Engineering Vascularized Organoid-on-a-Chip Models.

Recreating human organ-level function in vitro is a rapidly evolving field that integrates tissue engineering, stem cell biology, and microfluidic technology to produce 3D organoids. A critical component of all organs is the vasculature. Herein, we discuss general strategies to create vascularized organoids, including common source materials, and survey previous work using vascularized organoids to recreate specific organ functions and simulate tumor progression. Vascularization is not only an essential component of individual organ function but also responsible for coupling the fate of all organs and their functions. While some success in coupling two or more organs together on a single platform has been demonstrated, we argue that the future of vascularized organoid technology lies in creating organoid systems complete with tissue-specific microvasculature and in coupling multiple organs through a dynamic vascular network to create systems that can respond to changing physiological conditions.

Low levels of physiological interstitial flow eliminate morphogen gradients and guide angiogenesis.

Convective transport can significantly distort spatial concentration gradients. Interstitial flow is ubiquitous throughout living tissue, but our understanding of how interstitial flow affects concentration gradients in biological processes is limited. Interstitial flow is of particular interest for angiogenesis because pathological and physiological angiogenesis is associated with altered interstitial flow, and both interstitial flow and morphogen gradients (e.g., vascular endothelial growth factor, VEGF) can potentially stimulate and guide new blood vessel growth. We designed an in vitro microfluidic platform to simulate 3D angiogenesis in a tissue microenvironment that precisely controls interstitial flow and spatial morphogen gradients. The microvascular tissue was developed from endothelial colony forming cell-derived endothelial cells extracted from cord blood and stromal fibroblasts in a fibrin extracellular matrix. Pressure in the microfluidic lines was manipulated to control the interstitial flow. A mathematical model of mass and momentum transport, and experimental studies with fluorescently labeled dextran were performed to validate the platform. Our data demonstrate that at physiological interstitial flow (0.1-10 µm/s), morphogen gradients were eliminated within hours, and angiogenesis demonstrated a striking bias in the opposite direction of interstitial flow. The interstitial flow-directed angiogenesis was dependent on the presence of VEGF, and the effect was mediated by αvß3 integrin. We conclude that under physiological conditions, growth factors such as VEGF and fluid forces work together to initiate and spatially guide angiogenesis.

CD44 variant isoforms expressed by breast cancer cells are functional E-selectin ligands under flow conditions.

Adhesion of circulating tumor cells to vascular endothelium is mediated by specialized molecules that are functional under shear forces exerted by hematogenous flow. Endothelial E-selectin binding to glycoforms of CD44 mediates shear-resistant cell adhesion in numerous physiological and pathological conditions. However, this pathway is poorly understood in breast cancer and is the focus of the present investigation. All breast cancer cell lines used in this study strongly expressed CD44. In particular, BT-20 cells expressed CD44s and multiple CD44v isoforms, whereas MDA-MB-231 cells predominantly expressed CD44s but weakly expressed CD44v isoforms. CD44 expressed by BT-20, but not MDA-MB-231, cells possessed E-selectin ligand activity as detected by Western blotting and antigen capture assays. Importantly, CD44 expressed by intact BT-20 cells were functional E-selectin ligands, regulating cell rolling and adhesion under physiological flow conditions, as found by shRNA-targeted silencing of CD44. Antigen capture assays strongly suggest greater shear-resistant E-selectin ligand activity of BT-20 cell CD44v isoforms than CD44s. Surprisingly, CD44 was not recognized by the HECA-452 MAb, which detects sialofucosylated epitopes traditionally expressed by selectin ligands, suggesting that BT-20 cells express a novel glycoform of CD44v as an E-selectin ligand. The activity of this glycoform was predominantly attributed to N-linked glycans. Furthermore, expression of CD44v as an E-selectin ligand correlated with high levels of fucosyltransferase-3 and -6 and epithelial, rather than mesenchymal, cell phenotype. Together, these data demonstrate that expression of CD44 as a functional E-selectin ligand may be important in breast cancer metastasis.

A vascularized 3D model of the human pancreatic islet forex vivostudy of immune cell-islet interaction.

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively byßcells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of isletßcells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlyingßcell damage in diabetes rely onin vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing-key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel,ex vivoplatform for studying human islet biology in both health and disease.

Mitigating neutrophil trafficking and cardiotoxicity with DS-IkL in a microphysiological system of a cytokine storm.

A feature of severe COVID-19 is the onset of an acute and intense systemic inflammatory response referred to as the "cytokine storm". The cytokine storm is characterized by high serum levels of inflammatory cytokines and the subsequent transport of inflammatory cells to damaging levels in vital organs (e.g., myocarditis). Immune trafficking and its effect on underlying tissues (e.g., myocardium) are challenging to observe at a high spatial and temporal resolution in mouse models. In this study, we created a vascularized organ-on-a-chip system to mimic cytokine storm-like conditions and tested the effectiveness of a novel multivalent selectin-targeting carbohydrate conjugate (composed of DS - dermatan sulfate and IkL - a selectin-binding peptide, termed DS-IkL) in blocking infiltration of polymorphonuclear leukocytes (PMN). Our data shows that cytokine storm-like conditions induce endothelial cells to produce additional inflammatory cytokines and facilitate infiltration of PMNs into tissue. Treatment of tissues with DS-IkL (60 µM) reduced PMN accumulation in the tissue by >50%. We then created cytokine storm-like conditions in a vascularized cardiac tissue-chip and found that PMN infiltration increases the spontaneous beating rate of the cardiac tissue, and this effect is eliminated by treatment with DS-IkL (60 µM). In summary, we demonstrate the utility of an organ-on-a-chip platform to mimic COVID-19 related cytokine storm and that blocking leukocyte infiltration with DS-IkL could be a viable strategy to mitigate associated cardiac complications.

A microfluidic strategy to capture antigen-specific high affinity B cells.

Assessing B cell affinity to pathogen-specific antigens prior to or following exposure could facilitate the assessment of immune status. Current standard tools to assess antigen-specific B cell responses focus on equilibrium binding of the secreted antibody in serum. These methods are costly, time-consuming, and assess antibody affinity under zero-force. Recent findings indicate that force may influence BCR-antigen binding interactions and thus immune status. Here, we designed a simple laminar flow microfluidic chamber in which the antigen (hemagglutinin of influenza A) is bound to the chamber surface to assess antigen-specific BCR binding affinity of five hemagglutinin-specific hybridomas under 65- to 650-pN force range. Our results demonstrate that both increasing shear force and bound lifetime can be used to enrich antigen-specific high affinity B cells. The affinity of the membrane-bound BCR in the flow chamber correlates well with the affinity of the matched antibodies measured in solution. These findings demonstrate that a microfluidic strategy can rapidly assess BCR-antigen binding properties and identify antigen-specific high affinity B cells. This strategy has the potential to both assess functional immune status from peripheral B cells and be a cost-effective way of identifying individual B cells as antibody sources for a range of clinical applications.

Convection and extracellular matrix binding control interstitial transport of extracellular vesicles.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3ß1 and α6ß1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 µm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3ß1 and α6ß1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.

Receptor-ligand non-equilibrium kinetics (RLNEK) 1.0: An integrated Trackmate laminar flow chamber analysis.

Although parallel plate flow chamber assays are widely performed, extraction of kinetic parameters is limited to specialized labs with mathematical expertise and customized video-microscopy tracking tools. The recent development of Trackmate has increased researcher accessibility to tracking particles in video-microscopy experiments; however, there is a lack of tools that analyze this tracking information. We report a software tool, compatible with Trackmate, that extracts Receptor Ligand Non-Equilibrium Kinetic (RLNEK) parameters from video-microscopy data. This software should be of particular interest to the community of researchers and scientists interrogating the target-specific binding and release of immune cells.

An iPS-derived in vitro model of human atrial conduction.

Atrial fibrillation (AF) is the most common arrhythmia in the United States, affecting approximately 1 in 10 adults, and its prevalence is expected to rise as the population ages. Treatment options for AF are limited; moreover, the development of new treatments is hindered by limited (1) knowledge regarding human atrial electrophysiological endpoints (e.g., conduction velocity [CV]) and (2) accurate experimental models. Here, we measured the CV and refractory period, and subsequently calculated the conduction wavelength, in vivo (four subjects with AF and four controls), and ex vivo (atrial slices from human hearts). Then, we created an in vitro model of human atrial conduction using induced pluripotent stem (iPS) cells. This model consisted of iPS-derived human atrial cardiomyocytes plated onto a micropatterned linear 1D spiral design of Matrigel. The CV (34-41 cm/s) of the in vitro model was nearly five times faster than 2D controls (7-9 cm/s) and similar to in vivo (40-64 cm/s) and ex vivo (28-51 cm/s) measurements. Our iPS-derived in vitro model recapitulates key features of in vivo atrial conduction and may be a useful methodology to enhance our understanding of AF and model patient-specific disease.

Organ-on-a-chip model of vascularized human bone marrow niches.

Bone marrow niches (endosteal and perivascular) play important roles in both normal bone marrow function and pathological processes such as cancer cell dormancy. Unraveling the mechanisms underlying these events in humans has been severely limited by models that cannot dissect dynamic events at the niche level. Utilizing microfluidic and stem cell technologies, we present a 3D in vitro model of human bone marrow that contains both the perivascular and endosteal niches, complete with dynamic, perfusable vascular networks. We demonstrate that our model can replicate in vivo bone marrow function, including maintenance and differentiation of CD34+ hematopoietic stem/progenitor cells, egress of neutrophils (CD66b+), and niche-specific responses to doxorubicin and granulocyte-colony stimulating factor. Our platform provides opportunities to accelerate current understanding of human bone marrow function and drug response with high spatial and temporal resolution.

Gangliosides expressed on breast cancer cells are E-selectin ligands.

Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLe(x)) and sialyl Lewis A (sLe(a)) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin-Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin-Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis.

UVB-irradiation regulates VLA-4-mediated melanoma cell adhesion to endothelial VCAM-1 under flow conditions.

The major aspect contributing to the mortality of melanoma is its ability to spread, or metastasize. Ultraviolet B light (UVB) is considered an indirect cause of melanoma formation. However, little is known about the potential effects of UVB to melanoma metastasis. Integrins, a large family of cell adhesion molecules (CAMs) expressed on the melanoma cell surface, are important for cell signaling, growth, and migration during metastasis. Most critically, tumor cell tissue invasion is dependent on the initial interaction of tumor cells with vascular endothelium at the target organ, and there is increasing evidence for a prominent role of melanoma very late antigen-4 (VLA-4) integrin binding to its endothelial ligand vascular cell adhesion molecule-1 (VCAM-1) in this process. This research focuses on the quantitative modulation of VLA-4 integrin expression and function on melanoma cells after UVB irradiation. The present data show that at 3, 12, and 18 h post-UVB irradiation, VLA-4 expression was unchanged relative to untreated cells, but adhesion to VCAM-1 decreased significantly. Immunofluorescence studies implied that the spatial organization of VLA-4 on the melanoma cell surface contributed to the changes in avidity for VCAM-1 upon UVB irradiation. With increased understanding of the molecular mechanisms underlying melanoma-endothelial interactions upon UVB irradiation, clinical advances for melanoma may be developed.

Tumor-on-chip modeling of organ-specific cancer and metastasis.

Every year, cancer claims millions of lives around the globe. Unfortunately, model systems that accurately mimic human oncology - a requirement for the development of more effective therapies for these patients - remain elusive. Tumor development is an organ-specific process that involves modification of existing tissue features, recruitment of other cell types, and eventual metastasis to distant organs. Recently, tissue engineered microfluidic devices have emerged as a powerful in vitro tool to model human physiology and pathology with organ-specificity. These organ-on-chip platforms consist of cells cultured in 3D hydrogels and offer precise control over geometry, biological components, and physiochemical properties. Here, we review progress towards organ-specific microfluidic models of the primary and metastatic tumor microenvironments. Despite the field's infancy, these tumor-on-chip models have enabled discoveries about cancer immunobiology and response to therapy. Future work should focus on the development of autologous or multi-organ systems and inclusion of the immune system.

Human Induced Pluripotent Stem-Cardiac-Endothelial-Tumor-on-a-Chip to Assess Anticancer Efficacy and Cardiotoxicity.

Cancer remains a leading health threat in the United States, and cardiovascular drug toxicity is a primary cause to eliminate a drug from FDA approval. As a result, the demand to develop new anticancer drugs without cardiovascular toxicity is high. Human induced pluripotent stem (iPS) cell-derived tissue chips provide potentially a cost-effective preclinical drug testing platform, including potential avenues for personalized medicine. We have developed a three-dimensional microfluidic device that simultaneously cultures tumor cell spheroids with iPS-derived cardiomyocytes (iPS-CMs) and iPS-derived endothelial cells (iPS-EC). The iPS-derived cells include a GCaMP6 fluorescence reporter to allow real-time imaging to monitor intracellular calcium transients. The multiple-chambered tissue chip features electrodes for pacing of the cardiac tissue to assess cardiomyocyte function such as the maximum capture rate and conduction velocity. We measured the inhibition concentration (IC50) of the anticancer drugs, Doxorubicin (0.1 µM) and Oxaliplatin (4.2 µM), on the tissue chip loaded with colon cancer cells (SW620). We simultaneously evaluated the cardiotoxicity of these anticancer drugs by assessing the drug effect on the spontaneous beat frequency and conduction velocity of iPS-derived cardiac tissue. Consistent with in vivo observations, Doxorubicin reduced the spontaneous beating rate and maximum capture rate at or near the IC50 (0.04 and 0.22 µM, respectively), whereas the toxicity of Oxaliplatin was only observed at concentrations beyond the IC50 (33 and 9.9 µM, respectively). Our platform demonstrates the feasibility to simultaneously assess cardiac toxicity and antitumor effects of drugs and could be used to enhance personalized drug testing safety and efficacy. Impact statement Drug development using murine models for preclinical testing is no longer adequate nor acceptable both financially for the pharmaceutical industry as well as for generalized or personalized assessment of safety and efficacy. Innovative solutions using human cells and tissues provide exciting new opportunities. In this study, we report on the creation of a 3D microfluidic device that simultaneously cultures human tumor cell spheroids with cardiomyocytes and endothelial cells derived from the same induced pluripotent stem cell line. The platform provides the opportunity to assess efficacy of anticancer agents while simultaneously screening for potential cardiovascular toxicity in a format conducive for personalized medicine.

Quantitative design strategies for fine control of oxygen in microfluidic systems.

Hypoxia, or low oxygen (O2) tension, is a central feature of important disease processes including wound healing and cancer. Subtle temporal and spatial variations (≤1% change) in the concentration of O2 can profoundly impact gene expression and cellular functions. Sodium sulfite reacts rapidly with O2 and can be used to lower the O2 concentrations in PDMS-based tissue culture systems without exposing the cell culture to the chemical reaction. By carefully considering the mass transfer and reaction kinetics of sodium sulfite and O2, we developed a flexible theoretical framework to design an experimental microfluidic system that provides fine spatial and temporal control of O2 tension. The framework packages the dimensions, fluid flow, reaction rates, concentrations, and material properties of the fluidic lines and device into dimensionless groups that facilitate scaling and design. We validated the theoretical results by experimentally measuring O2 tension throughout the experimental system using phosphorescence lifetime imaging. We then tested the system by examining the impact of hypoxia inducible factor-1α (HIF-1α) on the proliferation and migration of MDA-MB-231 breast cancer cells. Using this system, we demonstrate that mild constant hypoxia (≤4%) induces HIF-1α mediated functional changes in the tumor cells. Furthermore, slow (>12 hours), but not rapid (<1 hour), fluctuations in O2 tension impact HIF-1α mediated proliferation and migration. Our results provide a generalized framework for fine temporal and spatial control of O2 and emphasize the need to consider mild spatial and temporal changes in O2 tension as potentially important factors in disease processes such as cancer.

Advances in Modeling the Immune Microenvironment of Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related death in the US. CRC frequently metastasizes to the liver and these patients have a particularly poor prognosis. The infiltration of immune cells into CRC tumors and liver metastases accurately predicts disease progression and patient survival. Despite the evident influence of immune cells in the CRC tumor microenvironment (TME), efforts to identify immunotherapies for CRC patients have been limited. Here, we argue that preclinical model systems that recapitulate key features of the tumor microenvironment-including tumor, stromal, and immune cells; the extracellular matrix; and the vasculature-are crucial for studies of immunity in the CRC TME and the utility of immunotherapies for CRC patients. We briefly review the discoveries, advantages, and disadvantages of current in vitro and in vivo model systems, including 2D cell culture models, 3D culture systems, murine models, and organ-on-a-chip technologies.

Tumor-on-a-chip platform to interrogate the role of macrophages in tumor progression.

Tumor-infiltrating leukocytes, in particular macrophages, play an important role in tumor behavior and clinical outcome. The spectrum of macrophage subtypes ranges from antitumor 'M1'-type to protumor 'M2'-type macrophages. Tumor-associated macrophages (TAMs) typically display phenotypic features of both M1 and M2, and the population distribution is thought to be dynamic and evolves as the tumor progresses. However, our understanding of how TAMs impact the tumor microenvironment remains limited by the lack of appropriate 3D in vitro models that can capture cell-cell dynamics at high spatial and temporal resolution. Using our recently developed microphysiological 'tumor-on-a-chip' (TOC) device, we present here our findings on the impact of defined macrophage subsets on tumor behavior. The TOC device design contains three adjacent and connected chambers in which both the upper and lower chambers are loaded with tumor cells, whereas the central chamber contains a dynamic, perfused, living microvascular network. Introduction of human pancreatic or colorectal cancer cells together with M1-polarized macrophages significantly inhibited tumor growth and tumor-induced angiogenesis. Protein analysis and antibody-based neutralization studies confirmed that these effects were mediated through production of C-X-C motif chemokines (CXCL9), CXCL10 and CXCL11. By contrast, M2-macrophages mediated increased tumor cell migration into the vascularized chamber and did not inhibit tumor growth or angiogenesis. In fact, single-cell RNA sequencing showed that M2 macrophages further segregated endothelial cells into two distinct subsets, corresponding to static cells in vessels versus active cells involved in angiogenesis. The impact of M2 macrophages was mediated mostly by production of matrix metalloproteinase 7 and angiopoietin 2. In summary, our data demonstrate the utility of the TOC device to mechanistically probe biological questions in a 3D in vitro microenvironment.

Microfluidic device to attain high spatial and temporal control of oxygen.

Microfluidic devices have been successfully used to recreate in vitro biological microenvironments, including disease states. However, one constant issue for replicating microenvironments is that atmospheric oxygen concentration (21% O2) does not mimic physiological values (often around 5% O2). We have created a microfluidic device that can control both the spatial and temporal variations in oxygen tensions that are characteristic of in vivo biology. Additionally, since the microcirculation is responsive to hypoxia, we used a 3D sprouting angiogenesis assay to confirm the biological relevance of the microfluidic platform. Our device consists of three parallel connected tissue chambers and an oxygen scavenger channel placed adjacent to these tissue chambers. Experimentally measured oxygen maps were constructed using phosphorescent lifetime imaging microscopy and compared with values from a computational model. The central chamber was loaded with endothelial and fibroblast cells to form a 3D vascular network. Four to six days later, fibroblasts were loaded into the side chambers, and a day later the oxygen scavenger (sodium sulfite) was flowed through the adjacent channel to induce a spatial and temporal oxygen gradient. Our results demonstrate that both constant chronic and intermittent hypoxia can bias vessel growth, with constant chronic hypoxia showing higher degrees of biased angiogenesis. Our simple design provides consistent control of spatial and temporal oxygen gradients in the tissue microenvironment and can be used to investigate important oxygen-dependent biological processes in conditions such as cancer and ischemic heart disease.

Tumor-on-a-chip platform to investigate progression and drug sensitivity in cell lines and patient-derived organoids.

Most cancer treatment strategies target cell proliferation, angiogenesis, migration, and intravasation of tumor cells in an attempt to limit tumor growth and metastasis. An in vitro platform to assess tumor progression and drug sensitivity could provide avenues to enhance our understanding of tumor metastasis as well as precision medicine. We present a microfluidic platform that mimics biological mass transport near the arterial end of a capillary in the tumor microenvironment. A central feature is a quiescent perfused 3D microvascular network created prior to loading tumor cells or patient-derived tumor organoids in an adjacent compartment. The physiological delivery of nutrients and/or drugs to the tumor then occurs through the vascular network. We demonstrate the culture, growth, and treatment of tumor cell lines and patient-derived breast cancer organoids. The platform provides the opportunity to simultaneously and dynamically observe hallmark features of tumor progression including cell proliferation, angiogenesis, cell migration, and tumor cell intravasation. Additionally, primary breast tumor organoids are viable in the device for several weeks and induce robust sprouting angiogenesis. Finally, we demonstrate the feasibility of our platform for drug discovery and personalized medicine by analyzing the response to chemo- and anti-angiogenic therapy. Precision medicine-based cancer treatments can only be realized if individual tumors can be rapidly assessed for therapeutic sensitivity in a clinically relevant timeframe (⪅14 days). Our platform indicates that this goal can be achieved and provides compelling opportunities to advance precision medicine for cancer.

Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for Three-Dimensional Microphysiological Systems.

Microphysiological systems (MPS), or "organ-on-a-chip" platforms, aim to recapitulate in vivo physiology using small-scale in vitro tissue models of human physiology. While significant efforts have been made to create vascularized tissues, most reports utilize primary endothelial cells that hinder reproducibility. In this study, we report the use of human induced pluripotent stem cell-derived endothelial cells (iPS-ECs) in developing three-dimensional (3D) microvascular networks. We established a CDH5-mCherry reporter iPS cell line, which expresses the vascular endothelial (VE)-cadherin fused to mCherry. The iPS-ECs demonstrate physiological functions characteristic of primary endothelial cells in a series of in vitro assays, including permeability, response to shear stress, and the expression of endothelial markers (CD31, von Willibrand factor, and endothelial nitric oxide synthase). The iPS-ECs form stable, perfusable microvessels over the course of 14 days when cultured within 3D microfluidic devices. We also demonstrate that inhibition of TGF-ß signaling improves vascular network formation by the iPS-ECs. We conclude that iPS-ECs can be a source of endothelial cells in MPS providing opportunities for human disease modeling and improving the reproducibility of 3D vascular networks.