Endothelial Responses to Curvature-Induced Flow Patterns in Engineered Cerebral Aneurysms.

Hemodynamic factors have long been associated with clinical outcomes in the treatment of cerebral aneurysms. Computational studies of cerebral aneurysm hemodynamics have provided valuable estimates of the mechanical environment experienced by the endothelium in both the parent vessel and aneurysmal dome walls and have correlated them with disease state. These computational-clinical studies have recently been correlated with the response of endothelial cells (EC) using either idealized or patient-specific models. Here, we present a robust workflow for generating anatomic-scale aneurysm models, establishing luminal cultures of ECs at physiological relevant flow profiles, and comparing EC responses to curvature mediated flow. We show that flow patterns induced by parent vessel curvature produce changes in wall shear stress (WSS) and wall shear stress gradients (WSSG) that are correlated with differences in cell morphology and cellular protein localization. Cells in higher WSS regions align better with the flow and display strong Notch1-extracellular domain (ECD) polarization, while, under low WSS, differences in WSSG due to curvature change were associated with less alignment and attenuation of Notch1-ECD polarization in ECs of the corresponding regions. These proof-of-concept results highlight the use of engineered cellularized aneurysm models for connecting computational fluid dynamics to the underlying endothelial biology that mediates disease.

Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues.

Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys. Animal models have provided insight into the mechanisms by which dystrophin protein deficiency causes cardiomyopathy, but there remains a need to develop human models of DMD to validate pathogenic mechanisms and identify therapeutic targets. Here, we have developed human engineered heart tissues (EHTs) from CRISPR-edited, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a truncated dystrophin protein lacking part of the actin-binding domain. The 3D EHT platform enables direct measurement of contractile force, simultaneous monitoring of Ca2+ transients, and assessment of myofibril structure. Dystrophin-mutant EHTs produced less contractile force as well as delayed kinetics of force generation and relaxation, as compared to isogenic controls. Contractile dysfunction was accompanied by reduced sarcomere length, increased resting cytosolic Ca2+ levels, delayed Ca2+ release and reuptake, and increased beat rate irregularity. Transcriptomic analysis revealed clear differences between dystrophin-deficient and control EHTs, including downregulation of genes related to Ca2+ homeostasis and extracellular matrix organization, and upregulation of genes related to regulation of membrane potential, cardiac muscle development, and heart contraction. These findings indicate that the EHT platform provides the cues necessary to expose the clinically-relevant, functional phenotype of force production as well as mechanistic insights into the role of Ca2+ handling and transcriptomic dysregulation in dystrophic cardiac function, ultimately providing a powerful platform for further studies in disease modeling and drug discovery.

Endothelial-derived von Willebrand factor accelerates fibrin clotting within engineered microvessels.

BACKGROUND: Von Willebrand factor (VWF) is classically associated with primary hemostasis and platelet-rich arterial thromboses, but recently has also been implicated in fibrin clotting and venous thrombosis. Direct interaction between fibrin and VWF may mediate these processes, although prior reports are conflicting. OBJECTIVES: We combined two complementary platforms to characterize VWF-fibrin(ogen) interactions and identify their potential physiologic significance. METHODS: Engineered microvessels were lined with human endothelial cells, cultured under flow, and activated to release VWF and form transluminal VWF fibers. Fibrinogen, fibrin monomers, or polymerizing fibrin were then perfused, and interactions with VWF evaluated. Thrombin and fibrinogen were perfused into living versus paraformeldahyde-fixed microvessels and the pressure drop across microvessels monitored. Separately, protein binding to tethered VWF was assessed on a single-molecule level using total internal reflection fluorescence (TIRF) microscopy. RESULTS: Within microvessels, VWF fibers colocalized with polymerizing fibrin, but not fibrinogen. TIRF microscopy showed no colocalization between VWF and fibrinogen or fibrin monomers in a microfluidic flow chamber across a range of shear rates and protein concentrations. Thrombin-mediated fibrin polymerization within living microvessels triggered endothelial VWF release, increasing the rate and amount of microvessel obstruction compared to fixed vessels with an inert endothelium. CONCLUSIONS: We did not identify specific binding between fibrin(ogen) and VWF at a single-molecule level. Despite this, our results suggest that rapid release of endothelial VWF during clotting may provide a physical support for fibrin polymerization and accelerate thrombosis. This interaction may be of fundamental importance for the understanding and treatment of human thrombotic disease.

Organ-on-a-chip systems for vascular biology.

Organ-on-a-chip (OOC) platforms involve the miniaturization of cell culture systems and enable a variety of novel experimental approaches. These range from modeling the independent effects of biophysical forces on cells to screening novel drugs in multi-organ microphysiological systems, all within microscale devices. As in living systems, the incorporation of vascular structure is a key feature common to almost all organ-on-a-chip systems. In this review we highlight recent advances in organ-on-a-chip technologies with a focus on the vasculature. We first present the developmental process of the blood vessels through which vascular cells assemble into networks and remodel to form complex vascular beds under flow. We then review self-assembled vascular models and flow systems for the study of vascular development and biology as well as pre-patterned vascular models for the generation of perfusable microvessels for modeling vascular and tissue function. We finally conclude with a perspective on developing future OOC approaches for studying different aspects of vascular biology. We highlight the fit for purpose selection of OOC models towards either simple but powerful testbeds for therapeutic development, or complex vasculature to accurately replicate human physiology for specific disease modeling and tissue regeneration.

Multiphoton-Guided Creation of Complex Organ-Specific Microvasculature.

Engineering functional human tissues in vitro is currently limited by difficulty replicating the small caliber, complex connectivity, cellularity, and 3D curvature of the native microvasculature. Multiphoton ablation has emerged as a promising technique for fabrication of microvascular structures with high resolution and full 3D control, but cellularization and perfusion of complex capillary-scale structures has remained challenging. Here, multiphoton ablation combined with guided endothelial cell growth from pre-formed microvessels is used to successfully create perfusable and cellularized organ-specific microvascular structures at anatomic scale within collagen hydrogels. Fabrication and perfusion of model 3D pulmonary and renal microvascular beds is demonstrated, as is replication and perfusion of a brain microvascular unit derived from in vivo data. Successful endothelialization and blood perfusion of a kidney-specific microvascular structure is achieved, using laser-guided angiogenesis. Finally, proof-of-concept hierarchical blood vessels and complex multicellular models are created, using multistep patterning with multiphoton ablation techniques. These successes open new doors for the creation of engineered tissues and organ-on-a-chip devices.

Engineering Heart Morphogenesis.

Recent advances in stem cell biology and tissue engineering have laid the groundwork for building complex tissues in a dish. We propose that these technologies are ready for a new challenge: recapitulating cardiac morphogenesis in vitro. In development, the heart transforms from a simple linear tube to a four-chambered organ through a complex process called looping. Here, we re-examine heart tube looping through the lens of an engineer and argue that the linear heart tube is an advantageous starting point for tissue engineering. We summarize the structures, signaling pathways, and stresses in the looping heart, and evaluate approaches that could be used to build a linear heart tube and guide it through the process of looping.

Soluble factors secreted by differentiating embryonic stem cells stimulate exogenous cell proliferation and migration.

INTRODUCTION: Stem cells are being investigated as catalysts of tissue regeneration to either directly replace or promote cellularity lost as a result of traumatic injury or degenerative disease. In many reports, despite low numbers of stably integrated cells, the transient presence of cells delivered or recruited to sites of tissue remodeling globally benefits functional recovery. Such findings have motivated the need to determine how paracrine factors secreted from transplanted cells may be capable of positively impacting endogenous repair processes and somatic cell responses. METHODS: Embryonic stem cells were differentiated as embryoid bodies (EBs) in vitro and media conditioned by EBs were collected at different intervals of time. Gene and protein expression analysis of several different growth factors secreted by EBs were examined by polymerase chain reaction and enzyme-linked immunosorbent assay analysis, respectively, as a function of time. The proliferation and migration of fibroblasts and endothelial cells treated with EB conditioned media was examined compared with unconditioned and growth media controls. RESULTS: The expression of several growth factors, including bone morphogenic protein-4, insulin-like growth factors and vascular endothelial growth factor-A, increased during the course of embryonic stem cell (ESC) differentiation as EBs. Conditioned media collected from EBs at different stages of differentiation stimulated proliferation and migration of both fibroblasts and endothelial cells, based on 5-bromo-2'-deoxyuridine incorporation and transwell assays, respectively. CONCLUSIONS: Overall, these results demonstrate that differentiating ESCs express increasing amounts of various growth factors over time that altogether are capable of stimulating mitogenic and motogenic activity of exogenous cell populations.