Name: vascular organoid generation from human-induced pluripotent stem cells
Uploaded: 2024-12-13T13:20:00.000+00:00
Duration: 4 min 41 s
Description: Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

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 organization16,17. The conventional EB generation method by lifting hiPSC colonies11,12 makes it difficult to control the cell numbers for individual EBs and usually results in heterogeneous EB formation. In this protocol, hiPSCs are dissociated into single-cell suspension and reaggregated in microwells16,18. By adjusting the seeding cell numbers, the size of EBs can be optimized to ensure consistent and effective mesodermal differentiation. For microwell seeding, 500-1,000 cells are used,&#160;and this can yield relatively small EBs (with a diameter of 100-200 &#181;m). This method could reproducibly generate uniform EBs with desired sizes from batch to batch (Figure 1F) and among various cell lines13. Since Day 0, the morphology of the EB has changed over time during the differentiation: the shape becomes less spherical, and the surface becomes rougher (Figure 1), but the volume does not increase significantly. The 3D vasculature morphology depends on the embedding density and the sizes of the organoids embedded. Too-large (&#62;500 &#181;m) organoids usually result in insufficient vascular cell differentiation, while too-small organoids tend to have underdeveloped vasculature.
The second modification is incorporating the CEPT cocktail to improve the hiPSC viability during the EB formation. The cell viability is crucial in addition to the quality of hiPSC colonies during this process. When hiPSCs are dissociated into a single-cell suspension, the ROCK inhibitor Y27632 is usually added to improve cell viability. However, Y27632 has been shown suboptimal for hiPSCs in low density and may cause undesired membrane stress, while the CEPT cocktail (composed of chroman 1, emricasan, polyamines&#160;and trans-ISRIB) could significantly reduce cell stress and improve the viability during this EB formation process with hiPSCs in a lower density15.
Prior to organoid embedding in the gel, the organoids are maintained in the microwell plate, and the medium change should be gentle to avoid possible disruption. It would be better to use a 200 &#181;L pipette to change the medium for the first 5 days. All the media used for vascular organoid generation must be prewarmed before use. Especially after gel embedding, the use of cold media could partially liquify the basement membrane matrix and affect the 3D hydrogel niche for vascular network formation. The embedding density and even distribution of the organoids are also critical factors for vascular network formation. Dense organoid embedding could potentially lead to excessive fusion of vascular networks, while low embedding density could lead to insufficient network formation. Seeding 60-80 organoids per well and gently moving the plate back and forth immediately after adding them to the first layer of the 3D gel can better distribute these organoids.
This protocol can be further modified for different applications. For example, blood-brain barrier (BBB) cues can be applied to further restrain the vascular endothelial cells from the formation of the BBB interface19,20,21,22,23,24,25. The 3D hydrogel composition can be adjusted as well. For example, fibrin and vitronectin are commonly used for vascular network formation in vitro25,26,27,28,29,30,31,32. The basement membrane matrix without collagen I may be used for embedding, which may simplify the embedding process and provide different mechanical stimuli.
Although the vascular organoids generated from this protocol could be potentially used for disease modeling or drug screening applications, one limitation is that these organoids only recapitulate mostly the capillary vasculature with few larger vessels. For research focusing on larger vessels, organoid generation may require additional mechanical stimuli24,31,32,33. Connecting the vascular organoids with recirculation, such as transplantation to animals34 or micro-/macro-fluidic devices with perfusion pumps33, could potentially address this.

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><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&#039;)&lt;sub&gt;2&lt;/sub&gt; 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,&amp;nbsp; store at -20 &amp;deg;C for up to 1 year, dilution ratio for use: 1:1000</td></tr><tr><td>Alexa Fluor 647 AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Donkey Anti-Goat IgG (H+L)&amp;nbsp;</td><td>Jackson Immuno Research Labs</td><td>705-606-147</td><td>reconstitute with 0.25 mL 50% glycerol,&amp;nbsp; store at -20&amp;deg;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 &amp;deg;C, aliquot and store at -20 &amp;deg;C, avoid freeze-thaw cycle</td></tr><tr><td>BMP4</td><td>ProSpec</td><td>CYT-081</td><td>reconstitute with sterile ddH&lt;sub&gt;2&lt;/sub&gt;O at a concentration of 100 ng/&amp;micro;L, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>CD31 antibody&amp;nbsp;</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 &amp;deg;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 &amp;micro;M with DMSO, and store at -20 &amp;deg;C for up to 1 year.</td></tr><tr><td>Collagen I&amp;nbsp;</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 ddH&lt;sub&gt;2&lt;/sub&gt;O and sterilize it with a 0.2 &amp;micro;m filter. Store at 4 &amp;deg;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 &amp;deg;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 &amp;deg;C&amp;nbsp; and aliquot, store at&amp;nbsp; -20 &amp;deg;C for up to five years, or store at 4 &amp;deg;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/&amp;micro;L, aliquot and store at -20 &amp;deg;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 &amp;deg;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 &amp;deg;C, aliquot and store at -20&amp;deg;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 &amp;deg;C, aliquot and store at -80&amp;deg;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 &amp;deg;C, aliquot and store at -20&amp;deg;C, avoid freeze-thaw cycle</td></tr><tr><td>N2 supplement (100x)</td><td>Gibco</td><td>17502048</td><td>thaw at 4 &amp;deg;C, aliquot and store at -20&amp;deg;C, avoid freeze-thaw cycle</td></tr><tr><td>NaOH solution (1 M)</td><td>Cytiva&amp;nbsp;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 &amp;deg;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&amp;beta; antibody</td><td>R&amp;amp;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&amp;deg;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/&amp;micro;L, aliquot and store at -20&amp;deg;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.

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 pericytes sprouted and outgrew to form complex vascular networks as early as Day 7 (Figure 1E). As the main purpose of this modified method is to minimize the variations between different generation batches, we measured the EB size from two independent batches, and both size measurements and the statistical analysis confirmed the consistency and&#160;uniformity with this method (Figure 1F).
To validate the vascular differentiation, organoids were collected on Day 5 before embedding and stained with vascular markers, including CD31, ERG1&#160;and PDGFR&#946;. The organoids expressed CD31, ERG1&#160;and PDGFR&#946; (Figure 2), indicating the presence of vascular endothelial cells and pericytes.
After tissue clearing, the vascular networks were analyzed with whole-mount immunofluorescence microscopy (Figure 3A). Mature organoids were stained with CD31, ERG1&#160;and PDGFR&#946; on Day 17 (Figure 3B, sample in the gel block) and Day 28 (Figure 3C, the sample was retrieved from the gel block and culture individually in a 96-well plate; note that the vascular network at the edges wrapped the spheroids instead of radially expanding after retrieved from the gel block).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67125/67125fig01.jpg" /> 
Figure 1: Vascular organoid generation. (A) Scheme of the steps and timeline for vascular organoid generation. (B-E) Representative bright field images of vascular organoids at different stages. Scale bars: 100 &#181;m. (F) Statistical analysis in EB size from two different EB generation batches using the microwell method presented in this work. For EB generation, 1,000 cells per microwell were used, and for Batches 1 and 2, n = 42 and n = 40, respectively. Significance is presented as ns, not significant. <a href="https://www.jove.com/files/ftp_upload/67125/67125fig01large.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/67125/67125fig02.jpg" /> 
Figure 2:&#160;Immunostaining of vascular organoids before gel embedding. Representative fluorescent images of organoids stained with the endothelial markers (A) CD31 (green) and (B) ERG1 (green)&#160;as well as (C) the pericyte marker, PDGFR&#946; (green), respectively. Nuclei were counterstained with DAPI (blue). Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67125/67125fig02large.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/67125/67125fig03.jpg" /> 
Figure 3:&#160;Immunofluorescent characterizations of the vascular network in vascular organoids. (A) A representative image shows gel-embedded vascular organoids in the glass dish after tissue clearing. The organoids were transparent but visible under a 385 nm UV light. (B) A representative 3D whole-mount image of vascular organoid on Day 17. The vascular organoid was stained with DAPI (blue), the vascular endothelial cell marker, CD31 (green)&#160;and the pericyte marker, PDGFR&#946; (magenta). (C) A representative whole-mount 3D imaging of a retrieved vascular organoid on Day 28, stained with the endothelial markers CD31 (red) and ERG1 (green). Scale bars: 150 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67125/67125fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>hiPSC expansion medium</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>mTeSR Plus basal medium</td>
			<td>400 mL</td>
		</tr>
		<tr>
			<td>mTeSR Plus supplement</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>1 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of hiPSC expansion medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>hiPSC seeding medium</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>PSC expansion medium</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>Chroman 1 (100 &#181;M)</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>Emricasan (1 mM)</td>
			<td>30 &#181;L</td>
		</tr>
		<tr>
			<td>polyamine supplement (1000x)</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>trans-ISRIB (1 mM)</td>
			<td>14 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 2: Composition of hiPSC seeding medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>N2B27 medium</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX supplement</td>
			<td>48.6 mL</td>
		</tr>
		<tr>
			<td>NeuroBasal medium</td>
			<td>48.6 mL</td>
		</tr>
		<tr>
			<td>GlutaMAX supplement (100x)</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (NEAA, 100x)</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (1000x)</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>N2 supplement (100x)</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>B27 supplement minus vitamin A (50x)</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Heparin, 4 kU/mL</td>
			<td>0.1 mL</td>
		</tr>
	</tbody>
</table>
Table 3: Composition of N2B27 medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mesoderm induction medium (MIM)</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>N2B27 medium</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>BMP4 (100 ng/&#181;L)</td>
			<td>0.9 &#181;L</td>
		</tr>
		<tr>
			<td>CHIR99021 (12 mM)</td>
			<td>3 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 4: Composition of mesoderm induction medium (MIM).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vascular differentiation medium 1 (VDM1)</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>N2B27 medium</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>Forsklin (12 mM)</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>VEGFA (100 ng/&#181;L)</td>
			<td>3 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 5: Composition of vascular differentiation medium 1 (VDM1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagen I&#160; mixture</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Collagen I&#160; (3.61g/mL)</td>
			<td>2.77 mL</td>
		</tr>
		<tr>
			<td>10x DMEM/F12 medium</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, 7.5%</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>HEPES, 1 M</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>0.73 mL</td>
		</tr>
		<tr>
			<td>NaOH, 1M</td>
			<td>75 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 6: Composition of collagen I mixture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagen I&#160; - basement membrane matrix mixture</td>
			<td>6.5 mL</td>
		</tr>
		<tr>
			<td>Collage I&#160; mixture</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Matrigel (10 mg/mL)</td>
			<td>1.5 mL</td>
		</tr>
	</tbody>
</table>
Table 7: Composition of collagen I-basement membrane matrix mixture.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vascular differentiation medium 2 (VDM2)</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX&#8482; supplement</td>
			<td>3.67 mL</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (NEAA, 100X)</td>
			<td>50 &#181;L</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (1000x)</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>Heparin, 4 kU/mL</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>0.75 mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>VEGFA (100 ng/&#181;L)</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>FGF2 (100 ng/&#181;L)</td>
			<td>5 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 8: Composition of vascular differentiation medium 2 (VDM2).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blocking buffer</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate solution (1%, wt/v)</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>46.5 mL</td>
		</tr>
	</tbody>
</table>
Table 9: Composition of blocking buffer for immunostaining.

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.5K Views.  Columbia University. This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

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

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Developmental Biology

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

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

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 ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

Using Induced Pluripotent Stem Cell Suspension for Kidney Organoid Production

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

Author Spotlight: Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids 

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid research.

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease ...

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

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.

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 ...

An In Vitro Method to Generate Human Brain Organoids

Source: Gabriel, E., et al., <a href="https://app.jove.com/v/55372">Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating human brain organoids from induced pluripotent stem cells. It shows how the neurosphere forms a neuroectodermal layer, which suppresses the formation of other germ layers. The neurospheres are embedded in a 3D gel matrix and form neuroepithelial buds. When incubated with organoid media, these neuroepithelial buds differentiate into specific brain regions, resulting in the formation of mature brain organoids.

Metoda in vitro do generowania organoidów ludzkiego mózgu

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Generation of Brain Organoids (23 ...