Primitive macrophages enable long-term vascularization of human heart-on-a-chip platforms.

The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues. In vivo neonatal studies highlight the key role of resident cardiac macrophages in post-injury regeneration and angiogenesis. Here, we integrate human pluripotent stem-cell-derived primitive yolk-sac-like macrophages within vascularized heart-on-chip platforms. Macrophage incorporation profoundly impacted the functionality and perfusability of microvascularized cardiac tissues up to 2 weeks of culture. Macrophages mitigated tissue cytotoxicity and the release of cell-free mitochondrial DNA (mtDNA), while upregulating the secretion of pro-angiogenic, matrix remodeling, and cardioprotective cytokines. Bulk RNA sequencing (RNA-seq) revealed an upregulation of cardiac maturation and angiogenesis genes. Further, single-nuclei RNA sequencing (snRNA-seq) and secretome data suggest that macrophages may prime stromal cells for vascular development by inducing insulin like growth factor binding protein 7 (IGFBP7) and hepatocyte growth factor (HGF) expression. Our results underscore the vital role of primitive macrophages in the long-term vascularization of cardiac tissues, offering insights for therapy and advancing heart-on-a-chip technologies.

Modeling human multi-lineage heart field development with pluripotent stem cells.

The cardiomyocyte (CM) subtypes in the mammalian heart derive from distinct lineages known as the first heart field (FHF), the anterior second heart field (aSHF), and the posterior second heart field (pSHF) lineages that are specified during gastrulation. We modeled human heart field development from human pluripotent stem cells (hPSCs) by using single-cell RNA-sequencing to delineate lineage specification and progression. Analyses of hPSC-derived and mouse mesoderm transcriptomes enabled the identification of distinct human FHF, aSHF, and pSHF mesoderm subpopulations. Through staged manipulation of signaling pathways identified from transcriptomics, we generated myocyte populations that display molecular characteristics of key CM subtypes. The developmental trajectory of the human cardiac lineages recapitulated that of the mouse, demonstrating conserved cardiovascular programs. These findings establish a comprehensive landscape of human embryonic cardiogenesis that provides access to a broad spectrum of cardiomyocytes for modeling congenital heart diseases and chamber-specific cardiomyopathies as well as for developing new therapies to treat them.

Therapeutic correction of hemophilia A by transplantation of hPSC-derived liver sinusoidal endothelial cell progenitors.

Liver sinusoidal endothelial cells (LSECs) form the predominant microvasculature in the liver where they carry out many functions including the secretion of coagulation factor VIII (FVIII). To investigate the early origins of this lineage, we develop an efficient and scalable protocol to produce human pluripotent stem cell (hPSC)-derived LSEC progenitors characterized as venous endothelial cells (VECs) from different mesoderm subpopulations. Using a sensitive and quantitative vascular competitive transplantation assay, we demonstrate that VECs generated from BMP4 and activin A-induced KDR+CD235a/b+ mesoderm are 50-fold more efficient at LSEC engraftment than venous cells from BMP4 and WNT-induced KDR+CD235a/b- mesoderm. When transplanted into immunocompromised hemophilia A mice (NSG-HA), these VECs engraft the liver, proliferate, and mature to functional LSECs that secrete bioactive FVIII capable of correcting the bleeding phenotype. Together, these findings highlight the importance of appropriate mesoderm induction for generating hPSC-derived LSECs capable of functioning in a preclinical model of hemophilia A.

Modeling human yolk sac hematopoiesis with pluripotent stem cells.

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac-derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

One-Step Formation of Protein-Based Tubular Structures for Functional Devices and Tissues.

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.

BMP10 Signaling Promotes the Development of Endocardial Cells from Human Pluripotent Stem Cell-Derived Cardiovascular Progenitors.

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.

Single-Cell Mechanical Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Testing and Pathophysiological Studies.

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. We present a method for directly measuring the active and passive forces generated by single-cell hPSC-CMs at different stretch levels. Utilizing this technique, single hPSC-CMs exhibited positive length-tension relationship and appropriate inotropic, klinotropic, and lusitropic changes in response to pharmacological treatments (isoproterenol and verapamil). The unique potential of the approach for drug testing and disease modeling was exemplified by doxorubicin and omecamtiv mecarbil drug studies revealing their known actions to suppress (doxorubicin) or augment (omecamtiv mecarbil at low dose) cardiomyocyte contractility, respectively. Finally, mechanistic 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.

Generation of Functional Liver Sinusoidal Endothelial Cells from Human Pluripotent Stem-Cell-Derived Venous Angioblasts.

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 ß (TGF-ß) 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.

Ultrasensitive and rapid quantification of rare tumorigenic stem cells in hPSC-derived cardiomyocyte populations.

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 Pluripotent Stem Cell-Derived Cardiovascular Cells: From Developmental Biology to Therapeutic Applications.

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.

A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations.

The ability to direct the differentiation of human pluripotent stem cells (hPSCs) to the different cardiomyocyte subtypes is a prerequisite for modeling specific forms of cardiovascular disease in vitro and for developing novel therapies to treat them. Here we have investigated the development of the human atrial and ventricular lineages from hPSCs, and we show that retinoic acid signaling at the mesoderm stage of development is required for atrial specification. Analyses of early developmental stages revealed that ventricular and atrial cardiomyocytes derive from different mesoderm populations that can be distinguished based on CD235a and RALDH2 expression, respectively. Molecular and electrophysiological characterization of the derivative cardiomyocytes revealed that optimal specification of ventricular and atrial cells is dependent on induction of the appropriate mesoderm. Together these findings provide new insights into the development of the human atrial and ventricular lineages that enable the generation of highly enriched, functional cardiomyocyte populations for therapeutic applications.

Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker.

The sinoatrial node (SAN) is the primary pacemaker of the heart and controls heart rate throughout life. Failure of SAN function due to congenital disease or aging results in slowing of the heart rate and inefficient blood circulation, a condition treated by implantation of an electronic pacemaker. The ability to produce pacemaker cells in vitro could lead to an alternative, biological pacemaker therapy in which the failing SAN is replaced through cell transplantation. Here we describe a transgene-independent method for the generation of SAN-like pacemaker cells (SANLPCs) from human pluripotent stem cells by stage-specific manipulation of developmental signaling pathways. SANLPCs are identified as NKX2-5- cardiomyocytes that express markers of the SAN lineage and display typical pacemaker action potentials, ion current profiles and chronotropic responses. When transplanted into the apex of rat hearts, SANLPCs are able to pace the host tissue, demonstrating their capacity to function as a biological pacemaker.

Hedgehog inhibits ß-catenin activity in synovial joint development and osteoarthritis.

Both the WNT/ß-catenin and hedgehog signaling pathways are important in the regulation of limb development, chondrocyte differentiation, and degeneration of articular cartilage in osteoarthritis (OA). It is not clear how these signaling pathways interact in interzone cell differentiation and synovial joint morphogenesis. Here, we determined that constitutive activation of hedgehog signaling specifically within interzone cells induces joint morphological changes by selectively inhibiting ß-catenin-induced Fgf18 expression. Stabilization of ß-catenin or treatment with FGF18 rescued hedgehog-induced phenotypes. Hedgehog signaling induced expression of a dominant negative isoform of TCF7L2 (dnTCF7L2) in interzone progeny, which may account for the selective regulation of ß-catenin target genes observed. Knockdown of TCF7L2 isoforms in mouse chondrocytes rescued hedgehog signaling-induced Fgf18 downregulation, while overexpression of the human dnTCF7L2 orthologue (dnTCF4) in human chondrocytes promoted the expression of catabolic enzymes associated with OA. Similarly, expression of dnTCF4 in human chondrocytes positively correlated with the aggrecanase ADAMTS4. Consistent with our developmental findings, activation of ß-catenin also attenuated hedgehog-induced or surgically induced articular cartilage degeneration in mouse models of OA. Thus, our results demonstrate that hedgehog inhibits selective ß-catenin target gene expression to direct interzone progeny fates and articular cartilage development and disease. Moreover, agents that increase ß-catenin activity have the potential to therapeutically attenuate articular cartilage degeneration as part of OA.

Generation of articular chondrocytes from human pluripotent stem cells.

The replacement of articular cartilage through transplantation of chondrogenic cells or preformed cartilage tissue represents a potential new avenue for the treatment of degenerative joint diseases. Although many studies have described differentiation of human pluripotent stem cells (hPSCs) to the chondrogenic lineage, the generation of chondrocytes able to produce stable articular cartilage in vivo has not been demonstrated. Here we show that activation of the TGFß pathway in hPSC-derived chondrogenic progenitors promotes the efficient development of articular chondrocytes that can form stable cartilage tissue in vitro and in vivo. In contrast, chondrocytes specified by BMP4 signaling display characteristics of hypertrophy and give rise to cartilage tissues that initiate the endochondral ossification process in vivo. These findings provide a simple serum-free and efficient approach for the routine generation of hPSC-derived articular chondrocytes for modeling diseases of the joint and developing cell therapy approaches to treat them.

Ankrd11 is a chromatin regulator involved in autism that is essential for neural development.

Ankrd11 is a potential chromatin regulator implicated in neural development and autism spectrum disorder (ASD) with no known function in the brain. Here, we show that knockdown of Ankrd11 in developing murine or human cortical neural precursors caused decreased proliferation, reduced neurogenesis, and aberrant neuronal positioning. Similar cellular phenotypes and aberrant ASD-like behaviors were observed in Yoda mice carrying a point mutation in the Ankrd11 HDAC-binding domain. Consistent with a role for Ankrd11 in histone acetylation, Ankrd11 was associated with chromatin and colocalized with HDAC3, and expression and histone acetylation of Ankrd11 target genes were altered in Yoda neural precursors. Moreover, the Ankrd11 knockdown-mediated decrease in precursor proliferation was rescued by inhibiting histone acetyltransferase activity or expressing HDAC3. Thus, Ankrd11 is a crucial chromatin regulator that controls histone acetylation and gene expression during neural development, thereby providing a likely explanation for its association with cognitive dysfunction and ASD.

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues.

Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function.

Specification of chondrocytes and cartilage tissues from embryonic stem cells.

Osteoarthritis primarily affects the articular cartilage of synovial joints. Cell and/or cartilage replacement is a promising therapy, provided there is access to appropriate tissue and sufficient numbers of articular chondrocytes. Embryonic stem cells (ESCs) represent a potentially unlimited source of chondrocytes and tissues as they can generate a broad spectrum of cell types under appropriate conditions in vitro. Here, we demonstrate that mouse ESC-derived chondrogenic mesoderm arises from a Flk-1(-)/Pdgfrα(+) (F(-)P(+)) population that emerges in a defined temporal pattern following the development of an early cardiogenic F(-)P(+) population. Specification of the late-arising F(-)P(+) population with BMP4 generated a highly enriched population of chondrocytes expressing genes associated with growth plate hypertrophic chondrocytes. By contrast, specification with Gdf5, together with inhibition of hedgehog and BMP signaling pathways, generated a population of non-hypertrophic chondrocytes that displayed properties of articular chondrocytes. The two chondrocyte populations retained their hypertrophic and non-hypertrophic properties when induced to generate spatially organized proteoglycan-rich cartilage-like tissue in vitro. Transplantation of either type of chondrocyte, or tissue generated from them, into immunodeficient recipients resulted in the development of cartilage tissue and bone within an 8-week period. Significant ossification was not observed when the tissue was transplanted into osteoblast-depleted mice or into diffusion chambers that prevent vascularization. Thus, through stage-specific manipulation of appropriate signaling pathways it is possible to efficiently and reproducibly derive hypertrophic and non-hypertrophic chondrocyte populations from mouse ESCs that are able to generate distinct cartilage-like tissue in vitro and maintain a cartilage tissue phenotype within an avascular and/or osteoblast-free niche in vivo.

Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage.

Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel preactivation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease.