Embryonic macrophages support endocrine commitment during human pancreatic differentiation.

Organogenesis is a complex process that relies on a dynamic interplay between extrinsic factors originating from the microenvironment and tissue-specific intrinsic factors. For pancreatic endocrine cells, the local niche consists of acinar and ductal cells as well as neuronal, immune, endothelial, and stromal cells. Hematopoietic cells have been detected in human pancreas as early as 6 post-conception weeks, but whether they play a role during human endocrinogenesis remains unknown.

Human liver sinusoidal endothelial cells support the development of functional human pluripotent stem cell-derived Kupffer cells.

In mice, the first liver-resident macrophages, known as Kupffer cells (KCs), are thought to derive from yolk sac (YS) hematopoietic progenitors that are specified prior to the emergence of the hematopoietic stem cell (HSC). To investigate human KC development, we recapitulated YS-like hematopoiesis from human pluripotent stem cells (hPSCs) and transplanted derivative macrophage progenitors into NSG mice previously humanized with hPSC-liver sinusoidal endothelial cells (LSECs). We demonstrate that hPSC-LSECs facilitate stable hPSC-YS-macrophage engraftment for at least 7 weeks.

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.

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.

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 KDRCD235a/b mesoderm are 50-fold more efficient at LSEC engraftment than venous cells from BMP4 and WNT-induced KDRCD235a/b mesoderm.

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.

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.

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.

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).

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 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.

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.

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.

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 Ca transients, as well as endpoint assessments of action potentials and conduction velocity.

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.

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.

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

Both the WNT/β-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 β-catenin-induced Fgf18 expression.

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 TGFβ pathway in hPSC-derived chondrogenic progenitors promotes the efficient development of articular chondrocytes that can form stable cartilage tissue in vitro and in vivo.

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.

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).

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.

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.

Metformin activates an atypical PKC-CBP pathway to promote neurogenesis and enhance spatial memory formation.

Although endogenous recruitment of adult neural stem cells has been proposed as a therapeutic strategy, clinical approaches for achieving this are lacking. Here, we show that metformin, a widely used drug, promotes neurogenesis and enhances spatial memory formation. Specifically, we show that an atypical PKC-CBP pathway is essential for the normal genesis of neurons from neural precursors and that metformin activates this pathway to promote rodent and human neurogenesis in culture.

An endothelial cell niche induces hepatic specification through dual repression of Wnt and Notch signaling.

Complex cross-talk between endoderm and the microenvironment is an absolute requirement to orchestrate hepatic specification and expansion. In the mouse, the septum transversum and cardiac mesoderm, through secreted bone morphogenetic proteins (BMP) and fibroblast growth factors (FGF), respectively, instruct the adjacent ventral endoderm to become hepatic endoderm. Consecutively, endothelial cells promote expansion of the specified hepatic endoderm.

Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture.

Background: The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu.

Methodology/principal Findings: We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes.