

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.

Standardizing designed and emergent quantitative features in microphysiological systems.

Microphysiological systems (MPSs) are cellular models that replicate aspects of organ and tissue functions in vitro. In contrast with conventional cell cultures, MPSs often provide physiological mechanical cues to cells, include fluid flow and can be interlinked (hence, they are often referred to as microfluidic tissue chips or organs-on-chips). Here, by means of examples of MPSs of the vascular system, intestine, brain and heart, we advocate for the development of standards that allow for comparisons of quantitative physiological features in MPSs and humans. Such standards should ensure that the in vivo relevance and predictive value of MPSs can be properly assessed as fit-for-purpose in specific applications, such as the assessment of drug toxicity, the identification of therapeutics or the understanding of human physiology or disease. Specifically, we distinguish designed features, which can be controlled via the design of the MPS, from emergent features, which describe cellular function, and propose methods for improving MPSs with readouts and sensors for the quantitative monitoring of complex physiology towards enabling wider end-user adoption and regulatory acceptance.

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.

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.

Three-Dimensional Vessels-on-a-Chip Based on hiPSC-derived Vascular Endothelial and Smooth Muscle Cells.

Blood vessels are composed of endothelial cells (ECs) that form the inner vessel wall and mural cells that cover the ECs to mediate their stabilization. Crosstalk between ECs and VSMCs while the ECs undergo microfluidic flow is vital for the function and integrity of blood vessels. Here, we describe a protocol to generate three-dimensional (3D) engineered vessels-on-chip (VoCs) composed of vascular cells derived from human induced pluripotent stem cells (hiPSCs). We first describe protocols for robust differentiation of vascular smooth muscle cells (hiPSC-VSMCs) from hiPSCs that are effective across multiple hiPSC lines. Second, we describe the fabrication of a simple microfluidic device consisting of a single collagen lumen that can act as a cell scaffold and support fluid flow using the viscous finger patterning (VFP) technique. After the channel is seeded sequentially with hiPSC-derived ECs (hiPSC-ECs) and hiPSC-VSMCs, a stable EC barrier covered by VSMCs lines the collagen lumen. We demonstrate that this 3D VoC model can recapitulate physiological cell-cell interaction and can be perfused under physiological shear stress using a microfluidic pump. The uniform geometry of the vessel lumens allows precise control of flow dynamics. We have thus developed a robust protocol to generate an entirely isogenic hiPSC-derived 3D VoC model, which could be valuable for studying vessel barrier function and physiology in healthy or disease states. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Differentiation of hiPSC-VSMCs Support Protocol 1: Characterization of hiPSC-NCCs and hiPSC-VSMCs Support Protocol 2: Preparation of cryopreserved hiPSC-VSMCs and hiPSC-ECs for VoC culture Basic Protocol 2: Generation of 3D VoC model composed of hiPSC-ECs and hiPSC-VSMCs Support Protocol 3: Structural characterization of 3D VoC model.

Pressure-Driven Perfusion System to Control, Multiplex and Recirculate Cell Culture Medium for Organs-on-Chips.

Organ-on-chip (OoC) devices are increasingly used to mimic the tissue microenvironment of cells in intact organs. This includes microchannels to mimic, for example, fluidic flow through blood vessels. Present methods for controlling microfluidic flow in these systems rely on gravity, rocker systems or external pressure pumps. For many purposes, pressure pumps give the most consistent flow profiles, but they are not well-suited for high throughput as might be required for testing drug responses. Here, we describe a method which allows for multiplexing of microfluidic channels in OoC devices plus the accompanying custom software necessary to run the system. Moreover, we show the approach is also suitable for recirculation of culture medium, an essential cost consideration when expensive culture reagents are used and are not "spent" through uptake by the cells during transient unidirectional flow.

Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro.

Sarcomeres are the structural units of the contractile apparatus in cardiac and skeletal muscle cells. Changes in sarcomere characteristics are indicative of changes in the sarcomeric proteins and function during development and disease. Assessment of sarcomere length, alignment, and organization provides insight into disease and drug responses in striated muscle cells and models, ranging from cardiomyocytes and skeletal muscle cells derived from human pluripotent stem cells to adult muscle cells isolated from animals or humans. However, quantification of sarcomere length is typically time consuming and prone to user-specific selection bias. Automated analysis pipelines exist but these often require either specialized software or programming experience. In addition, these pipelines are often designed for only one type of cell model in vitro. Here, we present an easy-to-implement protocol and software tool for automated sarcomere length and organization quantification in a variety of striated muscle in vitro models: Two dimensional (2D) cardiomyocytes, three dimensional (3D) cardiac microtissues, isolated adult cardiomyocytes, and 3D tissue engineered skeletal muscles. Based on an existing mathematical algorithm, this image analysis software (SotaTool) automatically detects the direction in which the sarcomere organization is highest over the entire image and outputs the length and organization of sarcomeres. We also analyzed videos of live cells during contraction, thereby allowing measurement of contraction parameters like fractional shortening, contraction time, relaxation time, and beating frequency. In this protocol, we give a step-by-step guide on how to prepare, image, and automatically quantify sarcomere and contraction characteristics in different types of in vitro models and we provide basic validation and discussion of the limitations of the software tool. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Staining and analyzing static hiPSC-CMs with SotaTool Alternate Protocol: Sample preparation, acquisition, and quantification of fractional shortening in live reporter hiPSC lines Support Protocol 1: Finding the image resolution Support Protocol 2: Advanced analysis settings Support Protocol 3: Finding sarcomere length in non-aligned cells.

On-chip analysis of glycolysis and mitochondrial respiration in human induced pluripotent stem cells.

Recent advances in microfluidic engineering allow the creation of microenvironments in which human cells can be cultured under (patho-)physiological conditions with greater reality than standard plastic tissue culture plates. Microfluidic devices, also called Organs-on-Chip (OoC), allow complex engineering of the cellular compartment, yielding designs in which microfluidic flow can be precisely controlled. However, it is important that cellular physiology is not only controlled but can also be monitored in these devices. Here, we integrated oxygen and pH sensors into microfluidics, allowing close monitoring of the extracellular flux from the cells, enabling constant assessment of features such as glycolysis and mitochondrial oxidative phosphorylation in situ. Using human-induced pluripotent stem cells (hiPSCs) as an exemplar of a highly metabolic and relatively challenging cell type to maintain, we showed that monitoring the extracellular environment allowed rapid optimization of the seeding protocol. Based on the measurements, we implemented earlier and more frequent media refreshment to counteract the rapid acidification and depletion of oxygen. The integrated sensors showed that hiPSCs in the devices exhibited mitochondrial and glycolytic capacity similar to that measured with the Seahorse extracellular flux system, the most widely used standard for these types of assays in conventional cell culture. Under both conditions, hiPSCs showed greater reliance on glycolysis than mitochondrial OXPHOS and the absolute values obtained were similar. These results thus pave the way for the assessment of cell metabolism in situ under conditions of fluidic flow with the same precision and relevance as current standard static cell cultures.

Multiplexed fluidic circuit board for controlled perfusion of 3D blood vessels-on-a-chip.

Three-dimensional (3D) blood vessels-on-a-chip (VoC) models integrate the biological complexity of vessel walls with dynamic microenvironmental cues, such as wall shear stress (WSS) and circumferential strain (CS). However, these parameters are difficult to control and are often poorly reproducible due to the high intrinsic diameter variation of individual 3D-VoCs. As a result, the throughput of current 3D systems is one-channel-at-a-time. Here, we developed a fluidic circuit board (FCB) for simultaneous perfusion of up to twelve 3D-VoCs using a single set of control parameters. By designing the internal hydraulic resistances in the FCB appropriately, it was possible to provide a pre-set WSS to all connected 3D-VoCs, despite significant variation in lumen diameters. Using this FCB, we found that variation of CS or WSS induce morphological changes to human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and conclude that control of these parameters using a FCB is necessary to study 3D-VOCs.