We thank all members of our laboratories for critical input and discussions. JMP received funding from the T. von Zastrow foundation, the FWF Wittgenstein award (Z 271-B19), the Austrian Academy of Sciences, the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 101005026, Leducq Transatlantic Networks of Excellence in Cardiovascular Research, Allen Institute Distinguished Investigator Program, and the Canada 150 Research Chairs Program F18-01336 as well as the Canadian Institutes of Health Research COVID-19 grants F20-02343 and F20-02015.

All experiments performed herein used the commercially available H9 human iPSC line. Common commercially and non-commercially available human pluripotent stem cell lines (i.e., H9, NC8) have also been tested and proven effective for the generation of human blood vessel organoids using this protocol. For details, please refer to our previously published reports8,10.
1. Media and reagent formulation for the generation of human blood vessel organoids
<ol>
	<li>Prepare the aggregation medium.
	<ol>
		<li>Mix 40 mL of knock-out DMEM/F12 (low osmolality medium without L-glutamine or HEPES buffer optimized for human ESC and iPSC growth), 10 mL of knock-out serum replacer (KOSR), 0.5 mL of 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl, 0.5 mL of non-essential amino acids (NEAA), 35 &#181;L of beta-mercaptoethanol (BME, 100 &#181;L of 2-mercaptoethanol in 10 mL of sterile PBS), and Y-27632 (cell-permeable and selective inhibitor of Rho-associated, coiled-coil containing protein kinase [ROCK]14 [10 mM] at 1:200) (see Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare N2B27 (N2 and B27 supplemented basic medium).
	<ol>
		<li>Mix 25 mL of DMEM/F12, 25 mL of neurobasal media, 1 mL of B27 supplement, 0.5 mL of N2 supplement, 250 &#181;L of 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl, and 35 &#181;L of beta-mercaptoethanol (see Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare the mesoderm induction medium.
	<ol>
		<li>Supplement N2B27 medium with ChiR (12 &#181;M, GSK3a/b inhibitor stimulating mesoderm induction) and BMP-4 (30 ng/mL, MSX2 activator inducing mesoderm lineage). 
		NOTE: Stock (ChiR): 10 mM (use 1:833, or 1.2 &#181;L/mL N2B27). Stock (BMP-4): 100 &#181;g/mL (use 1:3333, or 0.3 &#181;L/mL N2B27) (see Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare vascular induction medium.
	<ol>
		<li>Supplement N2B27 medium with VEGFA (100 ng/mL) and forskolin (2 &#181;M) (see Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare the extracellular matrix (ECM).
	<ol>
		<li>Use 1 mL of ECM for one well of a 12-well plate (for embedding 30-50 aggregates). Of the 1 mL of ECM solution used for each well, use 0.5 mL to create a bottom layer, &#34;layer 1&#34;, which will serve as an ECM foundation, and 0.5 mL plus aggregates for the top layer, &#34;layer 2&#34;. Using this approach provides, through the ECM suspension, sufficient space and support for the human blood vessel networks to sprout in all directions. 
		NOTE: Overall, 1 mL of ECM contains 500 &#181;L of purified bovine collagen type I, 250 &#181;L of solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (see Table of Materials), and 250 &#181;L of basic matrix solution (step 1.5.2). Prepare it fresh and keep on ice until ready to use.</li>
		<li>To prepare the basic matrix solution for four 12-well plates (48 wells), mix 5.627 mL of 0.1 N NaOH, 2.498 mL of 10x DMEM, 473 &#181;L of HEPES, 368 &#181;L of 7.5% sodium bicarbonate, 233 &#181;L of 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl, and 3.451 mL of Ham&#39;s F-12 (see Table of Materials). 
		NOTE: The leftover basic matrix solution can be placed in a capped 50 mL high-clarity polypropylene conical tube and stored at 4 &#730;C for use for up to 2 months.</li>
	</ol>
	</li>
	<li>Prepare the sprouting medium.
	<ol>
		<li>Specifically formulate 50 mL of flexible serum-free medium (SFM) to support the development of human hematopoietic cells, a 1.3 mL aliquot of the flexible serum-free medium nutrient supplement, 15% fetal bovine serum (FBS), 250 &#181;L of penicillin-streptomycin, 500 &#181;L of 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl, VEGFA (100 ng/mL), and FGF2 (100 ng/mL) (see Table of Materials).</li>
	</ol>
	</li>
	<li>Prepare the blocking buffer.
	<ol>
		<li>Mix 0.5 g of bovine serum albumin, 1.5 mL of fetal bovine serum, 250 &#181;L of Tween 20, 250 &#181;L of Triton X-100, 500 &#181;L of sodium deoxycholate (1% wt/vol stock), and 47.5 mL of 1x phosphate-buffered saline (see Table of Materials). Pipette up and down until all the components are well incorporated and the solution is clear.</li>
	</ol>
	</li>
</ol>
2. Maintenance and culture of human pluripotent and induced pluripotent stem cell
<ol>
	<li>Perform cell culture using LDEV-free reduced growth factor basement membrane matrix (1:50 dilution)-coated 6-well tissue culture plates with stem cell culture medium (see Table of Materials).</li>
	<li>Once the cells reach ~70% confluency, passage them using 1 mL of mammalian cell dissociating enzyme (see Table of Materials) for 3-4 min at 37 &#730;C. 
	NOTE: Splitting the cells at a ratio of 1:6 enhances the cell viability and achieves confluency within 2-4 days for most human stem cell lines. To increase the cell viability, ROCK inhibitor Y-27632-supplemented (10 mM, 1:1,000) culture medium is used for 24 h post passaging.</li>
	<li>Change the culture medium daily until a confluency of 70% is reached. 
	NOTE: For the present study, at 70% confluency, one well of a 6-well cell culture plate contains around 1 million H9 hPSCs.</li>
</ol>
3. Day 0 - Generation of pluripotent aggregates from a single-cell suspension
NOTE: A confluency of 70% in two wells of a 6-well culture plate will yield approximately 175 hBVOs.
<ol>
	<li>Using either a pipette or vacuum system, aspirate the culture medium, replace it with 1 mL of cell dissociation reagent (see Table of Materials), and incubate for 5 min at 37 &#730;C.</li>
	<li>While the cells are under the cell dissociation reagent, prepare the necessary volume of aggregation medium (step 1.1) in a 15 mL conical tube as needed for the desired number of wells in a 6-well ultra-low attachment culture plate (see Table of Materials).</li>
	<li>Aspirate the 1 mL of cell dissociation reagent, and suspend the cells in 1 mL of aggregation medium. Create a single-cell suspension with gentle up-down pipetting prior to counting the samples. 
	CAUTION: hPSCs are quite sensitive to mechanical stress and cannot tolerate aggressive pipetting.</li>
	<li>Count the cells using an automated cell counting device10 or a standardized system under a microscope. Calculate the cell number needed for the experiment. 
	NOTE: Depending on the cell line, 200,000 cells/well to 300,000 cells/well is considered ideal for aggregate formation (i.e., 4 wells = 800,000 to 1,200,000 cells in total).</li>
	<li>Add the appropriate volume of the cell suspension to the aggregation medium in the 15 mL high-clarity polypropylene conical tube. Gently pipette the diluted cell suspension up and down to ensure a homogenous cell distribution.</li>
	<li>Pipette 3 mL of the diluted cell suspension into each desired well of the 6-well ultra-low attachment culture plate.</li>
	<li>Place the plate in the incubator (37 &#730;C, 5% CO2, &#62;90% humidity), and avoid opening and closing the incubator doors as much as possible. 
	NOTE: This initial step is best completed in the evenings or during low incubator traffic. Even slight vibrations can affect the size and shape of the aggregates, which can impair the results.</li>
</ol>
4. Day 1 - Mesoderm induction of aggregates
<ol>
	<li>Ensure that 24 h post seeding, small aggregates comprising 2-10 cells are visible under the microscope. Collect the aggregates, and allow them to settle prior to changing the medium to the mesoderm induction medium (step 1.3).</li>
	<li>Set up one 15 mL high-clarity polypropylene conical tube per well of the 6-well ultra-low attachment culture plate.</li>
	<li>Use a circular motion (i.e., like an orbital shaker) to gather the aggregates in the center of each well, use a 1 mL pipette to gently collect the aggregates and the surrounding medium from each well, place them into their corresponding 15 mL high-clarity polypropylene conical tubes, and allow the aggregates to sediment at room temperature.</li>
	<li>Once collected, set a timer for 1 h, which is the time required for most cell lines/aggregates to sediment at this stage. 
	NOTE: If the time is less than 1 h, one might lose any aggregates that may not have settled to the bottom of the 15 mL conical tube.</li>
	<li>Once the hour has passed, with caution, aspirate the supernatant with a pipette or a high-sensitivity aspirating pump. Ensure the aggregates remain undisturbed at the bottom of the 15 mL conical tube.</li>
	<li>Resuspend the aggregates of each 15 mL conical tube in 2 mL of mesoderm induction medium, and place them back in their respective wells of the 6-well ultra-low attachment culture plate.</li>
	<li>Place the plate back in the incubator (37 &#730;C), and leave it until day 4. 
	NOTE: If the aggregates attach and grow onto or into one another, using a 1 mL pipette, gently pipette each well of aggregates up and down once per day to keep the aggregates similar in size.</li>
</ol>
5. Day 4 - Vascular induction and priming of the aggregates
<ol>
	<li>Collect the aggregates, and allow them to settle prior to changing the medium to the vascular induction medium (step 1.4).</li>
	<li>Set up one 15 mL conical tube per well of the 6-well ultra-low attachment culture plate.</li>
	<li>Use a circular motion (i.e., like an orbital shaker) to gather the aggregates in the center of each well, use a 1 mL pipette to gently collect the aggregates and the surrounding medium from each well, and place them into their corresponding 15 mL conical tubes.</li>
	<li>Once collected, set a timer for 30 min to allow the aggregates to sediment. 
	NOTE: A time of 30 min is required for most cell lines at this stage. If the time is less than 30 min, there is a risk of losing any aggregates that may not have settled to the bottom of the 15 mL conical tube.</li>
	<li>After 30 min, with caution, aspirate the supernatant with a pipette or a high-sensitivity aspirating pump. Ensure the aggregates remain undisturbed at the bottom of the 15 mL conical tube.</li>
	<li>Resuspend the aggregates of each 15 mL conical tube in 2 mL of vascular induction medium, and place them back in their respective wells of the 6-well ultra-low attachment culture plate.</li>
	<li>Place the plate back in the incubator (37 &#730;C), and leave it until day 6. 
	NOTE: Using a 1 mL pipette, gently pipette each well of aggregates up and down once per day to keep the aggregates similar in size and prevent them from growing into or attaching to one another.</li>
</ol>
6. Day 6 - Aggregate embedding and vessel sprout induction
<ol>
	<li>While working on ice, prepare the desired final volume of the ECM solution (step 1.5). 
	NOTE: The following is the protocol for one well of a 12-well plate (30-50 aggregates), which can be adjusted as necessary.
	<ol>
		<li>For one well of a 12-well plate, apply 0.5 mL of ECM as the bottom layer, and use 0.5 mL of ECM plus aggregates on the top layer. This creates the ECM &#34;sandwich&#34; necessary for effective 3D aggregate sprouting.</li>
	</ol>
	</li>
	<li>Pipette 500 &#181;L of ECM into one well of a 12-well plate. Ensure that no bubbles are formed and the well bottom and side meniscus are completely coated. This comprises layer 1 of the ECM sandwich.</li>
	<li>Place the plate back at 37 &#730;C for 2 h. 
	NOTE: A time of 2 h is necessary for effective polymerization. Any shorter time risks compromising the ECM integrity.</li>
	<li>Toward the end of layer 1 polymerization, use a circular motion to gather the aggregates in the center of each well, use a 1 mL pipette to gently collect the aggregates and surrounding medium from each well, and place them into their corresponding 15 mL conical tubes.</li>
	<li>Allow the aggregates to settle for 10-15 min, and aspirate the supernatant.</li>
	<li>Place the aggregates in the 15 mL conical tube on ice for 5 min. While cooling, take the 12-well plate with the now polymerized layer 1 out of the incubator. 
	NOTE: Cooling the aggregates helps prevent early polymerization of the layer 2 ECM.</li>
	<li>Working swiftly and carefully to prevent bubble formation, resuspend the aggregates in 500 &#181;L of ECM, and pipette the ECM-aggregate suspension atop the already polymerized layer 1. Being cautious not to touch layer 1, use a 200 &#181;L pipette tip to gently distribute layer 2 and the aggregates around the well.</li>
	<li>Place the plate back at 37 &#730;C for 2 h. 
	NOTE: An incubation time of 2 h is necessary for effective ECM polymerization. Any shorter time risks compromising the ECM integrity and may prevent strong adhesion between layer 1 and layer 2.</li>
	<li>Add 1 mL of pre-warmed (37 &#176;C) sprouting medium (step 1.6) to induce blood vessel differentiation. Sprouting vessels must appear 1 day to 3 days post embedding. Change the medium after 3 days and then every other day. 
	NOTE: The sprouting medium must be pre-warmed; otherwise, layer detachment may occur, and the ECM integrity may be impacted.</li>
</ol>
7. Day 11 - Isolation and maturation of the BVOs
<ol>
	<li>Working under sterile conditions, use the rounded end of a sterile spatula to loosen the ECM sprouting matrix containing the vascular networks. At this stage, the gel must resemble a free-floating disk.
	<ol>
		<li>Using sterile forceps and the rounded end of a sterile spatula, carefully transfer the gel disk (including the vascular networks) to the lid of a 10 cm culture dish.</li>
	</ol>
	</li>
	<li>Place the lid plus gel under a stereomicroscope adjusted to the desired magnification and focus, and use sterile needles to cut out single blood vessel networks, trying to limit the amount of non-vascularized ECM obtained in the process. 
	NOTE: The cells naturally degrade the surrounding ECM over time; however, reducing the amount cut out with each organoid increases the image quality and the freestanding vessel network integrity.</li>
	<li>Once all the organoids have been isolated from the gel, gently transfer them back into one well of an ultra-low attachment 6-well plate with 3 mL of sprouting medium . At this stage, the organoids can be left overnight, or one may immediately continue to step 7.4.</li>
	<li>Use a 1 mL pipette to transfer single organoids from the 6-well plate to the wells of an ultra-low attachment 96-well plate. Once transferred, add 200 &#181;L of pre-warmed (37 &#176;C) sprouting medium to each well of the 96-well plate. 
	NOTE: One well of vascular organoids from a 12-well plate must fill 30-40 wells of a 96-well plate.</li>
	<li>At 4-6 days post isolation in the 96-well plates, ensure that the organoids possess a round and healthy morphology. At this stage, the organoids are ready to be fixed and prepared for staining.</li>
</ol>
8. Day 15 - Fixation, blocking, and staining of the BVOs
<ol>
	<li>Using a cut 1 mL tip, transfer the organoids into a 1.5 mL microcentrifuge tube.</li>
	<li>Being careful to avoid the aspiration of any BVOs, use a 200 &#181;L or 1,000 &#181;L pipette to remove the remaining sprouting medium in the microcentrifuge tube, and then add 1 mL of 4% PFA in PBS for 1 h. 
	NOTE: Fixation on an orbital shaker (125 rpm) at room temperature is recommended. A maximum of 60 BVOs/microcentrifuge tube is acceptable. Any more BVOs will impair the fixation and wash efficacy. 
	CAUTION: PFA is a harmful chemical. Use it with caution in a fume hood, and follow the manufacturer&#39;s directions for use and appropriate disposal methods.</li>
	<li>Wash the newly fixed BVOs 3x for 15 min each time with 0.25% PBS-Tween.</li>
	<li>If the organoids are larger than 1 mm in diameter, permeabilize the BVOs with 1% Triton X-100 in PBS for 30-60 min at room temperature.</li>
	<li>Aspirate all the 0.25% PBS-Tween, and add 1 mL of blocking buffer (step 1.7). 
	NOTE: Although antibody-dependant, 2 h at room temperature on an orbital shaker (125 rpm) is sufficient to reduce non-specific antibody binding during the staining process. The organoids can be left in the blocking buffer at 4 &#730;C for up to 2 weeks.</li>
	<li>Remove the blocking buffer, and add the primary antibody (1:100) diluted in 1 mL of the blocking buffer. Keep overnight at 4 &#730;C on an orbital shaker (12 rpm), ensuring the organoid(s) remain submerged in the primary antibody solution. 
	NOTE: Overnight primary antibody incubation is suitable for the antibodies used for this study. The necessary primary and secondary antibodies and their respective dilutions are provided in the Table of Materials.</li>
	<li>Following overnight primary antibody incubation, remove the primary antibody solution and wash 3x for 15 min each time with 0.25% PBS-Tween.</li>
	<li>Add the secondary antibody (1:250) plus DAPI (1:1,000) diluted in 1 mL of blocking buffer, and incubate for 2 h at room temperature or at 4 &#730;C overnight. 
	NOTE: If the samples are prepared correctly (as described above), incubation for 2 h at room temperature or at 4 &#730;C overnight is suitable for the antibodies used in the present study.</li>
	<li>Following incubation, remove the secondary antibody solution, and wash 3x for 15 min each time with 0.25% PBS-Tween. 
	&#8203;NOTE: Following the staining, the organoids can be kept in PBS for up to 2 weeks.</li>
</ol>
9. Mounting of the blood vessel organoids (BVOs)
<ol>
	<li>Glue mounting spacers onto the desired imaging coverslips (see Table of Materials).</li>
	<li>Using a cut 1 mm pipette tip, use a 1 mL pipette to transfer single organoids into the spacer wells, and aspirate the remaining PBS.</li>
	<li>Fill the well with 150-200 &#181;L of clearing solution (see Table of Materials) heated to 75 &#730;C, fully submerging the organoid. 
	NOTE: The samples should become clear within 1 h of exposure to the warmed clearing solution.
	<ol>
		<li>Leave the organoids to become clear in a covered box in the dark at room temperature for 6 h to overnight if the 1 h clearing time is insufficient. If leaving the sample overnight, ensure it has enough clearing solution to avoid drying out.</li>
	</ol>
	</li>
	<li>After clearing, carefully aspirate the clearing solution with a 100-200 &#181;L pipette tip. Gently maneuver the organoid to the center of the spacer well, and add drops of mounting gel (see Table of Materials) (warmed to 75 &#730;C in a controlled heating block) until the well is full.</li>
	<li>Gently place the second coverslip on top of the mounting spacer to seal the samples in the space between the bottom and top coverslips. Let the mounting gel cure in the dark at 4 &#730;C overnight. 
	NOTE: Following the curing, topcoat nail polish can be used to further seal the mounted samples.</li>
</ol>

<ol>
	<li>Okwuosa, I. S., Lewsey, S. C., Adesiyun, T., Blumenthal, R. S., Yancy, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+disparities+in+cardiovascular+disease:+Challenges+and+solutions.">Worldwide disparities in cardiovascular disease: Challenges and solutions.</a> International Journal of Cardiology. 202, 433-440 (2016).</li><li>Page, R. L., Ghushchyan, V., Nair, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+to+action:+Responding+to+the+future+forecasting+of+cardiovascular+disease+in+America.">A call to action: Responding to the future forecasting of cardiovascular disease in America.</a> American Health &amp; Drug Benefits. 4 (5), 280-288 (2011).</li><li>Ferrara, N., Davis-Smyth, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+vascular+endothelial+growth+factor.">The biology of vascular endothelial growth factor.</a> Endocrine Reviews. 18 (1), 4-25 (1997).</li><li>Ishitobi, H., 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=Flk1-GFP+BAC+Tg+mice:+An+animal+model+for+the+study+of+blood+vessel+development.">Flk1-GFP BAC Tg mice: An animal model for the study of blood vessel development.</a> Experimental Animals. 59 (5), 615-622 (2010).</li><li>Campisi, 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=3D+self-organized+microvascular+model+of+the+human+blood-brain+barrier+with+endothelial+cells,+pericytes+and+astrocytes.">3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes.</a> Biomaterials. 180, 117-129 (2018).</li><li>Xu, Y., 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=A+novel+strategy+for+creating+tissue-engineered+biomimetic+blood+vessels+using+3D+bioprinting+technology.">A novel strategy for creating tissue-engineered biomimetic blood vessels using 3D bioprinting technology.</a> Materials. 11 (9), 1581 (2018).</li><li>Risau, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis.">Mechanisms of angiogenesis.</a> Nature. 386 (6626), 671-674 (1997).</li><li>Wimmer, R. 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=Human+blood+vessel+organoids+as+a+model+of+diabetic+vasculopathy.">Human blood vessel organoids as a model of diabetic vasculopathy.</a> Nature. 565 (7740), 505-510 (2019).</li><li>Fleischer, S., Tavakol, D. N., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+arteries+to+capillaries:+Approaches+to+engineering+human+vasculature.">From arteries to capillaries: Approaches to engineering human vasculature.</a> Advanced Functional Materials. 30 (37), 1910811 (2020).</li><li>Wimmer, R. A., Leopoldi, A., Aichinger, M., Kerjaschki, D., Penninger, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+blood+vessel+organoids+from+human+pluripotent+stem+cells.">Generation of blood vessel organoids from human pluripotent stem cells.</a> Nature Protocols. 14 (11), 3082-3100 (2019).</li><li>Monteil, 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=Inhibition+of+SARS-CoV-2+infections+in+engineered+human+tissues+using+clinical-grade+soluble+human+ACE2.">Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2.</a> Cell. 181 (4), 905-913 (2020).</li><li>Patsch, C., 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=generation+of+vascular+endothelial+and+smooth+muscle+cells+from+human+pluripotent+stem+cells.">generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.</a> Nature Cell Biology. 17 (8), 994-1003 (2015).</li><li>Bruveris, F. F., Ng, E. S., Stanley, E. G., Elefanty, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF,+FGF2,+and+BMP4+regulate+transitions+of+mesoderm+to+endothelium+and+blood+cells+in+a+human+model+of+yolk+sac+hematopoiesis.">VEGF, FGF2, and BMP4 regulate transitions of mesoderm to endothelium and blood cells in a human model of yolk sac hematopoiesis.</a> Experimental Hematology. 103, 30-39 (2021).</li><li>Julian, L., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-associated+coiled-coil+containing+kinases+(ROCK):+Structure,+regulation,+and+functions.">Rho-associated coiled-coil containing kinases (ROCK): Structure, regulation, and functions.</a> Small GTPases. 5, 29846 (2014).</li><li>Anitua, E., Prado, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addressing+reproducibility+in+stem+cell+and+PRP+therapies.">Addressing reproducibility in stem cell and PRP therapies.</a> Trends in Biotechnology. 37 (4), 340-344 (2019).</li><li>McMurtrey, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytic+models+of+oxygen+and+nutrient+diffusion,+metabolism+dynamics,+and+architecture+optimization+in+three-dimensional+tissue+constructs+with+applications+and+insights+in+cerebral+organoids.">Analytic models of oxygen and nutrient diffusion, metabolism dynamics, and architecture optimization in three-dimensional tissue constructs with applications and insights in cerebral organoids.</a> Tissue Engineering Part C: Methods. 22 (3), 221-249 (2016).</li><li>McKeown, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+normoxia,+physoxia+and+hypoxia+in+tumours-Implications+for+treatment+response.">Defining normoxia, physoxia and hypoxia in tumours-Implications for treatment response.</a> The British Journal of Radiology. 87 (1035), 20130676 (2014).</li><li>Thomlinson, R. H., Gray, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histological+structure+of+some+human+lung+cancers+and+the+possible+implications+for+radiotherapy.">The histological structure of some human lung cancers and the possible implications for radiotherapy.</a> British Journal of Cancer. 9 (4), 539-549 (1955).</li><li>Olive, P. L., Vikse, C., Trotter, 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=Measurement+of+oxygen+diffusion+distance+in+tumor+cubes+using+a+fluorescent+hypoxia+probe.">Measurement of oxygen diffusion distance in tumor cubes using a fluorescent hypoxia probe.</a> International Journal of Radiation Oncology. 22 (3), 397-402 (1992).</li><li>Torres Filho, I. P., Leunig, M., Yuan, F., Intaglietta, M., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+measurement+of+microvascular+and+interstitial+oxygen+profiles+in+a+human+tumor+in+SCID+mice.">Noninvasive measurement of microvascular and interstitial oxygen profiles in a human tumor in SCID mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (6), 2081-2085 (1994).</li><li>Lanzen, J., 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=Direct+demonstration+of+instabilities+in+oxygen+concentrations+within+the+extravascular+compartment+of+an+experimental+tumor.">Direct demonstration of instabilities in oxygen concentrations within the extravascular compartment of an experimental tumor.</a> Cancer Research. 66 (4), 2219-2223 (2006).</li><li>Ungrin, M. D., Joshi, C., Nica, A., Bauwens, C., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible,+ultra+high-throughput+formation+of+multicellular+organization+from+single+cell+suspension-derived+human+embryonic+stem+cell+aggregates.">Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.</a> PLoS One. 3 (2), 1565 (2008).</li><li>Sun, X. Y., 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=generation+of+vascularized+brain+organoids+to+study+neurovascular+interactions.">generation of vascularized brain organoids to study neurovascular interactions.</a> eLife. 11, 76707 (2022).</li></ol>

There is no relationship between the authors' financial interests and the research being presented. The blood vessel organoid technology has been licensed to Stemcell Technologies. J.M.P. is a founder and shareholder of Angios Biotech that develops blood vessel organoids as vascular transplant therapy. A patent application related to this work has been filed under Pat. No. US20200199541A1, listing Reiner A. Wimmer, Josef M. Penninger, and Dontscho Kerjaschki as inventors.

Recent breakthroughs in stem cell-derived organoid cultures have provided the framework for more advanced and physiologically relevant models of human vasculature. The human blood vessel organoid (hBVO) model presented here, developed in our laboratory8,10, provides a powerful means of exploring not only further aspects of human vasculogenesis but also new avenues of disease modeling and regenerative therapy8,10,11. Multiple in vivo models have been employed to explore the development and maturation of blood vessels, vascular disease, and endothelial dysfunction3,4. Varying approaches combine single and multiple lineage-defined cells either derived from stem cells or isolated from adult tissues in vivo to create replicative human vascular networks5,6. The protocol presented here leverages the principle of developmental biology and self-organization to yield the first ever multi-cell lineage human blood vessel networks that can be generated, in essence, from a single hPSC8,10.
Each stem cell line has a unique genetic makeup and differs from others in terms of its sourcing, function, and responsiveness15. Therefore, the protocol for human blood vessel organoids (hBVOs) was developed and optimized to ensure a robust and reproducible protocol compatibility with multiple (&#62;12) different hPSC lines8,10. The method described here generates hPSC-derived blood vessel organoids over 2 weeks. However, changes to the media composition and or the techniques in hBVO generation may lead to ineffective vessel network and organoid generation. The varying proliferation rates of individual stem cell lines also markedly impact the reproducibility of stem cell experimentation15 and, thus, organoid cultures. For example, in generating the BVOs, more proliferative cells or higher numbers of large day 1 aggregates are inherently subject to different metabolic environments and gas and nutrient diffusion parameters. This, in turn, changes the growth factor exposure times and efficiencies, the degree of differentiation and vascular priming, and, most importantly, the ability to form vessel networks upon embedding the aggregates in the collagen 1 and solubilized basement membrane matrix.
The passive diffusion of oxygen and the administration of essential nutrients from an external environment is not ideal for the long-term cell growth of 3D organoids and tissue morphogenesis in vitro16. Although dependant on several factors (i.e., the tissue metabolic rate, the nutrient and gas bioavailability, a static or dynamic environment), a general 150 &#181;m O2 and nutrient diffusion limit has been established for tissues cultured in vitro, considering that, physiologically, human tissues present cords of living cells within 150 &#181;m of perfused blood vessels17. Although effective gas and nutrient diffusion distances of 70-200 &#181;m have also been proposed18,19,20,21, the construct density, temperature, pH, and media composition significantly impact the diffusion efficacy. Due to the surface area optimization and integrin-beta receptor communication following day 6 embedding, aggregates of 250-300 &#181;m in diameter perform better than those &#62;500-600 &#181;m in diameter, resulting in a complete vessel sprouting process and a minimally condensed organoid core. Thus, the aggregate size is crucial and can be affected by both the number of cells used during the initial seeding and the time allotted for aggregate formation. Microwell plates that allow for control over the aggregate size and number22 are a viable alternative to the otherwise stochastic aggregate formation technique resulting from the use of ultra-low attachment 6-well plates in this protocol. Consistency in timing and managing the aggregate sizes during the first 6 days of the hBVO protocol is one of, if not the most, crucial indicators of successfully developing bona fide blood vessel organoids.
Medium changes on day 1 (mesoderm induction) and day 4 (vascular induction) must be completed in combination with the sedimentation of the aggregates. Although centrifugation is a tempting alternative, the additional forces applied to the weakly assembled aggerates can cause unwanted clumping, assembly, and shearing, which negatively impact differentiation, maturation, and sprouting efficacy in the later stages of the protocol. Working on ice during the day 6 embedding process is critical for preserving proper ECM polymerization and layer formation. During the aggregate embedding and sprouting induction, exposing the ECM to temperatures above 4&#730;C and/or an ECM pH other than 7.4 will affect not only the polymerization rates and ECM layer integrity but also the sprouting efficiency of the embedded aggregates. The elastic nature of the ECM sprouting matrix allows for easy detachment and transport from the 12-well culture dish to the sterile cutting surface. Individual organoids removed from the matrix and transferred to ultra-low attachment 96-well plates will consume the remaining surrounding ECM and retain self-organized mural cell-coated endothelial microvessel networks with a continuous basement membrane.
While not covered in this proposal, alterations to the media compositions can replicate diseases that, in turn, provoke pathological responses in hBVOs8,11. The boundaries of disease modeling using the hBVO system are far from well-known, and this is certainly an area that needs exploration. The application of our blood vessel organoid technology in the vascularization of previously avascular organoid constructs23 is also of significant impact and interest.

Vascular dysfunction and blood vessel diseases present with marked complications in organ functions. Cardiovascular disease (CVD) is the leading cause of death worldwide1 and is also the primary contributing factor to increasing healthcare costs in the United States. CVD case numbers are increasing annually, and rising numbers of these cases are occurring in younger age groups (20-45 years)2. Multiple in vivo models have been developed to explore the development and maturation of blood vessels, vascular disease, and endothelial dysfunction3,4. Currently, methods combining single and multiple lineage-defined cells either derived from stem cells or isolated from adult tissues in vivo can create vascular networks that replicate aspects of human vascular function and anatomy5,6. Blood vessels have emerged as one of the first functional systems during development from the mesoderm, and they organize either through a process of assembly called &#34;vasculogenesis&#34; or by expansion and branching from pre-existing vessels, which is termed &#34;angiogenesis&#34;7.
Leveraging the power of developmental biology and self-directed assembly, Wimmer et al. reported the first self-organizing 3D human blood vessel organoids from hPSCs that exhibit the functional, morphological, and molecular characteristics of human microvasculature8. Similar to human vasculature9, these hBVOs are generated and present with an endothelium, a continuous basement membrane, and surrounding mural cells8,10. The hBVOs can be transplanted in vivo and anastomosed with the endogenous circulation. They can also undergo maturation in vitro and serve as models for cardiovascular diseases (i.e., diabetes)8 or tissue tropism in virus infections such as SARS-CoV-211. While we previously published a written protocol10, there exists no available video protocol for this otherwise complex technique.
Through a concise stepwise progression, hBVO formation is accomplished through aggregate formation, mesoderm induction using a WNT agonist, Chiron (CHIR99021), and bone morphogenic protein - 4 (BMP4)12,13,&#160;vascular induction via vascular endothelial growth factor A (VEGFA) and Forskolin (Fors)12, and embedding in a custom sprouting matrix8,10. Vascular maturation and network formation follow the embedding of the aggregates in the sprouting matrix. These human vessel networks form within 2-3 weeks and can be removed from the sprouting matrix and further grown in scalable culture systems for up to 6 months. Here, optically guided procedures are provided for the formation and application of human stem cell-derived vasculature.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M HEPES&#160;</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Millipore</td>
			<td>60-24-2</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% sodium bicarbonate</td>
			<td>Gibco</td>
			<td>5080094</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase&#160;</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>cell dissociation reagent&#160;</td>
		</tr>
		<tr>
			<td>Albumin fraction V (BSA)</td>
			<td>AppliChem</td>
			<td>A1391, 0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488&#8211;anti-rabbit IgG (Fab&#8242;)2&#160;fragment</td>
			<td>&#8212;</td>
			<td>Jackson Immuno Research</td>
			<td>711-546-152</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488&#8211;anti-sheep IgG</td>
			<td>&#8212;</td>
			<td>Life Technologies</td>
			<td>A11015</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647&#8211;anti-goat IgG (Fab&#8242;)2&#160;fragment</td>
			<td>&#8212;</td>
			<td>Jackson Immuno Research</td>
			<td>705-606-147</td>
		</tr>
		<tr>
			<td>Automated cell counter&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Countess II</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet&#160;</td>
			<td>Faster</td>
			<td></td>
			<td>SafeFAST Premium 212</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>Miltenyi BioTec</td>
			<td>130-111-165</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Endothelial cell</td>
			<td>DAKO</td>
			<td>M0823</td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Endothelial cell</td>
			<td>R&#38;D</td>
			<td>AF806</td>
		</tr>
		<tr>
			<td>Cellulose wipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Heraeus</td>
			<td></td>
			<td>Multifuge 4 KR</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris Bioscience</td>
			<td>4423</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish (essence, the gel, 01 absolute pure)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator&#160;</td>
			<td>New Brunswick</td>
			<td></td>
			<td>Galaxy 170S</td>
		</tr>
		<tr>
			<td>Collagen type IV</td>
			<td>Basement membrane</td>
			<td>Millipore</td>
			<td>AB769</td>
		</tr>
		<tr>
			<td>Confocal microscope (10x, 20x, 63x objectives)</td>
			<td>Leica</td>
			<td></td>
			<td>SP8</td>
		</tr>
		<tr>
			<td>Counting chamber slides- including 0.1% Trypan blue</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips (22 x 50 mm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3&#8211;anti-mouse IgG (Fab&#8242;)2&#160;fragment</td>
			<td>&#8212;</td>
			<td>Jackson Immuno Research</td>
			<td>715-166-150</td>
		</tr>
		<tr>
			<td>DAPI&#160;</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modefied Eagle&#39;s Medium (DMEM)&#160;</td>
			<td>Sigma</td>
			<td>D5648-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovien Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Miltenyi BioTec</td>
			<td>130-093-841</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>FST</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Clipped Corner Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin&#160;</td>
			<td>Sigma</td>
			<td>F3917</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Fisher Scientific</td>
			<td>A1413302</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F12&#160;</td>
			<td>Gibco</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal laminar flow station, if stereomicroscope cannot fit in BSC&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Heraguard</td>
		</tr>
		<tr>
			<td>Inverted contrasting tissue culture microscope&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td>Vert.A1</td>
		</tr>
		<tr>
			<td>iSpacer&#160;</td>
			<td>SunJinLab</td>
			<td>IS009</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F12&#160;</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement&#160;</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement&#160;</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>non-essential amino acids (NEAAs)</td>
			<td>Gibco</td>
			<td>11140035</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (4%) in PBS</td>
			<td>Boston BioProducts</td>
			<td>BM-155</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFR-&#946;</td>
			<td>Pericyte</td>
			<td>R&#38;D</td>
			<td>AF385</td>
		</tr>
		<tr>
			<td>PDGFR-&#946;</td>
			<td>Pericyte</td>
			<td>Cell Signaling</td>
			<td>3169S</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator strips (6.5-10)</td>
			<td>Mquant, Millipore</td>
			<td>109543</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes (P1000, P200 and P20)</td>
			<td>Gilson, Integra Pipetboy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prime Surface-U 96-well plates&#160;</td>
			<td>Sumilon</td>
			<td>MS9096-U</td>
			<td></td>
		</tr>
		<tr>
			<td>PurCol&#160;</td>
			<td>Advanced BioMatrix</td>
			<td>5005</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiClear CS gel</td>
			<td>SunJinLab</td>
			<td>RCCS005</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiClear CS mounting solution</td>
			<td>SunJinLab</td>
			<td>RCCS002</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes (5, 10 and 25 mL)</td>
			<td>Falcon</td>
			<td>357529, 357530, 357515</td>
			<td></td>
		</tr>
		<tr>
			<td>SMA</td>
			<td>vSMC/Pericyte</td>
			<td>Sigma</td>
			<td>2547</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution (NaOH, 1.0 N)</td>
			<td>Sigma</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Solubilized Basement Membrane Matrix (i.e., Matrigel)</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel micro spatula (rounded end)</td>
			<td>Fisher Scientific</td>
			<td>21-401-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel spoon (double-ended)</td>
			<td>Fisher Scientific</td>
			<td>BelArt H367290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemflex medium</td>
			<td>Thermo Scientific</td>
			<td>A3349401</td>
			<td>stem cell culture medium&#160;</td>
		</tr>
		<tr>
			<td>StemPro-34 SFM</td>
			<td>Gibco</td>
			<td>10639011</td>
			<td>flexible serum-free medium&#160;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filter pipette tips (1,000, 300 and 20 &#956;L)</td>
			<td>Biozym, Surphob</td>
			<td>VT0270, VT0250, VT0220</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated 12-well plates TC&#160;</td>
			<td>BD Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated 6-well plates&#160;</td>
			<td>Eppendorf</td>
			<td>30720113</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryple Express Enzyme (1x), Phenol Red</td>
			<td>Thermo Scientific</td>
			<td>12605010</td>
			<td>mammalian cell dissociating enzyme&#160;</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low-attachment 6-well plates</td>
			<td>Corning&#160;</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGFA&#160;</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath (37 &#176;C)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Isotemp 210</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Calbiochem</td>
			<td>688000</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human blood vessel organoids from pluripotent stem cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

The steps described in this protocol were developed specifically to yield a controlled and precise method for the generation of human blood vessel organoids from hPSCs. The generation of aggregates of 30-100 &#181;m in diameter from hPSC cultures marks the starting point of the protocol (Figure 1, Figure 2B). The aggregates are led through stepwise inductions toward mesoderm (day 1-day 4) and vascular lineages (day 4-day 6) prior to embedding (day 6) (Figure 2A-D), which is necessary for vessel network formation. Near-radially symmetric vessel sprouting must be visible by d7 or d8 (Figure 2E) and continue through d10 (Figure 2F,G). The explantation of the hBVOs from the ECM to the 96-well ultra-low attachment plates reduces the fragility of the sprouting networks and allows for continued maintenance in suspension culture (Figure 2H)&#160;conditions for up to 6 months. By d15, the hBVOs exhibit an extensive and connected endothelial (CD31+) network surrounded by pericytes (PDGFR-&#223;+) and smooth muscle actin (SMA+) (Figure 3A-D). A continuous collagen IV (ColIV+) basement membrane envelopes the vessel networks (Figure 3E). Endothelial cells (CD31+, VE-Cadherin+) and pericytes (PDGFR-&#223;+) comprise approximately 30%-35% and 60%-65% of the organoid cell populations, respectively8. The active sprouting of the vessels occurs under the direction of an endogenously organized tip cell (CD31+) population that presents with typical tip cell morphology, such as excessive filipodia8,10. The presentation of PDGFR-&#223;+ and SMA+ mural cells encapsulating the endothelial vessel networks can be seen by d15 of the organoid maturation process (Figure 3A,C). Following removal from the ECM and maturation in suspension culture (i.e., d15), the hBVOs are tenable for respective analyses or transplantation under the mouse kidney capsule.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64715/64715fig01.jpg" /> 
Figure 1: Schematic of the hBVO protocol highlighting the timing and stepwise progression. From seemingly homogenous hPSC or hiPSC cultures, aggregate formation occurs in the presence of the Rho-kinase inhibitor Y27632. BMP4 and CHiR-supplemented medium is used to instruct the aggregates toward the mesodermal fate. Mesoderm induction medium is replaced by VEGFA and forskolin-supplemented medium to stimulate the aggregates toward a vascular lineage. Mechanical and chemical queues achieved through the embedding of the aggregates in a collagen I and solubilized basement membrane matrix and exposure to VEGFA and FGF2-containing medium results in near-radially symmetric vascular sprouting from the aggregate body. The vessel networks generated can be removed from the collagen I and solubilized basement membrane and placed in suspension culture for further maturation, analysis, or transplantation in vivo. <a href="https://www.jove.com/files/ftp_upload/64715/64715fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64715/64715fig02.jpg" /> 
Figure 2: Stepwise progression of the hBVO generation captured under brightfield. (A)&#160;Typical morphology of hPSC (H9) colonies at day &#8722;1. (B) Generation of hPSC aggregates (day 0) on 6-well ultra-low attachment plates in the presence of Y-27632. (C) The mesodermal induction of aggregates using BMP4 and ChiR-supplemented medium (day 1). Subtle changes in the aggregate size and shape can also be observed. (D) Day 4 aggregates primed toward a vascular lineage using VEGFA and forskolin-supplemented medium. (E)&#160;Early vessel sprouting on day 7, one day after embedding the aggregates in the sprouting matrix and exposure to VEGFA and FGF2-supplemented sprouting medium (day 7). (F)&#160;Healthy organoid morphology and continued vessel sprouting on day 9. (G) Late-stage vessel sprouting at day 10. Ideally, the dense cell structures at the organoid center have disappeared by this time. (H)&#160;Typical morphology of mature (day 15) human blood vessel organoids. The removal of the surrounding matrix by cutting and maturation in ultra-low attachment 96-well culture plates shapes the organoids spherically, with the vessel networks housed internally. Scale bars: (A,B,E,F) 250 &#181;m; (C,D) 500 &#181;m; (G,H) 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64715/64715fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64715/64715fig03.jpg" /> 
Figure 3: Whole-mount staining of mature (day 15) human blood vessel organoids. (A) Presentation of mature (day 15) human blood vessel organoids with self-organized endothelial (CD31+, magenta) networks and surrounding pericyte (PDGFR-&#946;+, cyan) and alpha-smooth muscle actin (SMA+, yellow) coverage. (B) Higher magnification of A detailing the SMA+ (yellow) and PDGFR-&#946;+ (cyan) expression of the vessel networks. (C)&#160;Detailed presentation of endothelial (CD31+, magenta), pericyte (PDGFR-&#946;+, cyan), and alpha-smooth muscle actin (SMA+, yellow) interaction. (D)&#160;Whole-mount staining of the vessel endothelial (CD31+, magenta)-smooth muscle (SMA+, yellow) interaction in a cross-section (left) and a higher magnification (right) of mature blood vessel organoids. (E) Self-directed formation of a vascular basement membrane (Col IV+, grayscale) through the close association of the mural cells and endothelial tubes. Scale bars: (A,D[left]) 100 &#181;m; (B,D[right],E) 25 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64715/64715fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells at JoVE.com

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

generation-of-human-blood-vessel-organoids-from-pluripotent-stem-cells

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Bioengineering

5.7K Views.  University of British Columbia. This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

Video: Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Developmental Biology

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

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 ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

Biochemistry

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate.
The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain&#39;s inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain.
The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson&#39;s disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.

This protocol describes a novel method for creating 3D midbrain organoids from human induced pluripotent stem cells, guiding their formation to mimic native midbrain tissue, thereby aiding in the study of development and disorders.

The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain ...

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 ...