This work was supported by the American Heart Association (grant number 15SDG25740035, awarded to J.Z.), the National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (grant number EB007507, awarded to C.C.), and the Alliance for Regenerative Rehabilitation Research &#38; Training (AR3T, grant number 1 P2C HD086843-01, awarded to J.Z.). We would like to acknowledge Prof. Jeanne Stachowiak (The University of Texas at Austin) for her technical advice on confocal microscopy. We are also grateful for discussions with Samuel Mihelic (The University of Texas at Austin), Dr. Alicia Allen (The University of Texas at Austin), Dr. Julie Rytlewski (Adaptive Biotech), and Dr. Leon Bellan (Vanderbilt University) for their insight on the computational analysis of 3D networks. Finally, we thank Dr. Xiaoping Bao (University of California, Berkeley) for his advice on differentiating iPSCs into iPSC-EPs.

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The authors have nothing to disclose.

This protocol involves the long-term culture of cells in three types of cell culture media: E8, LaSR Basal, and EGM-2. Therefore, great care should be taken to appropriately sterilize all materials. Additionally, lab coats and ethanol-cleaned gloves should always be worn when working in the laboratory&#8217;s laminar flow hood. It is recommended to frequently test for mycoplasma contamination; if a large amount of debris is observed during iPSC culture or a sudden drop in differentiation efficiency is noted, mycoplasma c...

Human umbilical vein endothelial cells (HUVECs) and other primary endothelial cell types have been utilized for two decades to model blood vessel sprouting and development in vitro1. Such vascular platforms promise to illuminate molecular and tissue-level mechanisms of cardiovascular disease and may present physiological insight into the development of primitive vascular networks2,3. Though the field of vascular modeling has witnessed significant advances, a &#8220;gold standard&#8221; assay that can quantitatively model and assess physiological vascular development remains elusive. Most published protocols do not adequately recapitulate the vascular niche to encourage the formation of mature, functional blood vessels or do not have a method to quantitatively compare the vasculogenic potential of the assessed cell types in three dimensions (3D).
Many current vascular models are limited in their ability to mimic the physiological vascular niche. One of the most commonly employed in vitro platforms is the gelatinous protein mixture-based tube formation assay. Briefly, HUVECs are seeded as single cells on a thin layer of gel that consists of proteins harvested from murine sarcoma extracellular matrix (ECM); within one to two days, the HUVECs self-assemble into primitive tubes4. However, this process occurs in two-dimensions (2D) and the endothelial cells (ECs) utilized in this assay do not form enclosed, hollow lumens, thereby limiting the physiological significance of these studies. More recently, ECs and supporting cells (e.g., mesenchymal stem cells (MSCs) and pericytes) have been co-cultured in 3D microenvironments that simulate the fibrous architecture of the native ECM, such as collagen or fibrin hydrogels5. To model vascular development in this microenvironment, polymeric beads coated with ECs are typically employed6. The addition of exogenous growth factors and/or growth factors secreted by other cells interstitially embedded in the hydrogel can induce the ECs, coating the polymeric beads, to sprout and form single lumen; the number and diameter of sprouts and vessels can then be computed. However, these sprouts are singular and do not form an enclosed, connected network as is seen in physiological conditions and thus is more reminiscent of a tumor vasculature model. Microfluidic devices have also been utilized to mimic the vascular niche and to promote the formation of vasculature in EC-laden hydrogels7,8. Typically, an angiogenic growth factor-gradient is applied to the circulating cell culture medium to induce EC migration and sprouting. ECs that constitute the lumen of developed vessels are sensitive to the shear stress induced by the application of fluid flow through the microfluidic device; thus, these microfluidic devices capture key physiological parameters that are not accessible in the static models. However, these devices require costly microfabrication abilities.
Most importantly, all three vascular models (2D, 3D, microfluidic) overwhelmingly utilize primary ECs as well as primary supporting cell types. Primary cells cannot be developed into an effective cardiovascular therapy because the cells would engender an immune response upon implantation; furthermore, HUVECs and similar primary cell types are not patient-specific and do not capture vascular abnormalities that occur in patients with a genetic disposition or pre-existing health conditions, e.g., diabetes mellitus. Induced pluripotent stem cells (iPSCs) have emerged in the past decade as a patient-specific, proliferative cell source that can be differentiated into all somatic cells in the human body9. In particular, protocols have been published that outline the generation and isolation of iPSC-derived endothelial progenitors (iPSC-EPs)10,11; iPSC-EPs are bipotent and can, therefore, be further differentiated into endothelial cells and smooth muscle cells/pericytes, the building blocks of mature, functional vasculature. Only one study has convincingly detailed the development of a primary capillary plexus from iPSC-EPs in a 3D microenvironment12; though this study is critical to an understanding of iPSC-EP assembly and differentiation in natural and synthetic hydrogels, it did not quantitatively compare the network topologies of the resulting vasculature. Another recent study has used the polymeric bead model to compare the sprouting of HUVECs and iPSC-derived ECs5. Therefore, there is a clear need to further elucidate the physical and chemical signaling mechanisms that regulate iPSC-EP vasculogenesis in 3D microenvironments and to determine if these cells are suitable for ischemic therapy and cardiovascular disease modeling.
In the past decade, different open-source computational pipelines and skeletonization algorithms have been developed to quantify and compare vascular network length and connectivity. For example, Charwat et al. developed a Photoshop-based pipeline to extract a filtered, binarized image of vascular networks derived from a co-culture of adipose-derived stem cells and outgrowth ECs in a fibrin matrix13,14. Perhaps the most widely used topology comparison tool is AngioTool, a program published online by the National Cancer Institute15; despite the program&#8217;s widespread adoption and well-documented fidelity, the program is limited to analyzing vessel-like structures in two dimensions and other programs, including AngioSys and Wimasis, share the same dimensionality limitation16. Powerful software suites such as Imaris, Lucis, and Metamorph have been developed to analyze the network topology of engineered microvasculature; however, these suites are cost-prohibitive for most academic labs and limit access to the source code, which may hinder the ability of the end-user to customize the algorithm to their specific application. 3D Slicer, an open-source magnetic resonance imaging/computed tomography package, contains a Vascular Modeling Toolkit that can effectively analyze the topology of 3D vascular networks17; however, the analysis is dependent on the user manually placing the end-points of the network, which may become tedious when analyzing a large dataset and can be influenced by the user&#8217;s subconscious biases. In this manuscript, a computational pipeline that can quantify 3D vascular networks is described in detail. To overcome the above-outlined limitations, this open source computational pipeline utilizes ImageJ to pre-process acquired confocal images to load the 3D volume into a skeleton analyzer. The skeleton analyzer uses a parallel medial axis thinning algorithm, and was originally developed by Kerschnitzki et al. to analyze the length and connectivity of osteocyte networks18; this algorithm can be effectively applied to characterize the length and connectivity of engineered microvasculature.
Altogether, this protocol outlines the creation of microvascular networks in 3D microenvironments and provides an open-source and user-bias free computational pipeline to readily compare the vasculogenic potential of iPSC-EPs.

<table><tbody><tr><td>&amp;micro;-Slide Angiogenesis</td><td>Ibidi</td><td>N/A</td><td>A flat, glass bottom tissue-culture plate with side walls enables facile confocal imaging</td></tr><tr><td>96 well, round bottom, ultra low attachment microplate, sterile</td><td>Corning</td><td>7007</td><td>Prevents the binding of cell-laden collagen hydrogels to the cell culture dish</td></tr><tr><td>Accutase</td><td>STEMCELL Technologies</td><td>7920</td><td>Gentle cell detachment solution; does not degrade extracellular epitopes vital for FACS</td></tr><tr><td>Advanced DMEM/F12</td><td>Thermo Scientific</td><td>12634010</td><td>The base media for iPSC-EP differentiation.&amp;nbsp;</td></tr><tr><td>Barnstead GenPure xCAD Plus&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>50136165</td><td>Water purification system; others can be readily substituted</td></tr><tr><td>Bovine Serum Albumin solution,7.5% in DPBS, sterile-filtered, BioXtra, suitable for cell culture</td><td>Fisher Scientific</td><td>A8412</td><td>To preserve cell viability when FACs sorting</td></tr><tr><td>CD34-PE, human (clone: AC136)</td><td>Miltenyi Biotec</td><td>130-098-140</td><td>Antibody used for FACs isolation of iPSC-EPs</td></tr><tr><td>CHIR99021</td><td>LC Laboratories</td><td>C-6556</td><td>Induces the formation of mesoderm from pluripotent stem cells</td></tr><tr><td>Collagen I Rat Tail High Protein 100 mg</td><td>VWR</td><td>354249</td><td>Main component of the 3D microenvironment</td></tr><tr><td>Conical centrifuge tubes (15/50 mL)</td><td>Fisher Scientific</td><td>14-959-49D/A</td><td>Used to store and mix relatively large volumes of reagents and cell culture media</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td>To counterstain and visualize cell nuclei</td></tr><tr><td>DMEM/F12</td><td>Thermo Fisher Scientific</td><td>11320-082</td><td>For dilution of Matrigel and thawing of pluripotent stem cells</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>ThermoFisher</td><td>14190-250</td><td>To wash monolayer cultures</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E8008</td><td>For passaging of pluripotent stem cell colonies and to prevent cell aggregation when FACs sorting</td></tr><tr><td>Endothelial Cell Growth Medium 2</td><td>PromoCell</td><td>C-22011</td><td>Promotes endothelial cell viability and proliferation</td></tr><tr><td>Essential 8 Medium</td><td>Thermo Fisher Scientific</td><td>A1517001&amp;nbsp;&amp;nbsp;</td><td>For maintenance of pluripotent stem cells</td></tr><tr><td>Glycine,BioUltra, for molecular biology, &gt;=99.0% (NT)</td><td>Sigma-Aldrich</td><td>50046</td><td>Neutralizes remaining detergent</td></tr><tr><td>L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate,&gt;=95%</td><td>Sigma-Aldrich</td><td>A8960</td><td>Component of iPSC-EP differentiation medium</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>1.8.0_152</td><td>Multi-paradigm numerical computing environment (free available at most academic institutions)</td></tr><tr><td>Matrigel Matrix GFR PhenolRF Mouse 10 mL (gelatinous protein mixture)</td><td>Fisher Scientific</td><td>356231</td><td>Diluted in DMEM/F12 to coat plates for iPSC-EP differentiation</td></tr><tr><td>Medium-199 10X</td><td>Thermo Fisher Scientific</td><td>1825015</td><td>Used to balance final hydrogel osmolarity and pH</td></tr><tr><td>Microcentrifuge tubes (1.7 mL)</td><td>VWR</td><td>87003-294</td><td>Stores small volumes of reagents</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P3813</td><td>The main ingredient of the immunostaining solutions</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Antibiotic used after sorting to remove possible contamination from FACS instrument</td></tr><tr><td>Recombinant Human VEGF 165 Protein</td><td>R&amp;amp;D Systems</td><td>293-VE</td><td>Mitogen that stimulates endothelial cell proliferation and tubulogenesis</td></tr><tr><td>Rhodamine phalloidin</td><td>Themo Fisher Scientific</td><td>R415</td><td>To identify F-actin deposition and therfore outline the borders of the vascular networks</td></tr><tr><td>Triton X-100 (nonionic surfactant)</td><td>Sigma-Aldrich</td><td>X-100</td><td>Detergent used to gently permeabilize cells</td></tr><tr><td>Tween-20 (emulsifying reagent)</td><td>Fisher Scientific</td><td>BP337</td><td>Increases the binding specificity of the added antibodies</td></tr><tr><td>VE-Cadherin (F-8)</td><td>Santa Cruz Biotechnology</td><td>sc-9989&amp;nbsp;</td><td>To identify 3D endothelial lumen in collagen hydrogels</td></tr><tr><td>Vitronectin</td><td>ThermoFisher</td><td>A14700</td><td>For maintenance of pluripotent stem cells</td></tr><tr><td>Y-27632</td><td>Selleck Chemicals</td><td>S1049</td><td>Preserves pluripotent stem cell and iPSC-EP viability when dissociated and re-seeded</td></tr></tbody></table>

an in vitro 3d model and computational pipeline to quantify the vasculogenic potential of ipsc-derived endothelial progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

After differentiation (Figure 1), FACS&#160;and encapsulation of iPSC-EPs in collagen hydrogels, the cells will typically remain rounded for 24 h before beginning to migrate and form initial lumens. After about 6 days of culture, a primitive capillary plexus will be visible in the hydrogel when viewed with brightfield microscopy (Figure 2). After imaging the fixed, stained cell-laden hydrogels on a confocal microscope (Mo...

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An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

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6.0K Views.  University of Texas at Austin. Ons protocol maakt unieke inzichten in de moleculaire en bulk weefsel niveau mechanismen van vasculogenese die kunnen helpen bij de engineering functionele vasculatuur voor hart-en vaatziekten therapie in modellering. Dit protocol maakt het mogelijk om robuuste, driedimensionale vasculaire netwerken te creëren voor het evalueren van het vasculogene potentieel van verschillende platforms, en biedt ook een gratis, open source computationele pijplijn voor het analyseren van de uiteindelijke net...

Video: An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Cancer Research

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Bioengineering

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Author Spotlight: Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

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