This work was supported by grants from the Nederlandse organisatie voor gezondheidsonderzoek en zorginnovatie (ZonMW 446002501), Health Holland (LSHM19057-H040), Leading Fellows Programme Marie Sk&#322;odowska-Curie COFUND, and by the Association Maladie de Rendu-Osler (AMRO).

1. Media preparation and culture of mESC
<ol>
	<li>Prepare conditioned medium +/- (CM+/-) using the supplement 1x Glasgow MEM (G-MEM BHK-21) medium with 10% (vol/vol) heat-inactivated Fetal Bovine Serum (FBS), 0.05 mM &#946;-mercaptoethanol, 1x non-essential amino acids (NEAA 1x), 2 mM L-glutamine, and 1 mM sodium pyruvate.</li>
	<li>Prepare conditioned medium +/+ (CM+/+) using the supplement CM+/- medium with Leukemia Inhibitory Factor (LIF) (1,500 U/mL) and 0.1 mM &#946;-mercaptoethanol.</li>
	<li>Prepare conditioned medium +/+ in the presence of two inhibitors (CM+/+ 2i) using the supplement CM+/+ medium with 1 &#181;M PD0325901 and 3 &#181;M CHIR99021.</li>
	<li>Prepare 0.1% gelatin solution by mixing 25 mL of pre-warmed 2% gelatin solution in 500 mL of phosphate-buffered saline without calcium and magnesium (DPBS).</li>
	<li>Prepare 10x Trypsin Versene Phosphate (TVP 10x) buffer by mixing Trypsin (2.5%) with TVP 1x (9.5% 10x DPBS, 1 mM EDTA, 0.01% chicken serum, 0.01% Trypsin (2.5%) in H2O) (1:10 ratio, vol/vol). 
	NOTE: Filter all the solutions through a 0.22 &#181;m pore filter. Store the culture medium at -20 &#176;C for long term and keep other reagents at 4 &#176;C for up to 3 weeks (see Table of Materials).</li>
	<li>Coat two 12-well cell culture plates with 0.1% gelatin solution (500 &#181;L per well) and incubate them for 30 min in a CO2 incubator (37 &#176;C, 5% CO2, humid atmosphere).</li>
	<li>Wash the gelatin-coated plates with PBS and add 500 &#181;L of CM+/- medium.</li>
	<li>Thaw two vials of 1 x 106 cryopreserved irradiated mouse embryonic fibroblasts (MEFs) at 37 &#176;C and transfer the cell suspension to a conical tube with 5 mL of CM+/- medium.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at room temperature (RT). Aspirate the medium and gently resuspend the cell pellet in 12 mL of CM+/- medium at a concentration of 1.67 x 105 cells per mL.</li>
	<li>Seed 500 &#181;L of MEF suspension in a 12-well cell culture plate (2.4 x 105 cells per cm2) and incubate the plate overnight (o/n) in a CO2 incubator.</li>
	<li>Thaw one vial of cryopreserved mESCs (1 x 106) in CM+/+ 2i medium.</li>
	<li>Seed the mESC suspension on a pre-washed 12 well plate with MEFs in 1 mL of CM+/+ 2i medium and transfer the plate to a CO2 incubator. Refresh the medium daily.</li>
	<li>At 70% confluency, wash the mESC colonies with DPBS. Add 150 &#181;L of warm 10x TVP buffer and incubate at RT for 30 s to initiate enzymatic dissociation.</li>
	<li>Carefully remove the TVP buffer, resuspend the cells in 1 mL of CM+/+ 2i medium and dissociate the colonies into single cells by gentle pipetting.</li>
	<li>Passage the cells by transferring them to a new 12-well cell culture plate with MEFs (splitting ratio: 1:3-1:5). Mix gently to distribute the cells and incubate in a CO2 incubator.</li>
	<li>Refresh the culture medium (2-3 mL) and observe the cell growth/morphology daily. Repeat serial passaging at 70% confluency every 2 days. Switch to CM+/+ medium for two passages before initiating cell differentiation.</li>
</ol>
2. EB formation in hanging drop
<ol>
	<li>Prepare fresh mesoderm differentiation medium by supplementing the CM+/- medium with basic Fibroblast Growth Factor (bFGF) (50 ng&#183;mL-1) and with Bone Morphogenetic Protein 4 (BMP-4) (5 ng&#183;mL-1) and keep it at 4 &#176;C until use.</li>
	<li>Coat one well of the 6-well cell culture plate with 500 &#181;L of 0.1% gelatin solution and place it in a CO2 incubator for 30 min.</li>
	<li>Wash the gelatin-coated plates with PBS and add 500 &#181;L of CM+/- medium.</li>
	<li>To obtain a pure population of mESCs, trypsinize the cell culture plate with 10x TVP buffer for 30 s at RT, resuspend the cells in 1 mL mesoderm differentiation medium and then transfer them to the gelatin-coated 6-well plate for 30 min allowing the MEFs to attach while the mESCs stay in suspension.</li>
	<li>Collect the cell suspension in a 50 mL conical tube and count the cells using a Neubauer hemocytometer and Trypan blue dye for a live/dead cell exclusion.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at RT. Remove the supernatant and resuspend the cell pellet in mesoderm differentiation medium to reach 4.55 x 104 cells per mL.</li>
	<li>Fill the bottom of 94 mm low attachment polystyrene dishes with 15 mL of sterile water. 
	&#8203;NOTE: Four dishes containing hanging drops (1.6 x 105 cells in 3.52 mL of medium) will be required for testing one particular condition using the 3D sprouting angiogenesis assay.</li>
	<li>Transfer the cell suspension into a sterile plastic reservoir and load four positions of a multichannel pipette with 22 &#181;L of cell suspension per channel (1 x 103 cells per 22 &#181;L drop).</li>
	<li>Lift and invert the lid of the 94 mm dish and place it on the clean surface of the flow cabinet with the inner side facing upwards.</li>
	<li>Deposit 40 drops of the cell suspension on the inner surface of each lid. Carefully invert the lid without disturbance and place it back on the dish, so that the drops face the water.</li>
	<li>Incubate the dishes in a CO2 incubator. Consider this as differentiation day 0. Maintain the plates for 4 days to form EBs.</li>
</ol>
3. Competition assay for the tip cell position
<ol>
	<li>Culture one fluorescent and one non-fluorescent mESC&#160;line as previously described13. As an example, 7ACS/EYFP mESCs labeled in yellow and R1 mESCs are used.</li>
	<li>Prepare mosaic EBs by mixing equal amounts of the two mESC lines (1:1 ratio) and incubate the hanging drop dishes in a CO2 incubator as described in step 2.</li>
</ol>
4. Floating EBs culture for vascular differentiation
<ol>
	<li>Before collecting EBs from the hanging drops, prepare the following.
	<ol>
		<li>Prepare 5% agar solution in H2O and sterilize it by autoclaving (20 min at 120 &#176;C).</li>
		<li>Use the warm 5% agar solution to prepare G-MEM BHK-21 medium containing 1% agar and quickly pour 3 mL in one of the 60 mm polystyrene dishes. Allow the agar to solidify for 1 h at RT. Store the dishes at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Prepare fresh 2x vascular differentiation medium by supplementing the CM+/- medium with bFGF (100 ng&#183;mL-1) and VEGF-A (50 ng&#183;mL-1). Store the medium at 4 &#176;C until use.</li>
	<li>Collect the hanging drops in a 15 mL conical tube using a P1000 pipette and remove the supernatant after a few minutes of EB sedimentation.</li>
	<li>Resuspend the EBs in 3 mL of 2x vascular differentiation medium, transfer the EB suspension to one agar-coated dishand distribute the EBs homogenously to avoid aggregation.</li>
	<li>Incubate the dishes in CO2 incubator and refresh the medium every 2 days until day 9 using 1x vascular differentiation medium in the presence of bFGF (50 ng&#183;mL-1) and VEGF-A (25 ng&#183;mL-1).</li>
	<li>Alternatively, add Platelet Derived Growth Factor-BB (PDGF-BB) (10 ng&#183;mL-1 on day 4 and 5 ng&#183;mL-1 on day 6 and day 8) to the vascular differentiation medium to promote mural cell differentiation.</li>
</ol>
5. Flow cytometry analysis
<ol>
	<li>Collect the 9-day-old EBs in a 15 mL conical tube using a P1000 pipette and wash them once with warm PBS.</li>
	<li>Add 1mL of G-MEM BHK-21 medium containing 0.2 mg&#183;mL-1 of collagenase A and incubate the cells in a CO2 incubator for 5 min.</li>
	<li>Dissociate the EBs by gently pipetting up and down with a P1000 pipette.</li>
	<li>Stop the collagenase activity by adding 1 mL of cold G-MEM BHK-21 medium with 10% FBS.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at RT.</li>
	<li>Resuspend the cells in 500 &#181;L of PBS with 2% FBS.</li>
	<li>Count the cells using a Neubauer hemocytometer.</li>
	<li>Resuspend 400,000 cells in 100 &#181;L of PBS with 2% FBS per staining condition.</li>
	<li>Incubate the cells for 45 min at 4 &#176;C with the following antibodies: APC conjugated rat anti-mouse PECAM-1 antibody (clone MEC13.3) and FITC conjugated rat anti-mouse CD45 (clone 30-F11) or isotype control antibodies.</li>
	<li>Wash the cells twice with 1 mL of PBS containing 2% FBS.</li>
	<li>Resuspend the cells to reach a final concentration of 5 x 106 cells per mL.</li>
	<li>Filter the cells using a round bottom polystyrene test tube, with a cell strainer snap cap.</li>
	<li>Analyze 20,000 PECAM-1 (+) events by flow cytometry.</li>
</ol>
6. 3D sprouting angiogenesis assay and immunofluorescence staining
<ol>
	<li>At day 9, prepare a sprouting medium by adding 10% FBS (vol/vol), bFGF (50 ng&#183;mL-1), VEGF-A (25 ng&#183;mL-1), human recombinant Erythropoietin (hEPO) (20 ng&#183;mL-1), human interleukin-6 (IL-6) (10 ng&#183;mL-1), 0.05 mM &#946;-mercaptoethanol, NEAA (1x), L-glutamine (1x), sodium pyruvate (1x), type I rat tail collagen (1.25 mg&#183;mL-1), and NaOH (3.1 mM) to GMEM BHK-1 medium.</li>
	<li>To avoid collagen gelation, maintain the sprouting medium on ice until use.</li>
	<li>To evaluate the effect of pro/anti-angiogenic molecules, add the selected drug or vehicle to the sprouting medium at the selected concentration.</li>
	<li>Collect 9-day-old EBs from one 60 mm agar dish in a 15 mL conical tube (equivalent to one condition) and remove the supernatant after a few minutes of sedimentation.</li>
	<li>Cover the bottom of a 35 mm culture dish with 1 mL of the sprouting medium and incubate at 37 &#176;C for 5 min to induce gelation.</li>
	<li>Resuspend the EBs in 2 mL of cold sprouting medium.</li>
	<li>Transfer the suspension to the 35 mm culture dish coated with the first layer of sprouting medium.</li>
	<li>Distribute the EBs all over the plate and ensure they are at an equal distance from each other. Incubate the dish in a CO2 incubator. The first sprout formation happens within 24-48 h.</li>
	<li>At day 12, the collagen gel containing EBs is carefully transferred to a glass slide (75 x 26 mm) using a spatula.</li>
	<li>Remove the excess liquid using a pipette (P1000) and dehydrate the gel by placing a gauze sheet of nylon linen and absorbent filter cards (gel blotting paper) on top of the gel. Apply pressure by placing a weight (250 g) for 2 min. Carefully remove the nylon/filter papers and allow the slides to air dry for 30 min at RT.</li>
	<li>Wash the slides three times with PBS for 5 min at RT.</li>
	<li>Fix the EBs using zinc solution (see Table of Materials) o/n at 4 &#176;C. Alternatively, fix the mosaic fluorescent EBs with Paraformaldehyde (PFA) (4%) o/n at 4 &#176;C in the dark.</li>
	<li>Remove the fixative. Wash the slides five times with PBS for 5 min at RT.</li>
	<li>Permeabilize the EBs in PBS containing 0.1% Triton-X100 for 15 min at RT.</li>
	<li>Remove the permeabilization solution. Wash the slides five times with PBS for 5 min.</li>
	<li>Incubate the EBs in the blocking buffer (PBS with 2% Bovine Serum Albumin, BSA) for 1 h at RT.</li>
	<li>To stain the endothelial sprouts, use a rat anti-mouse anti-PECAM-1 primary antibody (1:100 dilution) in blocking buffer o/n at 4 &#176;C.</li>
	<li>Wash the slides five times with PBS for 5 min.</li>
	<li>Incubate the slides with goat anti-rat Alexa 555 secondary antibody in blocking buffer (1:250 dilution) and when required, with FITC-conjugated anti-&#945;-SMA antibody in blocking buffer (1:250 dilution) to stain mural cells for 2 h at RT in the dark.</li>
	<li>Wash the slides three times with PBS for 5 min and one time with H20 before mounting them.</li>
</ol>
7. Confocal imaging, morphometric, and quantitative analysis of EB endothelial sprouts
<ol>
	<li>Acquire high-resolution images of the immunostained EBs by focal plane merging (z-stacking) using a confocal microscope. Use 10x magnification objective to image whole EBs.</li>
	<li>Analyze the images acquired using ImageJ to evaluate the morphology and quantify the characteristics of PECAM-1 positive endothelial sprouts according to established quantification methods13,14,21.
	<ol>
		<li>Calculate the mean number of endothelial sprouts per EBs by manually counting the total sprout number per individual EBs.</li>
		<li>Measure the individual sprout lengths using the ImageJ drawing tool. Define the base of the endothelial sprout starting at the EB core area and manually draw a line until the sprout tip end.</li>
		<li>Calculate the mean number of tip cells per sprout by manually counting the number of tip cells per individual sprout and then calculate the mean per EB.</li>
		<li>Calculate the filopodia orientation by manually determining the axis of the parent sprout and measure the acute angle between them using the ImageJ software angle tool. Calculate the number of sprouts with an orientation &#62;50&#176; and divide it by the total number of sprouts of the EB of interest. 
		NOTE: The angles always ranged from 0&#176; to 90&#176;.</li>
	</ol>
	</li>
	<li>Acquire high-resolution images of the immunostained EBs using a confocal microscope. Use 40x magnification objective to realize high-resolution images of single endothelial sprouts.</li>
	<li>Quantify the vessel coverage of PECAM-1 positive EC sprouts surrounded by &#945;-SMA positive MCs using the ImageJ software.
	<ol>
		<li>Split the merged images into separate red and green channels.</li>
		<li>Convert the images into its binary form.</li>
		<li>Measure the total cellular area of the sprout occupied separately by PECAM-1 (endothelial cells labeled in red) and by &#945;-SMA (mural cells labeled in green) positive cells.</li>
		<li>Generate the merged image using the image calculator function and the AND operator. Measure the total cellular area of the image. To calculate the coverage, divide the area of the colocalized image by the area of the PECAM-1 binary image.</li>
	</ol>
	</li>
	<li>To analyze the cell competition for endothelial tip/stalk cell position of sprouts developed by mosaic EBs, manually score the number of tip cells and mark their genotypic origin based on the fluorescence signal. Calculate the mean values per EB.</li>
</ol>

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(186), (2007).</li><li>Gau, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+intervention+of+MKL/SRF+signaling+by+CCG-1423+impedes+endothelial+cell+migration+and+angiogenesis.">Pharmacological intervention of MKL/SRF signaling by CCG-1423 impedes endothelial cell migration and angiogenesis.</a> Angiogenesis. 20 (4), 663-672 (2017).</li><li>Torres-Estay, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgens+modulate+male-derived+endothelial+cell+homeostasis+using+androgen+receptor-dependent+and+receptor-independent+mechanisms.">Androgens modulate male-derived endothelial cell homeostasis using androgen receptor-dependent and receptor-independent mechanisms.</a> Angiogenesis. 20 (1), 25-38 (2017).</li><li>Merjaneh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-angiogenic+capacities+of+microvesicles+produced+by+skin+wound+myofibroblasts.">Pro-angiogenic capacities of microvesicles produced by skin wound myofibroblasts.</a> Angiogenesis. 20 (3), 385-398 (2017).</li><li>Rezzola, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+ex+vivo+retina+angiogenesis+assays.">In vitro and ex vivo retina angiogenesis assays.</a> Angiogenesis. 17 (3), 429-442 (2014).</li><li>Wang, X., Phan, D. T. T., George, S. C., Hughes, C. C. W., Lee, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Anastomosed+microvascular+network+model+with+living+capillary+networks+and+endothelial+cell-lined+microfluidic+channels.">3D Anastomosed microvascular network model with living capillary networks and endothelial cell-lined microfluidic channels.</a> Methods in Molecular Biology. 1612, 325-344 (2017).</li><li>Nicosia, R. F., Ottinetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+microvessels+in+serum-free+matrix+culture+of+rat+aorta.+A+quantitative+assay+of+angiogenesis+in+vitro.">Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.</a> Laboratory Investigation; A Journal of Technical Methods and Pathology. 63 (1), 115-122 (1990).</li><li>Nicosia, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aortic+ring+model+of+angiogenesis:+a+quarter+century+of+search+and+discovery.">The aortic ring model of angiogenesis: a quarter century of search and discovery.</a> Journal of Cellular and Molecular Medicine. 13 (10), 4113-4136 (2009).</li><li>Nowak-Sliwinska, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+guidelines+for+the+use+and+interpretation+of+angiogenesis+assays.">Consensus guidelines for the use and interpretation of angiogenesis assays.</a> Angiogenesis. 21 (3), 425 (2018).</li><li>Belair, D. G., Schwartz, M. P., Knudsen, T., Murphy, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+endothelial+cell+sprouting+assay+in+synthetic+hydrogel+arrays.">Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays.</a> Acta Biomaterialia. 39, 12-24 (2016).</li><li>Bezenah, J. R., Kong, Y. P., Putnam, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+potential+of+endothelial+cells+derived+from+human+induced+pluripotent+stem+cells+to+form+microvascular+networks+in+3D+cultures.">Evaluating the potential of endothelial cells derived from human induced pluripotent stem cells to form microvascular networks in 3D cultures.</a> Scientific Reports. 8 (1), 2671 (2018).</li><li>Henderson, A. R., Choi, H., Lee, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+and+lymphatic+vasculatures+on-chip+platforms+and+their+applications+for+organ-specific+in+vitro+modeling.">Blood and lymphatic vasculatures on-chip platforms and their applications for organ-specific in vitro modeling.</a> Micromachines (Basel). 11 (2), 147 (2020).</li><li>Lin, D. S. Y., Guo, F., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+organ-specific+vasculature+with+organ-on-a-chip+devices.">Modeling organ-specific vasculature with organ-on-a-chip devices.</a> Nanotechnology. 30 (2), 024002 (2019).</li><li>Pollet, A., den Toonder, J. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+the+vasculature+using+organ-on-chip+technology.">Recapitulating the vasculature using organ-on-chip technology.</a> Bioengineering. 7 (1), 17 (2020).</li><li>Cochrane, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+in+vitro+models+of+vascular+biology:+Human+induced+pluripotent+stem+cells+and+organ-on-chip+technology.">Advanced in vitro models of vascular biology: Human induced pluripotent stem cells and organ-on-chip technology.</a> Advanced Drug Delivery Reviews. 140, 68-77 (2019).</li></ol>

The authors have nothing to disclose.

This protocol describes an unbiased, robust, and reproducible 3D EB-based vascular sprouting assay that is amenable to screening for drugs and genes modulating angiogenesis. This method offers advantages over many widely used two dimensional (2D) assays using endothelial cell cultures such as Human Umbilical Vein Endothelial Cells (HUVECs) to monitor migration (lateral scratch assay or the Boyden chamber assay)22,23&#160;or proliferation (counting cell number, de...

In the past three decades, target-based drug discovery (TDD) has been widely employed in drug discovery by the pharmaceutical industry. TDD incorporates a defined molecular target playing an important role in a disease and relies on the development of relatively simple cell culture systems and readouts for drug screening1. Most typical disease models used in TDD programs include traditional cell culture methods such as cancer cells or immortalized cell lines grown within artificial environments and non-physiological substrates. Although many of these models have provided viable tools for identifying successful drug candidates, the use of such systems can be questionable owing to their poor disease relevance2.
For most diseases, the underlying mechanisms are indeed complex and various cell types, independent signaling pathways, and multiple sets of genes are often found to contribute to a specific disease phenotype. This is also true for inherited diseases where the primary cause is a mutation in one single gene. With the recent advent of human induced pluripotent stem cell (iPSC) technologies and gene editing tools, it is now possible to generate 3D organoids and organ-on-chip disease models that could better recapitulate the in vivo human complexity3,4. The development of such technologies is associated with a resurgence in interest in phenotypic drug discovery (PDD) programs1. PDD can be compared to empirical screening, as they do not rely on knowledge of the identity of a specific drug target or a hypothesis about its role in disease. The PDD approach is now increasingly recognized to strongly contribute to the discovery of first-in-class drugs5. Because the development of human organoid and organ-on-chip technologies is still in its infancy, it is expected that iPSC models (complemented with innovative imaging and machine-learning tools6,7) will provide, in the near future, multiple novel complex cell-based disease models for drug screening and associated PDD programs to overcome the poor productivity of the TDD approach8,9.
While human organoid and organ-on-chip models can provide important insights into disease complexity and to the identification of novel drugs, bringing drugs into new clinical practice also strongly relies on data from animal models to assess their efficacy and safety. Among them, genetically modified mice are certainly the most preferred mammalian models. They have many advantages as they have a relatively short generation time for mammals, have many similar phenotypes to human diseases, and can be easily genetically manipulated. They are therefore extensively used in drug discovery programs10. However, bridging the gap between mice and humans remains an important challenge11. The development of in vitro mouse models equivalent to human organoid and organ-on-chip models could at least partially fill this gap as it will allow direct drug efficacy and safety comparisons between in vivo mouse and in vitro human data.
Here, a vascular sprouting assay in mouse embryoid bodies (EBs) is described. Blood vessels are composed of endothelial cells (inner lining of vessel walls), mural cells (vascular smooth muscle cells&#160;and pericytes)12. This protocol is based on the differentiation of mouse embryonic stem cells (mESCs) into vascularized EBs using hanging droplets that recapitulate de novo endothelial cell and mural cell differentiation13,14. Mouse ESCs can be easily established in culture from isolated day 3.5 mouse blastocysts having different genetic backgrounds15. They also provide possibilities for clonal analysis, lineage tracing, and can be easily genetically manipulated to generate disease models13,16.
As blood vessels nourish all organs, it is not surprising that many diseases if not all, are associated with changes in the microvasculature. In pathological conditions, endothelial cells can adopt an activated state or can become dysfunctional resulting in mural cell death or migration away from blood vessels. These can result in excessive angiogenesis or in vessel rarefaction, can induce abnormal blood flow and defective blood vessel barrier leading to immune cell extravasation, and inflammation12,17,18,19. Research for the development of drugs modulating blood vessels is, therefore&#160;high, and multiple molecular players and concepts have already been identified for therapeutic targeting. In this context, the protocol described is particularly suitable for building disease models and for drug testing as it recapitulates key features of in vivo sprouting angiogenesis, including endothelial tip and stalk cell selection, endothelial cell migration and proliferation, endothelial cell guidance, tube formation, and mural cell recruitment. It also shows similarities with recently described 3D vascular assays based on human iPSC technologies20.

<table><tbody><tr><td>2-mercaptoethanol</td><td>Milipore, Merck</td><td>805740</td><td>Biohazard: adequate safety instructions should be taken when handling</td></tr><tr><td>Agar Noble</td><td>Difco, BD Pharmigen</td><td>214220</td><td></td></tr><tr><td>Alexa Fluo 555 goat anti rat IgG</td><td>Life technologies</td><td>A21434</td><td></td></tr><tr><td>APC conjugated rat anti-mouse PECAM-1 antibody (clone MEC13.3)</td><td>BD Biosciences</td><td>551262</td><td></td></tr><tr><td>APC Rat IgG2a &amp;kappa; Isotype Control (Clone&amp;nbsp; R35-95)</td><td>BD Biosciences</td><td>553932</td><td></td></tr><tr><td>Axiovert 25 inverted phase contrast tissue culture microscope</td><td>ZEISS</td><td></td><td></td></tr><tr><td>Basic Fibroblast Growth Factor-2 (bFGF)</td><td>Peprotech</td><td>450-33</td><td></td></tr><tr><td>Benchtop Centrifuge, Allegra X-15R</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>Biosafety cabinet BioVanguard (Green Line)</td><td>Telstar</td><td>133H401001</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Cell counting chamber, Buerker, 0.100mm</td><td>Marienfeld</td><td>640211</td><td></td></tr><tr><td>Cell culture dishes 60 x 15mm</td><td>Corning</td><td>353802</td><td></td></tr><tr><td>Cell culture dishes, 35 x 10 mm</td><td>Corning</td><td>353801</td><td></td></tr><tr><td>Cell culture plates 12-well</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>CFX96 Touch Real-Time PCR Detection System</td><td>Biorad</td><td>1855196</td><td></td></tr><tr><td>Chicken serum</td><td>Sigma-Aldrich</td><td>C5405</td><td></td></tr><tr><td>CHIR-99021 (CT99021) HCl</td><td>Selleckchem</td><td>S2924</td><td></td></tr><tr><td>Collagen I, High Concentration, Rat Tail, 100mg</td><td>Corning</td><td>354249</td><td></td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103586001</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope, TCS SP5</td><td>Leica</td><td></td><td></td></tr><tr><td>Cover glasses, 24 &amp;times; 50 mm</td><td>Vwr</td><td>631-0146</td><td></td></tr><tr><td>DAPT &amp;gamma;?secretase inhibitor</td><td>Sigma Aldrich</td><td>D5942</td><td></td></tr><tr><td>DC101 anti mouse VEGFR-2 Clone</td><td>BioXcell</td><td>BP0060</td><td></td></tr><tr><td>DC101 isotype rat IgG1</td><td>BioXcell</td><td>BP0290</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2438-5X</td><td>Biohazard: adequate safety instructions should be taken when handling</td></tr><tr><td>DPBS (10x), no calcium, no magnesium</td><td>Gibco, Thermofisher scientific</td><td>14200067</td><td></td></tr><tr><td>EDTA 40 mM</td><td>Gibco, Thermofisher scientific</td><td>15575-038</td><td></td></tr><tr><td>Embryonic stem-cell Fetal Bovine Serum</td><td>Gibco, Thermofisher scientific</td><td>16141-079</td><td>Should be lot-tested for maximum ES cell viability and growth. Heat inactivate at 60&amp;deg;C and store at &amp;minus;20 &amp;deg;C for up to 1 year</td></tr><tr><td>Eppendorf Microcentrifuge 5415R</td><td>Eppendorf AG</td><td>&amp;nbsp;Z605212</td><td></td></tr><tr><td>Erythropoietin, human (hEPO), 250 U (2.5 &amp;micro;g) (1 mL)</td><td>Roche</td><td>11120166001</td><td></td></tr><tr><td>ESGRO Recombinant Mouse LIF Protein (10? units&amp;nbsp;&amp;nbsp;1 mL)</td><td>Milipore, Merck</td><td>ESG1107</td><td></td></tr><tr><td>Falcon tubes 15 mL</td><td>Greiner Bio-One</td><td>188271</td><td></td></tr><tr><td>Falcon tubes 50 mL</td><td>Greiner Bio-0ne</td><td>227270</td><td></td></tr><tr><td>Filter tip ,clear ,sterile F.Gilson, P-200</td><td>Greiner Bio-One</td><td>739288</td><td></td></tr><tr><td>Filter tip ,clear ,sterile F.Gilson, P10</td><td>Greiner Bio-One</td><td>771288</td><td></td></tr><tr><td>Filter tip ,clear ,sterile F.Gilson, P1000</td><td>Greiner Bio-One</td><td>740288</td><td></td></tr><tr><td>FITC conjugated anti-&amp;alpha; Smooth Muscle Actin (SMA) (clone 1A4)</td><td>Sigma Aldrich</td><td>F3777</td><td></td></tr><tr><td>FITC conjugated rat anti-mouse CD45 (clone 30-F11)</td><td>Biolegend</td><td>103107</td><td></td></tr><tr><td>FITC Rat IgG2b, &amp;kappa; Isotype Ctrl Antibody (clone RTK4530)</td><td>Biolegend</td><td>400605</td><td></td></tr><tr><td>Fluorscent mounting media</td><td>DAKO</td><td>S3023</td><td></td></tr><tr><td>Gascompress</td><td>Cutisoft</td><td>45846</td><td></td></tr><tr><td>Gauze Cutisoft 10 x 10 cm</td><td>Bsn Medical</td><td>45844_00</td><td></td></tr><tr><td>Gel blotting paper, Grade GB003</td><td>Whatman</td><td>WHA10547922</td><td></td></tr><tr><td>Gelatin solution, type B</td><td>Sigma-Aldrich</td><td>G1393-100 ml</td><td></td></tr><tr><td>Glasgow&#039;s MEM (GMEM)</td><td>Gibco, Thermofisher scientific</td><td>21710082</td><td></td></tr><tr><td>IHC Zinc Fixative</td><td>BD Pharmigen</td><td>550523</td><td></td></tr><tr><td>IncuSafe CO2 Incubator</td><td>PHCBi</td><td>MCO-170AICUV-PE</td><td></td></tr><tr><td>Interleukin-6, human (hIL-6)</td><td>Roche</td><td>11138600001</td><td></td></tr><tr><td>L-Glutamine 200 mM</td><td>Gibco, Thermofisher scientific</td><td>25030-024</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Gibco, Thermofisher scientific</td><td>11140035</td><td></td></tr><tr><td>Microscope slide box</td><td>Kartell Labware</td><td>278</td><td></td></tr><tr><td>Microscope slide, Starfrost</td><td>Knittel glass</td><td>VS113711FKB.0</td><td></td></tr><tr><td>Mm_Cdh5_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00110467</td><td></td></tr><tr><td>Mm_Eng_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00148981</td><td></td></tr><tr><td>Mm_Epha4_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00093576</td><td></td></tr><tr><td>Mm_Ephb2_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00154014</td><td></td></tr><tr><td>Mm_Flt1_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00096292</td><td></td></tr><tr><td>Mm_Flt4_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00099064</td><td></td></tr><tr><td>Mm_Gapdh_3_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT01658692</td><td></td></tr><tr><td>Mm_Kdr_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00097020</td><td></td></tr><tr><td>Mm_Notch1_1_SG QuantiTect Primer</td><td>Qiagen</td><td>QT00156982</td><td></td></tr><tr><td>Mm_Nr2f2_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00153104</td><td></td></tr><tr><td>Mm_Pecam1_1_SG QuantiTect Primer</td><td>Qiagen</td><td>QT01052044</td><td></td></tr><tr><td>Mm_Tek_1_SG QuantiTect Primer Assay</td><td>Qiagen</td><td>QT00114576</td><td></td></tr><tr><td>Mouse (ICR) Inactivated Embryonic Fibroblasts&amp;nbsp; (2 M)</td><td>Gibco, Thermofisher scientific</td><td>A24903</td><td>Store vials in liquid nitrogen (195.79 &amp;deg;C) indefinitely</td></tr><tr><td>Mouse embryonic stem cell line 7AC5/EYFP (ATCC SCRC-1033)</td><td>ATCC</td><td>SCRC-1033</td><td>Generated by Dr A Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, Ontario, M5G 1X5, Canada. [Hadjantonakis, A. K., et al. Mechanisms of Development. 76 (1&amp;ndash;2), 79&amp;ndash;90 (1998)].</td></tr><tr><td>Mouse embryonic stem cell lines Acvrl1 +/- and Acvrl1 +/+</td><td></td><td></td><td>Generated at Leiden University Medical Centre [Thalgott, J.H. et al. Circulation. 138 (23), 2698&amp;ndash;2712 (2018)].</td></tr><tr><td>Mouse embryonic stem cells line E14</td><td></td><td></td><td>Provided by M Letarte laboratory and generated according to Cho, S. K., et al. Blood. 98 (13), 3635&amp;ndash;3642 (2001).</td></tr><tr><td>Mouse embryonic stem cells line R1 (ATCC SCRC-1011)</td><td>ATCC</td><td>SCRC-1011</td><td>Generated by Dr A Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, Ontario, M5G 1X5, Canada. [Nagy, A., et al. Procedings of the National Academy of Sciences of the United States of America. 90 (18), 8424&amp;ndash;8428 (1993)].</td></tr><tr><td>Mouse embryonic stem cells line Z/Red (strain 129/Ola)</td><td></td><td></td><td>Generated by Dr A Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, Ontario, M5G 1X5, Canada [Vintersten, K., et al. Genesis. 40 (4), 241&amp;ndash;246 (2004)].</td></tr><tr><td>NanoDrop 1000 UV/VIS Spectrophotometer</td><td>Thermo Fischer Scientific</td><td>ND-1000</td><td></td></tr><tr><td>PD0325901</td><td>Selleckchem</td><td>S1036</td><td></td></tr><tr><td>PDGF-BB, Recombinant Human</td><td>Peprotech</td><td>100-14B</td><td></td></tr><tr><td>Pecam-1 antibody, Rat Anti-Mouse</td><td>BD Biosciences</td><td>550274</td><td></td></tr><tr><td>Penicillin-streptomycin (10,000 U/mL)</td><td>Gibco, Thermofisher scientific</td><td>15140122</td><td></td></tr><tr><td>Petri dish, PS, 94/16 mm, standard ,with vents, sterile</td><td>Greiner Bio-One</td><td>633181</td><td></td></tr><tr><td>Pipetboy acu 2</td><td>Integra-Biosciences</td><td>155 019</td><td></td></tr><tr><td>Pipetman G Multichannel P8 x 200G</td><td>Gilson</td><td>F144072</td><td></td></tr><tr><td>Pipetman G Starter Kit, 4 Pipette Kit, P2G, P20G, P200G, P1000G</td><td>Gilson</td><td>F167360</td><td></td></tr><tr><td>Recombinant Human BMP-4 Protein</td><td>R&amp;amp;D Systems</td><td>314-BP</td><td></td></tr><tr><td>RNeasy Plus mini Kit</td><td>QIAGEN</td><td>74134</td><td></td></tr><tr><td>Serological pipettes, 10 mL</td><td>Greiner Bio-One</td><td>607 180</td><td></td></tr><tr><td>Serological pipettes, 25 mL</td><td>Greiner Bio-One</td><td>760 180</td><td></td></tr><tr><td>Serological pipettes, 5 mL</td><td>Greiner Bio-One</td><td>606 180</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Merck</td><td>106498</td><td></td></tr><tr><td>Sodium pyruvate 100 mM</td><td>Gibco, Thermofisher scientific</td><td>11360039</td><td></td></tr><tr><td>Test tubes 5ml round-bottom with cell-strainer cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Thermal cycler, T100</td><td>Biorad</td><td>1861096</td><td></td></tr><tr><td>Triton X-100 (BioXtra)</td><td>Sigma Aldrich</td><td>T9284</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco, Thermofisher scientific</td><td>15250061</td><td></td></tr><tr><td>Trypsin (2.5%)</td><td>Gibco, Thermofisher scientific</td><td>15090046</td><td></td></tr><tr><td>Vacuum Filter/Storage Bottle System, 500 mL</td><td>Corning</td><td>430758</td><td></td></tr><tr><td>VEGFA&lt;sup&gt;165&lt;/sup&gt; , recombinant murine</td><td>Peprotech</td><td>450-32</td><td></td></tr><tr><td>Water, Sterile</td><td>Fresenius-Kabi</td><td>B230531</td><td></td></tr><tr><td>Waterbath, Lab-Line Digital</td><td>Thermo Fischer Scientific</td><td>18052A</td><td></td></tr></tbody></table>

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.

The protocol overview of the blood vessel sprouting assay is shown in Figure 1. Nine-day-old EBs derived from three independent 129/Ola mESC lines (Z/Red, R1, and E14) were enzymatically dissociated into single cells using collagenase A. Cells were stained for PECAM-1 and analyzed by Fluorescence-activated cell sorting (FACS) as described. All the cell lines exhibited robust endothelial differentiation, and no differences in their ability to differentiate into endothelial cells were observed...

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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.

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 ...

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Research

JoVE Journal

Biology

2.7K Views.  Leiden University Medical Center. Dit protocol beschrijft een in vitro kiemende angiogenesetest die veel kenmerken van de vorming van bloedvaten samenvat en kan worden gebruikt voor het testen van geneesmiddelen. Deze methode is eenvoudig te implementeren in een lab, robuust en schaalbaar. Het heeft veel overeenkomsten met testen met menselijke endotheelcellen en microsferen.Dit model bootst op robuuste wijze het fenotype na met defecte endotheelcelselectie en georiënteerde endotheelcelmigratie die leidt tot pathologi...

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

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

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Developmental Biology

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Neuroscience

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.

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 ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

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 ...

Bioengineering

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Study of Angiogenesis Using Scratch Wound Migration and Spheroid Sprouting Assays

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

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

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