Search

Background: Human pluripotent stem cell (hPSC)–derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved through culture on polydimethylsiloxane (PDMS)-lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations. Methods: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic substrates. All hPSC-CMs were generated from a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, and bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction using end points including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic monitoring. Results: We demonstrated the economic generation of >1×10 8 mature hPSC-CMs per PDMS-lined roller bottle. Compared with their counterparts generated on tissue culture plastic substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More important, intracardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less proarrhythmic behavior, and greater beneficial effects on contractile function. Conclusions: We describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs., Cardiomyocytes of heart ventricles consist of subpopulations of trabecular and compact subtypes. Here the authors describe the generation of structurally, metabolically and functionally mature compact ventricular cardiomyocytes as well as mature atrial cardiomyocytes from human pluripotent stem cells.

A typical problem for magnetic surveys with small Unmanned Aerial Systems (sUAS) is the heading error caused by undesired magnetic signals that originate from the aircraft. This can be addressed by suspending the magnetometers on sufficiently long tethers. However, tethered payloads require skilled pilots and are difficult to fly safely. Alternatively, the magnetometer can be fixed on the aircraft. In this case, aircraft magnetic signals are removed from the recordings with a process referred to as magnetic compensation, which requires parameters estimated from calibration flights flown in an area with magnetically low���gradients prior to the survey. We present open���source software fully written in Python to process data and compute compensations for two fundamentally different magnetometer systems (scalar and vector). We used the software to compare the precision of two commercially available systems by flying dense grid patterns over a 135 �� 150 m area using different suspension configurations. The accuracy of the magnetic recordings is assessed using both standard deviations of the calibration pattern and tie���line cross���over differences from the survey. After compensation, the vector magnetometer provides the lowest heading error. However, the magnetic field intensity recovered with this system is relative and needs to be adjusted with absolute data if absolute intensity values are needed. Overall, the highest accuracy of all suspension configurations tested was obtained by fixing the magnetometer 0.5 m below the sUAS onto a self���built carbon���fiber frame, which also offered greater stability and allowed fully autonomous flights in a wide range of conditions., Plain Language Summary: Mapping the strength of the Earth's magnetic field is widely used for imaging the subsurface in geophysical exploration, detecting unexploded ordnance, and investigating archaeological sites. Small Unmanned Aerial Systems (sUAS), commonly referred to as drones, offer fast, flexible, and affordable aeromagnetic surveys. The main challenge in using sUAS in magnetic surveys is the magnetic signals generated by the sUAS itself, which may swamp out natural geologic signals. In some setups the sensors are attached to the sUAS with long tethers, which are difficult to fly safely. We developed a compact system in which the sensor is attached to the aircraft with a 0.5 m carbon���fiber frame. We correct the magnetic signals caused by the sUAS using a process called magnetic compensation. This requires specific calibration maneuvers performed at the beginning of a survey. We developed software for processing the magnetic data and computing the magnetic compensation. We tested this configuration with two magnetometer systems that are commercially available and compared it to a tethered configuration and a setup where the sensor systems were fixed to the landing gear of the sUAS. The fixed���frame system combined with compensation offers higher flight stability and more accurate magnetic recordings than the other configurations., Key Points: This study compares the accuracy of Unmanned Aerial Systems (UAS) scalar and vector magnetometer systems with different suspension designs and compensation methods. We provide software to process, perform magnetic compensation, and assess the accuracy of UAS scalar and vector magnetometers data. A fixed frame suspension method combined with magnetic compensation offers flight stability and highly accurate magnetic recordings., DOI, U.S. Geological Survey (USGS) http://dx.doi.org/10.13039/100000203

Summary: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show considerable promise for regenerating injured hearts, and we therefore tested their capacity to stably engraft in a translationally relevant preclinical model, the infarcted pig heart. Transplantation of immature hESC-CMs resulted in substantial myocardial implants within the infarct scar that matured over time, formed vascular networks with the host, and evoked minimal cellular rejection. While arrhythmias were rare in infarcted pigs receiving vehicle alone, hESC-CM recipients experienced frequent monomorphic ventricular tachycardia before reverting back to normal sinus rhythm by 4 weeks post transplantation. Electroanatomical mapping and pacing studies implicated focal mechanisms, rather than macro-reentry, for these graft-related tachyarrhythmias as evidenced by an abnormal centrifugal pattern with earliest electrical activation in histologically confirmed graft tissue. These findings demonstrate the suitability of the pig model for the preclinical development of a hESC-based cardiac therapy and provide new insights into the mechanistic basis of electrical instability following hESC-CM transplantation. : In this article, Laflamme and colleagues show that the transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) partially remuscularizes the scar of infarcted and appropriately immunosuppressed pigs. hESC-CM recipients exhibited frequent monomorphic ventricular tachycardia before reverting back to normal sinus rhythm by 4 weeks post transplantation. These graft-related tachyarrhythmias were found to be due to focal mechanisms rather than macro-reentry. Keywords: human embryonic stem cell-derived cardiomyocytes, pluripotent stem cells, myocardial infarction, ventricular tachyarrhythmias, electroanatomical mapping, MRI

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) would provide a model for studying the pathogenesis of cholangiopathies and for developing novel therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we describe a new strategy to generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of CFTR and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of new cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies showed that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice providing the first indication that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

Tubular biological structures consisting of extracellular matrix (ECM) proteins and cells are basic functional units of all organs in animals and humans. ECM protein solutions at low concentrations (5-10 milligrams per milliliter) are abundantly used in 3D cell culture. However, their poor "printability" and minute-long gelation time have made the direct extrusion of tubular structures in bioprinting applications challenging. Here, this limitation is overcome and the continuous, template-free conversion of low-concentration collagen, elastin, and fibrinogen solutions into tubular structures of tailored size and radial, circumferential and axial organization is demonstrated. The approach is enabled by a microfabricated printhead for the consistent circumferential distribution of ECM protein solutions and lends itself to scalable manufacture. The attached confinement accommodates minute-long residence times for pH, temperature, light, ionic and enzymatic gelation. Chip hosted ECM tubular structures are amenable to perfusion with aqueous solutions and air, and cyclic stretching. Predictive collapse and reopening in a crossed-tube configuration promote all-ECM valves and pumps. Tissue level function is demonstrated by factors secreted from cells embedded within the tube wall, as well as endothelial or epithelial barriers lining the lumen. The described approaches are anticipated to find applications in ECM-based organ-on-chip and biohybrid structures, hydraulic actuators, and soft machines.

Animal models have been instrumental in providing insight into the molecular basis of disease. While such information has been successfully applied to the study of human disease, this translation would be significantly strengthened by the availability of models based on human cells. This would be particularly important for cardiovascular disease, as the physiology of human cardiomyocytes (CMs) differs significantly from rodents. Here, we have generated a three-dimensional human engineered cardiac tissue, termed biowire, from human embryonic stem cell-derived CMs to investigate the effects of chronic (7 day) treatment with isoproterenol, endothelin-1, or angiotensin II. We show that biowires chronically treated with either isoproterenol, endothelin-1, or angiotensin II have disrupted myofibril alignment and significantly reduced force of contraction. Isoproterenol-treated biowires have upregulated brain natriuretic peptide and atrial natriuretic peptide gene expression. Endothelin-1 and angiotensin II-treated biowires demonstrated a significantly increased cell size. Endothelin-1-treated biowires exhibited increased cardiac troponin secretion into the culture media. This demonstrates that human biowires treated for 7 days with isoproterenol, angiotensin II, or endothelin-1 exhibit some changes compatible with hypertrophic cardiomyopathy.

Background The advent of human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) provided exciting tools for cardiovascular physiological studies, disease modeling and drug testing applications. Current platforms for studying the mechanical properties of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as single-cells do not measure forces directly, require numerous assumptions, and cannot study cell mechanics at different loading conditions. Objective To establish a novel platform to assess the active and passive mechanical properties of single-cell hPSC-CMs at different loading conditions and to demonstrate the potential of this approach for drug testing and disease modeling applications. Methods and results To allow morphological maturation, hPSC-CMs were treated with Tri-iodo-thyronine hormone, dexamethasone and Insulin-like growth factor-1. The hPSC-CM were then lifted and attached to a highly sensitive optical-force transducer and a piezoelectric length controller and electrically-stimulated. The attached hPSC-CM remained intact and contractile allowing evaluation of their passive and active mechanical properties. Utilizing this technique, single-cell hPSC-CMs exhibited positive length-tension (Frank-Starling) relationships, and appropriate inotropic, klinotropic, and lusitropic changes in response to treatment with isoproterenol. The unique potential of the approach for drug testing and disease modeling was exemplified by treating the cells with doxorubicin (a potential cardiotoxic anti-cancer agent) and omecamtiv mecarbil (a positive ionotropic drug currently in stage 3 clinical trial). The results of these studies recapitulated the drugs' known actions to suppress (doxorubicin) and augment (omecamtiv mecarbil at low dose) cardiomyocyte contractility. Finally, novel insights were gained regarding the cellular effects of these drugs as doxorubicin treatment led to cellular mechanical alternans and high doses of omecamtiv mecarbil suppressed contractility and worsened the cellular diastolic properties. Conclusion A novel method that allows direct active and passive force measurements from single hPSC-CMs at different loading conditions for the first time was established and validated. Our results highlight the potential implications of this novel approach for pharmacological studies and disease modeling studies. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): European Research Council

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom 3D-print-molded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28–36), a neutral endopeptidase–generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28–36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10–dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28–36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC(–/–) cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28–36) and demonstrate that the ability of GLP-1(28–36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28–36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.

The functions of the heart are achieved through coordination of different cardiac cell subtypes (e.g., ventricular, atrial, conduction-tissue cardiomyocytes). Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer unique opportunities for cardiac research. Traditional studies using these cells focused on single-cells and utilized mixed cell populations. Our goal was to develop clinically-relevant engineered heart tissues (EHTs) comprised of chamber-specific hPSC-CMs. Here we show that such EHTs can be generated by directing hPSCs to differentiate into ventricular or atrial cardiomyocytes, and then embedding these cardiomyocytes in a collagen-hydrogel to create chamber-specific, ring-shaped, EHTs. The chamber-specific EHTs display distinct atrial versus ventricular phenotypes as revealed by immunostaining, gene-expression, optical assessment of action-potentials and conduction velocity, pharmacology, and mechanical force measurements. We also establish an atrial EHT-based arrhythmia model and confirm its usefulness by applying relevant pharmacological interventions. Thus, our chamber-specific EHT models can be used for cardiac disease modeling, pathophysiological studies and drug testing., The cellular composition of previous engineered heart tissue is often heterogeneous. Here, the authors create chamber-specific human pluripotent stem cell-derived cardiomyocytes to form both ventricular and atrial cells before embedding in collagen-based matrix to form ring-shaped engineered heart tissue.

The liver is the largest solid organ in the body and is critical for metabolic and immune functions. However, little is known about the cells that make up the human liver and its immune microenvironment. Here we report a map of the cellular landscape of the human liver using single-cell RNA sequencing. We provide the transcriptional profiles of 8444 parenchymal and non-parenchymal cells obtained from the fractionation of fresh hepatic tissue from five human livers. Using gene expression patterns, flow cytometry, and immunohistochemical examinations, we identify 20 discrete cell populations of hepatocytes, endothelial cells, cholangiocytes, hepatic stellate cells, B cells, conventional and non-conventional T cells, NK-like cells, and distinct intrahepatic monocyte/macrophage populations. Together, our study presents a comprehensive view of the human liver at single-cell resolution that outlines the characteristics of resident cells in the liver, and in particular provides a map of the human hepatic immune microenvironment.

Summary The identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. Here, using an integrated mass spectrometry- and microarray-based approach, we analyzed the surface proteome and transcriptome of cardiac progenitor cells (CPCs) generated from the stage-specific differentiation of mouse and human pluripotent stem cells. Through bioinformatics analysis, we have identified and characterized FZD4 as a marker for lateral plate mesoderm. Additionally, we utilized FZD4, in conjunction with FLK1 and PDGFRA, to further purify CPCs and increase cardiomyocyte (CM) enrichment in both mouse and human systems. Moreover, we have shown that NORRIN presented to FZD4 further increases CM output via proliferation through the canonical WNT pathway. Taken together, these findings demonstrate a role for FZD4 in mammalian cardiac development., Highlights • Identified and characterized FZD4 as a new marker for lateral plate mesoderm • FZD4, in conjunction with FLK1 and PDGFRA, increases cardiomyocyte enrichment • FZD4 is expressed in the human system and shows enrichment in cardiomyocytes • NORRIN addition shows increase in cardiomyocyte output from FZD4 progenitor cells, In this article, Zandstra and colleagues have identified FZD4 as a new marker for lateral plate mesoderm. Using FZD4, they showed an increase in cardiac progenitor cell purity and a subsequent increase in cardiomyocyte induction in both the mouse and human systems. These findings demonstrate a role for FZD4 in mammalian cardiac development.

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

Summary The embryonic endocardium is essential for early heart development as it functions to induce trabecular myocardium, the first heart tissue to form, and is the source of the cells that make up the valves and a portion of the coronary vasculature. With this potential, human endocardial cells could provide unique therapeutic opportunities that include engineering biological valves and cell-based therapy strategies to replace coronary vasculature in damaged hearts. To access human endocardial cells, we generated a human pluripotent stem cell (hPSC)-derived endothelial population that displays many characteristics of endocardium, including expression of the cohort of genes that identifies this lineage in vivo, the capacity to induce a trabecular fate in immature cardiomyocytes in vitro, and the ability to undergo an endothelial-to-mesenchymal transition. Analyses of the signaling pathways required for development of the hPSC-derived endocardial cells identified a novel role for BMP10 in the specification of this lineage from cardiovascular mesoderm.

Advances in our understanding of cardiovascular development have provided a roadmap for the directed differentiation of human pluripotent stem cells (hPSCs) to the major cell types found in the heart. In this Perspective, we review the state of the field in generating and maturing cardiovascular cells from hPSCs based on our fundamental understanding of heart development. We then highlight their applications for studying human heart development, modeling disease-performing drug screening, and cell replacement therapy. With the advancements highlighted here, the promise that hPSCs will deliver new treatments for degenerative and debilitating diseases may soon be fulfilled.

Rare tumorigenic stem cells identified in hPSC-derived cardiomyocytes using stem cell quantitative cytometry., The ability to detect rare human pluripotent stem cells (hPSCs) in differentiated populations is critical for safeguarding the clinical translation of cell therapy, as these undifferentiated cells have the capacity to form teratomas in vivo. The detection of hPSCs must be performed using an approach compatible with traceable manufacturing of therapeutic cell products. Here, we report a novel microfluidic approach, stem cell quantitative cytometry (SCQC), for the quantification of rare hPSCs in hPSC-derived cardiomyocyte (CM) populations. This approach enables the ultrasensitive capture, profiling, and enumeration of trace levels of hPSCs labeled with magnetic nanoparticles in a low-cost, manufacturable microfluidic chip. We deploy SCQC to assess the tumorigenic risk of hPSC-derived CM populations in vivo. In addition, we isolate rare hPSCs from the differentiated populations using SCQC and characterize their pluripotency.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an unprecedented opportunity to remuscularize infarcted human hearts. However, studies have shown that most hiPSC-CMs do not survive after transplantation into the ischemic myocardial environment, limiting their regenerative potential and clinical application. We established a method to improve hiPSC-CM survival by cotransplanting ready-made microvessels obtained from adipose tissue. Ready-made microvessels promoted a sixfold increase in hiPSC-CM survival and superior functional recovery when compared to hiPSC-CMs transplanted alone or cotransplanted with a suspension of dissociated endothelial cells in infarcted rat hearts. Microvessels showed unprecedented persistence and integration at both early (~80%, week 1) and late (~60%, week 4) time points, resulting in increased vessel density and graft perfusion, and improved hiPSC-CM maturation. These findings provide an approach to cell-based therapies for myocardial infarction, whereby incorporation of ready-made microvessels can improve functional outcomes in cell replacement therapies.

Summary Liver sinusoidal endothelial cells (LSECs) form a highly specialized microvasculature that plays a critical role in liver function and disease. To better understand this role, we developed a strategy to generate LSECs from human pluripotent stem cells (hPSCs) by first optimizing the specification of arterial and venous angioblasts and derivative endothelial populations. Induction of a LSEC-like fate by hypoxia, cyclic AMP (cAMP) agonism, and transforming growth factor β (TGF-β) inhibition revealed that venous endothelial cells responded more rapidly and robustly than the arterial cells to upregulate LSEC markers and functions in vitro. Upon intrahepatic transplantation in neonates, venous angioblasts engrafted the liver and generated mature, fenestrated LSECs with scavenger functions and molecular profiles of primary human LSECs. When transplanted into the liver of adult mice, angioblasts efficiently gave rise to mature LSECs with robust factor VIII (FVIII) production. Humanization of the murine liver with hPSC-derived LSECs provides a tractable system for studying the biology of this key liver cell type.

Summary: Ibrutinib (IB) is an oral Bruton's tyrosine kinase (BTK) inhibitor that has demonstrated benefit in B cell cancers, but is associated with a dramatic increase in atrial fibrillation (AF). We employed cell-specific differentiation protocols and optical mapping to investigate the effects of IB and other tyrosine kinase inhibitors (TKIs) on the voltage and calcium transients of atrial and ventricular human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). IB demonstrated direct cell-specific effects on atrial hPSC-CMs that would be predicted to predispose to AF. Second-generation BTK inhibitors did not have the same effect. Furthermore, IB exposure was associated with differential chamber-specific regulation of a number of regulatory pathways including the receptor tyrosine kinase pathway, which may be implicated in the pathogenesis of AF. Our study is the first to demonstrate cell-type-specific toxicity in hPSC-derived atrial and ventricular cardiomyocytes, which reliably reproduces the clinical cardiotoxicity observed. : The authors employ cell-specific cardiac differentiation protocols, RNA-seq, and optical mapping to demonstrate atrial-specific toxicity of ibrutinib, a first-in-class BTK inhibitor. Other tyrosine kinase inhibitors (TKIs) with the same drug target do not affect atrial electrophysiology. Nilotinib and vandetanib, two TKIs known to be associated with QT prolongation and risk of sudden death, demonstrated ventricular-specific electrophysiologic dysregulation. Keywords: cardiac electrophysiology, tyrosine kinase inhibitors, atrial fibrillation, drug screening, optical mapping, RNA-seq

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be amenable to standard multiwell-plate manipulations and be capable of quantifying tissue-level functions such as contractile force. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom printed multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a ramp. The wells are seeded with cell-laden collagen which, in response to the slope of the ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells and we used it to determine an optimal tissue formulation. We validated contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

Pluripotent stem cells (PSCs) can differentiate into all somatic cell lineages, but fail to form functional macro-scale heart muscle organoids in monolayer, suspension, or aggregate cultures. Here we show that human and non-human primate (NHP) PSCs have the capacity to self-organize into bioengineered heart muscle (BHM) if exposed to a serum-free cardio-instructive collagen-hydrogel environment with defined temporarily restricted growth factor, small molecule, and mechanical stimulation. BHMs traversed through distinct developmental stages with molecular signatures representative for: (i) mesoderm induction (3 days), (ii) cardiac specification (+10 days), and (iii) cardiac maturation (+9 days). Matured BHM responded to electrical pacing, preloading, inotropic stimuli and pharmacological agents similar to bona fide myocardium. We further demonstrate the applicability of BHM in modelling of Noonan Syndrome associated with Raf1 mutations. Collectively, we provide proof-of-concept for directed assembly of human and NHP PSCs into force-generating macro-scale myocardial organoids for applications in simulations of heart development and disease.

Human pluripotent stem cells (hPSCs) provide an unparalleled opportunity to establish in vitro differentiation models that will transform our approach to the study of human development. In the case of the blood system, these models will enable investigation of the earliest stages of human embryonic haematopoiesis that was previously not possible. In addition, they will provide platforms for studying the origins of human blood cell diseases and for generating de novo haematopoietic stem and progenitor cell populations for cell-based regenerative therapies.

The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration., The use of stem cell-derived cardiomyocytes for heart repair is hampered by their immature structural and contractile properties that may cause arrhythmia. Here, Eng et al. show that electrical conditioning of human cardiomyocytes in 3D culture can enhance connectivity and provide resistance to arrhythmia.

Cholangiocytes form an intricate network of bile ducts to enable proper liver function; yet, recapitulating human stem cell differentiation to cholangiocytes in vitro requires Notch signaling and s ...

Summary: Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology. : Mair et al. establish a robust, inducible CRISPR screening platform for forward genetics in human pluripotent stem cells (hPSCs). Genome-wide proliferation screens identified core essential genes for hPSCs and revealed context-dependent genetic requirements on different substrates. This underlines hPSC plasticity and helps us to understand the genetic wiring of hPSCs. Keywords: human pluripotent stem cells, genome-wide CRISPR screen, functional genomics, essential genes, DRICH1

Adult articular cartilage lacks significant regenerative capacity, and damage to this tissue often leads to progressive joint degeneration (osteoarthritis). We developed strategies to generate articular cartilage from human pluripotent stem cells (hPSCs) as a source of clinically relevant tissues for joint repair1. Previously, we demonstrated that these chondrocytes retain cartilage forming potential following subcutaneous implantation in mice. In this report, we evaluated the potential of human embryonic stem cell (hESC)-derived articular cartilage tissue to repair osteochondral defects created in the rat knee. Following implantation, the hESCderived cartilage maintained a proteoglycan and type II collagen-rich matrix, and was well integrated with native rat tissue at the basal and lateral surfaces. The ability to generate cartilage tissue with integrative and reparative properties from an unlimited and robust cell source represents a significant clinical advance for cartilage repair that can be applied to large animal models and ultimately to patient care.

Summary Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) can improve the contractility of injured hearts. We hypothesized that mesodermal cardiovascular progenitors (hESC-CVPs), capable of generating vascular cells in addition to cardiomyocytes, would provide superior repair by contributing to multiple components of myocardium. We performed a head-to-head comparison of hESC-CMs and hESC-CVPs and compared these with the most commonly used clinical cell type, human bone marrow mononuclear cells (hBM-MNCs). In a nude rat model of myocardial infarction, hESC-CMs and hESC-CVPs generated comparable grafts. Both similarly improved systolic function and ventricular dilation. Furthermore, only rare human vessels formed from hESC-CVPs. hBM-MNCs attenuated ventricular dilation and enhanced host vascularization without engrafting long-term or improving contractility. Thus, hESC-CMs and CVPs show similar efficacy for cardiac repair, and both are more efficient than hBM-MNCs. However, hESC-CVPs do not form larger grafts or more significant numbers of human vessels in the infarcted heart., Highlights • Transplantation of hBM-MNCs can halt the negative remodeling of the infarcted heart • Both hESC-derived cardiovascular progenitors and definitive cardiomyocytes improve contractility • hBM-MNCs lead to greater vessel number than hESC-derived cells, In the present study, Murry and colleagues compared the impact of three promising cellular sources for cardiac repair on host cardiac remodeling and contractile function of the infarcted rat heart. After transplantation, human bone marrow mononuclear cells halt the deterioration of left ventricular contractile function. In contrast, human embryonic stem cell (hESC)-derived cardiovascular progenitors and definitive cardiomyocytes both improved systolic function and negative remodeling of the infarcted hearts.