We would like to acknowledge the technical support from the Confocal and Specialized Microscopy Shared Resource of Herbert Irving Comprehensive Cancer Center at Columbia University, funded in part through NIH Center Grant (P30CA013696). This work is supported by NIH (R21NS133635, Y.-H.L.; UH3TR002151, K. W. L.). Figure 1A was created using BioRender.

Figure 1 presents an overview of the steps involved in this protocol. Detailed information regarding the reagents and materials utilized in this protocol is given in the Table of Materials. It is recommended that all the media should be prewarmed at 37 &#176;C before use unless specified otherwise. All the cell culture materials and supplies should be either autoclaved or sterile, and cell handling should be done in a biosafety cabinet. Additionally, the pH value of the collagen I mixture should be carefully adjusted to pH 7.3 to avoid possible solidification issues.
1. Human iPSC maintenance
<ol>
	<li>Thaw the basement membrane matrix extract on ice, gently shake the bottle to mix the solution, and make 0.5 mL aliquots in 1.5 mL microcentrifuge tubes using a P1000 pipette and pre-refrigerated tips. Store the aliquots at -20 &#176;C for up to 1 year.</li>
	<li>Thaw one aliquot of matrix extract (0.5 mL) on ice. Mix with 49.5 mL of pre-cooled DMEM/F12 medium in a 50 mL tube. Distribute the mixture to each well (1.5 mL/well) in 6-well plates and then gently shake the plate to ensure that the liquid solution covers the entire well. Seal the plates with paraffin film and store them at 4 &#176;C for up to 1 week.</li>
	<li>Prepare the hiPSC expansion medium as described in Table 1. Make aliquots of the complete medium (40 mL/tube) and store at -20 &#176;C for up to 1 year.</li>
	<li>Put a pre-coated plate in the 37 &#176;C incubator for 1 h prior to hiPSC culture.</li>
	<li>Prewarm hiPSC expansion medium (40 mL) at 37 &#176;C and prepare the hiPSC seeding medium as described in Table 2.</li>
	<li>Transfer the coated plate from the 37 &#176;C incubator to the biosafety cabinet. Replace the medium in the coated plate with hiPSC seeding medium (1 mL/well).</li>
	<li>Thaw a cryopreserved vial of hiPSCs from the cryotank in a 37 &#176;C water bath for 1 min.</li>
	<li>Transfer the cells to a 15 mL tube and then add 5 mL of DMEM/F12 medium, followed by centrifugation at 100 x g for 3 min at room temperature to collect the cells.</li>
	<li>Aspirate the supernatant and resuspend the cells with 1.5 mL of hiPSC seeding medium. Gently resuspend cells in the 15 mL tube.</li>
	<li>Aliquot 10 &#181;L of the cell suspension and mix with 10 &#181;L of trypan blue solution. Transfer 10 &#181;L mixture to the hemacytometer slide and cover it with a coverslip.</li>
	<li>Mount the slide on the holder and insert it into the automated cell counter. Wait for the instrument to find the focus or manually adjust the focus by pressing the focus +/- button on the screen. Afterward, use the default setting and press the Capture button. Use the LIVE cell density reading as the concentration of the hiPSC suspension prepared from step 1.9.</li>
	<li>Seed 2 &#215; 105 cells in each well and ensure that the cells are evenly distributed in the well.</li>
	<li>Culture the cells at 37 &#176;C with 5% CO2 and replace the medium with hiPSC expansion medium (1.5 mL/well) after 24 h.</li>
	<li>Change the medium daily until the cells reach a confluency of 80%. Check the cell morphology and colony size routinely to ensure no spontaneous differentiation. Discard the culture and restart with a new batch of hiPSCs if extensive (&#62;5%) spontaneous differentiation is observed.</li>
</ol>
2. EB formation (Day 0)
<ol>
	<li>On Day 0, prepare and prewarm the DMEM/F12 medium, hiPSC seeding medium&#160;and cell detachment solution at 37 &#176;C for 20-30 min.</li>
	<li>Remove the culture medium from the 6-well plate and add prewarmed cell detachment solution (1.5 mL/well). Incubate the plate at 37 &#176;C for 5-7 min.</li>
	<li>Transfer the plate to the biosafety cabinet, tap the plate to detach hiPSC colonies, and break up the aggregates.</li>
	<li>Transfer the cell suspension into a 15 mL tube. Add 4.5 mL of DMEM/F12 medium. Gently pipet the mixture up and down using a 10 mL serological pipet five times to dissociate cells into a single-cell suspension. Avoid generating bubbles.</li>
	<li>Centrifuge at 100 x g for 3 min at room temperature to collect the cells.</li>
	<li>Aspirate the supernatant and gently resuspend the cells with 1 mL of hiPSC seeding medium using a P1000 pipette.</li>
	<li>Mix 10 &#181;L of cell suspension with 10 &#181;L of trypan blue solution for cell counting. Transfer 10 &#181;L of the mixture to the hemacytometer slide and cover it with a coverslip. Mount the slide on the holder and insert it into the automated cell counter. After the instrument finds the focus, press the Capture button and use the LIVE cell density reading to determine the concentration of the hiPSC suspension.</li>
	<li>Seed the cells in the ultra low-attachment round bottom microwell plate with a density of 2.7 &#215; 105 cells/well to yield 500-1,000 cells per EB.</li>
	<li>Add 2 mL of hiPSC seeding medium to each well and mix the solution gently to distribute cells evenly.</li>
	<li>Culture the cells at 37 &#176;C with 5% CO2 for 24 h.</li>
</ol>
3. Vascular differentiation (Day 1 - 5)
<ol>
	<li>On Day 1, prepare the mesoderm induction medium (MIM) as described in Table 3 and Table 4 and prewarm it at 37 &#176;C for 20 min. The MIM medium must be made freshly.</li>
	<li>Use a P200 pipette to carefully aspirate the medium from the microwell plate without disturbing the EBs at the bottom.</li>
	<li>Add 2.5 mL of MIM slowly using a P200 pipette. Culture the EBs in MIM at 37 &#176;C with 5% CO2 for 2 days.</li>
	<li>On Day 3, gently replace the medium with prewarmed, freshly made 2.5 mL of the vascular differentiation medium 1 (VDM1, Table 5) without disturbing the EBs at the bottom of the microwell. Culture the cells at 37 &#176;C with 5% CO2 for another 2 days.</li>
</ol>
4. Hydrogel embedding (Day 5)
<ol>
	<li>Prepare the collagen I mixture as described in Table 6. Place a 50 mL tube on ice and add collagen I solution, H2O, 10&#215; DMEM/F12 medium, sodium bicarbonate&#160;and HEPES subsequently. Mix thoroughly by shaking the tube gently. Add 1 M NaOH solution dropwise while shaking the mixed solution to adjust the pH value to 7.3 and check the pH value of the mixture with a pH meter. 
	NOTE: All solutions should be pre-cooled on ice before use. Perform the procedure on ice in the biosafety cabinet all the time. Adding the NaOH solution is critical, and quick dispersion to make a homogenous solution is needed to avoid local curing of collagen I. The volume of NaOH may vary based on the lot number of collagen I solution used. Do not pipet vigorously for mixing, as shear stress could cause undesired pre-curing.</li>
	<li>Prepare the collagen I-basement membrane matrix mixture as described in Table 7. Add 1.25 mL of basement membrane matrix to the 5 mL collagen I mixture prepared from step 4.1. Mix the solution gently by shaking the tube.</li>
	<li>Place a 12-well plate on ice for 2 min.</li>
	<li>Add collagen I-basement membrane matrix mixture slowly to the 12-well plate (1.5 mL/well) using the wide-bore tips. Make sure the mixture is distributed evenly.</li>
	<li>Put the plate in the 37 &#176;C incubator for 2 h to solidify the first layer of hydrogel.</li>
	<li>Keep the remaining collagen I-basement membrane matrix mixture on ice for later use.</li>
	<li>Transfer ~60 cell aggregates into a 1.5 mL microcentrifuge tube and cool the tube on ice for 5 min. Carefully remove the medium with a P200 pipette tip. 
	NOTE: Start this step 1.5h after Step 4.5.</li>
	<li>Dropwise, add 1.5 mL of collagen I-basement membrane mixture to the aggregates and gently mix them using wide-bore P200 pipette tips.</li>
	<li>Transfer the plate with the first layer of hydrogel from the incubator (step 4.5) to the biosafety cabinet.</li>
	<li>Add 1.5 mL of the aggregate suspension onto the cured layer. Gently shake the plate to distribute the aggregates evenly. Avoid generating any bubbles.</li>
	<li>Incubate the plate at 37 &#176;C with 5% CO2 for another 2 h.</li>
	<li>Prepare the vascular differentiation medium 2 (VDM2; Table 8) and prewarm the medium in a 37 &#176;C water bath for 20 min prior to use.</li>
	<li>Add 2 mL of VDM2 to each well and culture the cells at 37 &#176;C with 5% CO2.</li>
	<li>Change the medium every 3 days with prewarmed, freshly-made VDM2. 
	NOTE: (Optional) Vascular organoids could be released from the gel mixture by removing the gel with a syringe needle. The vascular organoids retrieved from the gel can be maintained individually in a 96-well plate with VDM2 (200 &#181;L/well). Change the medium every 2-3 days.</li>
</ol>
5. 3D Immunofluorescent assay
<ol>
	<li>Vascular organoids should become mature roughly on Day 13. For organoids embedded in the hydrogel, aspirate the medium carefully and wash with 2 mL PBS three times (10 min each time). For organoids retrieved from the gel (after Step 4.14), collect 6-10 organoids in a 2 mL microcentrifuge tube, aspirate the media, and wash with 2 mL PBS three times (10 min each).</li>
	<li>Add 2 mL of 4% paraformaldehyde (PFA) to the sample and incubate at 4 &#176;C overnight with paraffin film sealing.</li>
	<li>Remove the PFA solution and wash the sample with 2 mL PBS four times (15 min each time).</li>
	<li>Add 2 mL of blocking buffer (Table 9) and incubate at room temperature for 2 h.</li>
	<li>Remove the blocking buffer and add 1.5 mL of primary antibodies (see Table of Materials for antibody information; antibodies should be diluted in the blocking buffer). Incubate the sample with primary antibodies at 4 &#176;C for 2 days with gentle shaking. 
	NOTE: The primary antibody (CD31, PDGFR&#946;, and ERG1) is recommended to be diluted in the blocking buffer at a ratio of 1:400 (see Table of Materials for more information).</li>
	<li>Remove the primary antibody solution and wash with 2 mL PBST for 1 h six times.</li>
	<li>Add 2 mL of secondary antibodies (see Table of Materials for the antibody information; antibodies should be diluted in PBST) to the sample. Wrap the plate with aluminum foil and incubate at 4 &#176;C for 2 days with gentle shaking. 
	NOTE: The secondary antibody is recommended to be diluted in PBST at a ratio of 1:1000 (see Table of Materials for more information). Keep the plate from the light during incubation and wash from this step afterward.</li>
	<li>Remove the secondary antibody solution and wash with 2 mL TBST six times (1 h each time). Add DAPI (1 &#181;g/mL) in the wash buffer used for the last wash step if nuclei staining is needed.</li>
	<li>For these gel-embedded organoids, cut the gel into 4 pieces and transfer them carefully to 2 mL microcentrifuge tubes with a spatula. For the retrieved organoids, remove the wash buffer.</li>
	<li>Use wipes to absorb the remaining solution without touching the gel or the organoids.</li>
	<li>Add 2 mL of water-soluble tissue-clearing reagent (Refraction Index = 1.49) to each tube. Incubate the samples with clearing reagent at room temperature in the dark until the organoids become clear (usually overnight). Do not shake the samples.</li>
	<li>Transfer the organoids with the clearing reagent carefully to the center of the glass-bottom dish using a spatula.</li>
	<li>Cover the organoids or gel piece with a 24 mm &#215; 24 mm coverslip.</li>
	<li>Push the coverslip down gently until it lays flat, and sandwich the samples with the glass bottom.</li>
	<li>Remove the redundant clearing reagent in the dish and seal the coverslip with nail polish.</li>
	<li>For 3D imaging, use a confocal microscope with a 10&#215; objective. Use tile scan and Z-stack scan to obtain whole-mount images of organoids.</li>
</ol>

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

<ol>
	<li>Park, S. E., Georgescu, A., Huh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids-on-a-chip.">Organoids-on-a-chip.</a> Science. 364 (6444), 960-965 (2019).</li><li>Takebe, T., Wells, 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=Organoids+by+design.">Organoids by design.</a> Science. 364 (6444), 956-959 (2019).</li><li>Griffith, L. G., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+complex+3d+tissue+physiology+in+vitro.">Capturing complex 3d tissue physiology in vitro.</a> Nat Rev Mol Cell Bio. 7 (3), 211-224 (2006).</li><li>Lancaster, M. 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Pasca, A. 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=Functional+cortical+neurons+and+astrocytes+from+human+pluripotent+stem+cells+in+3d+culture.">Functional cortical neurons and astrocytes from human pluripotent stem cells in 3d culture.</a> Nat Methods. 12 (7), 671-678 (2015).</li><li>Takasato, 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=Kidney+organoids+from+human+iPS+cells+contain+multiple+lineages+and+model+human+nephrogenesis.">Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.</a> Nature. 526 (7574), 564-568 (2015).</li><li>Koike, 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=Modelling+human+hepato-biliary-pancreatic+organogenesis+from+the+foregut-midgut+boundary.">Modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary.</a> Nature. 574 (7776), 112-116 (2019).</li><li>Takebe, T., 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=Vascularized+and+functional+human+liver+from+an+iPSC-derived+organ+bud+transplant.">Vascularized and functional human liver from an iPSC-derived organ bud transplant.</a> Nature. 499 (7459), 481-484 (2013).</li><li>Osakada, F., 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=Toward+the+generation+of+rod+and+cone+photoreceptors+from+mouse,+monkey+and+human+embryonic+stem+cells.">Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.</a> Nat Biotechnol. 26 (2), 215-224 (2008).</li><li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146 (6), 873-887 (2011).</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> Nat Protoc. 14 (11), 3082-3100 (2019).</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>He, 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=Mapping+morphological+malformation+to+genetic+dysfunction+in+blood+vessel+organoids+with+22q11.2+deletion+syndrome.">Mapping morphological malformation to genetic dysfunction in blood vessel organoids with 22q11.2 deletion syndrome.</a> Biorxiv. , (2021).</li><li>He, 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=Human+vascular+organoids+with+a+mosaic+akt1+mutation+recapitulate+proteus+syndrome.">Human vascular organoids with a mosaic akt1 mutation recapitulate proteus syndrome.</a> Biorxiv. , (2024).</li><li>Chen, 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+versatile+polypharmacology+platform+promotes+cytoprotection+and+viability+of+human+pluripotent+and+differentiated+cells.">A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.</a> Nat Methods. 18 (5), 528-541 (2021).</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), e1565 (2008).</li><li>Hwang, Y. -. 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=Microwell-mediated+control+of+embryoid+body+size+regulates+embryonic+stem+cell+fate+via+differential+expression+of+wnt5a+and+wnt11.">Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of wnt5a and wnt11.</a> Proc Natl Acad Sci. 106 (40), 16978-16983 (2009).</li><li>Antonchuk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+embryoid+bodies+from+human+pluripotent+stem+cells+using+aggrewell+plates.">Formation of embryoid bodies from human pluripotent stem cells using aggrewell plates.</a> Methods Mol Biol. 946, 523-533 (2013).</li><li>Lippmann, E. 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=Derivation+of+blood-brain+barrier+endothelial+cells+from+human+pluripotent+stem+cells.">Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.</a> Nat Biotechnol. 30 (8), 783-791 (2012).</li><li>Nishihara, 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=Differentiation+of+human+pluripotent+stem+cells+to+brain+microvascular+endothelial+cell-like+cells+suitable+to+study+immune+cell+interactions.">Differentiation of human pluripotent stem cells to brain microvascular endothelial cell-like cells suitable to study immune cell interactions.</a> Star Protoc. 2 (2), 100563 (2021).</li><li>Qian, T., 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=Directed+differentiation+of+human+pluripotent+stem+cells+to+blood-brain+barrier+endothelial+cells.">Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells.</a> Sci Adv. 3 (11), e1701679 (2017).</li><li>Stebbins, M. 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=Human+pluripotent+stem+cell-derived+brain+pericyte-like+cells+induce+blood-brain+barrier+properties.">Human pluripotent stem cell-derived brain pericyte-like cells induce blood-brain barrier properties.</a> Sci Adv. 5 (3), eaau7375 (2019).</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>Osaki, T., Sivathanu, V., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+3D+vascular+and+neuronal+networks+in+a+microfluidic+platform.">Engineered 3D vascular and neuronal networks in a microfluidic platform.</a> Sci Rep. 8 (1), 5168 (2018).</li><li>Osaki, T., Uzel, S. G. M., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microphysiological+3D+model+of+amyotrophic+lateral+sclerosis+(ALS)+from+human+iPS-derived+muscle+cells+and+optogenetic+motor+neurons.">Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons.</a> Sci Adv. 4 (10), eaat5847 (2018).</li><li>Sakurai, 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+microengineered+vascularized+bleeding+model+that+integrates+the+principal+components+of+hemostasis.">A microengineered vascularized bleeding model that integrates the principal components of hemostasis.</a> Nat Commun. 9 (1), 509 (2018).</li><li>Kim, 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=Engineering+of+a+biomimetic+pericyte-covered+3d+microvascular+network.">Engineering of a biomimetic pericyte-covered 3d microvascular network.</a> PLoS ONE. 10 (7), e0133880 (2015).</li><li>Kosyakova, N., 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=Differential+functional+roles+of+fibroblasts+and+pericytes+in+the+formation+of+tissue-engineered+microvascular+networks+in+vitro.">Differential functional roles of fibroblasts and pericytes in the formation of tissue-engineered microvascular networks in vitro.</a> Biorxiv. , (2019).</li><li>Whisler, J. A., Chen, M. B., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+perfusable+microvascular+network+morphology+using+a+multiculture+microfluidic+system.">Control of perfusable microvascular network morphology using a multiculture microfluidic system.</a> Tissue Eng Part C Methods. 20 (7), 543-552 (2013).</li><li>Haase, K., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+on-chip+vascularization.">Advances in on-chip vascularization.</a> Regen Med. 12 (3), 285-302 (2017).</li><li>Wong, K. H. K., Chan, J. M., Kamm, R. D., Tien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+models+of+vascular+functions.">Microfluidic models of vascular functions.</a> Annu Rev Biomed Eng. 14 (1), 205-230 (2012).</li><li>Boerckel, J. D., Uhrig, B. A., Willett, N. J., Huebsch, N., Guldberg, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+vascular+growth+and+tissue+regeneration+in+vivo.">Mechanical regulation of vascular growth and tissue regeneration in vivo.</a> Proc Natl Acad Sci. 108 (37), E674-E680 (2011).</li><li>Homan, K. 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=Flow-enhanced+vascularization+and+maturation+of+kidney+organoids+in+vitro.">Flow-enhanced vascularization and maturation of kidney organoids in vitro.</a> Nat Methods. 16 (3), 255 (2019).</li><li>Mansour, A. 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=An+in+vivo+model+of+functional+and+vascularized+human+brain+organoids.">An in vivo model of functional and vascularized human brain organoids.</a> Nat Biotechnol. 36 (5), 432-441 (2018).</li></ol>

The authors declare no competing financial interest.

The described protocol includes two key modifications for homogenous and reproducible generation of high-quality vascular organoids. The first modification is the use of a microwell plate to enable better control of the EB uniformity. As a starting point of vascular differentiation, the size of EBs is critical as it regulates the stem cell fate and differentiation efficacy towards different lineages. Variations in EB size usually lead to heterogeneous organoids with inconsistent structure and organization<sup class="xref...

In vitro microphysiological models, including organoid and tissue-on-a-chip systems, have emerged over the past decade1,2,3. These three-dimensional (3D), human cell-derived systems address the limitations in conventional two-dimensional (2D) cell culture and animal models, such as the lack of many components in the physiological microenvironment and genetic differences across species, respectively. They provide more physiological insights and are more suitable for disease modeling and drug screening than conventional models. Among the microphysiological models, organoids derived from human induced pluripotent stem cells (hiPSCs) have been proven to recapitulate the architectural features of native human tissues effectively and have been developed to mimic different human organs, including the brain4,5, kidney6, liver7,8&#160;and retina9. Here, we particularly focus on the generation of vascular organoids from hiPSCs and outline a streamlined protocol that aims to minimize the variations between different organoid samples and batches, thereby improving the overall reproducibility.
Vasculature plays a crucial role in maintaining physiological homeostasis across all human organs. The formation of vasculature involves the processes of vasculogenesis and angiogenesis, orchestrated through the interactions between endothelial and mural (e.g., pericytes and vascular smooth muscle cells) cells and influenced by various stimuli10. Penninger, Wimmer&#160;and colleagues developed the first vascular organoid generation method11,12 that mimics these two processes. With the organoids generated from this method, their group12 and us13,14 have demonstrated the potential of vascular organoids for modeling vascular malfunctions in diseases. For example, in our recent works13,14, distinct phenotypes were observed, such as vessel sprouting and mural cell composition variations, between disease-mimicking vascular organoids and their wild-type counterparts. These examples highlight that the vascular&#160;organoid model could serve as a platform for drug screening by mimicking disease-related vascular malfunctions.
Built upon the initial vascular organoid generation works11,12, a revised protocol is presented that includes the use of microwell to facilitate homogenous embryoid body (EB) formation, a critical factor in minimizing the variations often seen within the same organoid generation batch. Additionally, the CEPT cocktail, a combination of chroman 1, emricasan, polyamines&#160;and the integrated stress response inhibitor (trans-ISRIB)15, is employed&#160;to improve the viability of hiPSCs and differentiated cells during the EB formation process. This modification is mainly to reduce the variations between different organoid samples and batches. Uniform vascular organoids can be produced with this roughly two-week-long protocol, where vasculogenesis is induced to help endothelium organize into primitive tubular networks on Day 1, followed by angiogenesis induction on Day 5 to develop complex vascular networks in organoids. On Day 12-15, the generated organoids should show mature vascular networks composed of both endothelium and mural cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol (1000x)</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immuno Research Labs</td>
			<td>711-546-152</td>
			<td>reconstitute with 0.25 mL 50% glycerol,&#160; store at -20 &#176;C for up to 1 year, dilution ratio for use: 1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Goat IgG (H+L)&#160;</td>
			<td>Jackson Immuno Research Labs</td>
			<td>705-606-147</td>
			<td>reconstitute with 0.25 mL 50% glycerol,&#160; store at -20&#176;C for up to 1 year, dilution ratio for use: 1:1000</td>
		</tr>
		<tr>
			<td>B27 supplement minus vitamin A (50x)</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20 &#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>ProSpec</td>
			<td>CYT-081</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>CD31 antibody&#160;</td>
			<td>abcam</td>
			<td>ab28364</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>022626001</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris Bioscience</td>
			<td>4423/10</td>
			<td>make the stock solution at 12 mM with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Chroman 1</td>
			<td>Tocris</td>
			<td>7163/10</td>
			<td>make the stock solution at 100 &#181;M with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Collagen I&#160;</td>
			<td>Corning</td>
			<td>40236</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>AXRMP/Ti2</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Elplasia Plates</td>
			<td>Corning</td>
			<td>4441</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL automated cell counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, powder</td>
			<td>Gibco</td>
			<td>12500062</td>
			<td>make 10x stock solution with biograde ddH2O and sterilize it with a 0.2 &#181;m filter. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Edi042A</td>
			<td>Cedars Sinai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emricasan</td>
			<td>Medchemexpress LLC</td>
			<td>HY1039610MG</td>
			<td>make the stock solution at 1 mM with DMSO, and store at -20 &#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>ERG antibody</td>
			<td>Cell Singali Technology</td>
			<td>97249</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td>thaw at 4 &#176;C&#160; and aliquot, store at&#160; -20 &#176;C for up to five years, or store at 4 &#176;C for up to a month</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>ProSpec</td>
			<td>CYT557</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Fluorodish Cell Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Tocris Bioscience</td>
			<td>1099</td>
			<td>reconstitute with DMSO at a concentration of 12 mM, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement (100x)</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin, 100 kU</td>
			<td>MilliporeSigma</td>
			<td>375095100KU</td>
			<td>reconstitute with sterile ddH2O at a concentration of 4 kU/mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>13998123</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>Matrigel, GFR Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356230</td>
			<td>thaw at 4 &#176;C, aliquot and store at -80&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (NEAA, 100x)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46000CV</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus kit</td>
			<td>STEMCELL Technologies</td>
			<td>1001130</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>N2 supplement (100x)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>thaw at 4 &#176;C, aliquot and store at -20&#176;C, avoid freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>NaOH solution (1 M)</td>
			<td>Cytiva&#160;HyClone</td>
			<td>SH31088.01</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuroBasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements, ver 5.42</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson Immuno Research Labs</td>
			<td>17000121</td>
			<td>reconstitute with 10 mL sterile ddH2O, store at 4 &#176;C for up to two weeks</td>
		</tr>
		<tr>
			<td>paraformaldehyde, 20%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713S</td>
			<td>dilute with PBS</td>
		</tr>
		<tr>
			<td>PBST, 20x</td>
			<td>Thermofisher</td>
			<td>28352</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFR&#946; antibody</td>
			<td>R&#38;D</td>
			<td>AF385</td>
			<td>dilution ratio: 1:400</td>
		</tr>
		<tr>
			<td>polyamine supplement (1000x)</td>
			<td>Sigma-Aldrich</td>
			<td>P8483</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapiclear clearing reagent, 1.49</td>
			<td>SUNJIN LAB</td>
			<td>RC149001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (7.5%)</td>
			<td>Gibco</td>
			<td>25080094</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate monohydrate</td>
			<td>Thermofisher</td>
			<td>J6228822</td>
			<td>dissolve with ddH2O for 1% (wt/v) stock solution</td>
		</tr>
		<tr>
			<td>TBST, 20x</td>
			<td>Thermofisher</td>
			<td>28360</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-ISRIB</td>
			<td>Tocris Bioscience</td>
			<td>5284/10</td>
			<td>make the stock solution at 1 mM with DMSO, and store at -20&#176;C for up to 1 year.</td>
		</tr>
		<tr>
			<td>Tritin X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF-A</td>
			<td>ProSpec</td>
			<td>CYT-116</td>
			<td>reconstitute with sterile ddH2O at a concentration of 100 ng/&#181;L, aliquot and store at -20&#176;C</td>
		</tr>
	</tbody>
</table>

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

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Figure 1A presents the scheme of this protocol, including the key components used in each stage. The differentiation started with the EB formation, followed by mesoderm induction and vascular lineage specification (Figure 1B-D). The aggregates grew larger and less spherical over time. Organoids started showing rough edges on Day 5. After embedded in the collagen I-basement membrane matrix mixture, vascular endothelial cells and ...

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 ...

vascular-organoid-generation-from-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Bioengineering

Consistent Generation of Human Vascular Organoids Using Microwell Technology

905 Views.  Columbia University.  This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

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