Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Summary

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

Abstract

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.

Introduction

In vitro microphysiological models, including organoid and tissue-on-a-chip systems, have emerged over the past decade^1,2,3. 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 brain^4,5, kidney⁶, liver^7,8 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 and colleagues developed the first vascular organoid generation method^11,12 that mimics these two processes. With the organoids generated from this method, their group¹² and us^13,14 have demonstrated the potential of vascular organoids for modeling vascular malfunctions in diseases. For example, in our recent works^13,14, 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 organoid model could serve as a platform for drug screening by mimicking disease-related vascular malfunctions.

Built upon the initial vascular organoid generation works^11,12, 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 and the integrated stress response inhibitor (trans-ISRIB)¹⁵, is employed 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.

Protocol

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 °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

1. 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 °C for up to 1 year.
2. 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 °C for up to 1 week.
3. Prepare the hiPSC expansion medium as described in Table 1. Make aliquots of the complete medium (40 mL/tube) and store at -20 °C for up to 1 year.
4. Put a pre-coated plate in the 37 °C incubator for 1 h prior to hiPSC culture.
5. Prewarm hiPSC expansion medium (40 mL) at 37 °C and prepare the hiPSC seeding medium as described in Table 2.
6. Transfer the coated plate from the 37 °C incubator to the biosafety cabinet. Replace the medium in the coated plate with hiPSC seeding medium (1 mL/well).
7. Thaw a cryopreserved vial of hiPSCs from the cryotank in a 37 °C water bath for 1 min.
8. 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.
9. Aspirate the supernatant and resuspend the cells with 1.5 mL of hiPSC seeding medium. Gently resuspend cells in the 15 mL tube.
10. Aliquot 10 µL of the cell suspension and mix with 10 µL of trypan blue solution. Transfer 10 µL mixture to the hemacytometer slide and cover it with a coverslip.
11. 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.
12. Seed 2 × 10⁵ cells in each well and ensure that the cells are evenly distributed in the well.
13. Culture the cells at 37 °C with 5% CO₂ and replace the medium with hiPSC expansion medium (1.5 mL/well) after 24 h.
14. 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 (>5%) spontaneous differentiation is observed.

2. EB formation (Day 0)

1. On Day 0, prepare and prewarm the DMEM/F12 medium, hiPSC seeding medium and cell detachment solution at 37 °C for 20-30 min.
2. Remove the culture medium from the 6-well plate and add prewarmed cell detachment solution (1.5 mL/well). Incubate the plate at 37 °C for 5-7 min.
3. Transfer the plate to the biosafety cabinet, tap the plate to detach hiPSC colonies, and break up the aggregates.
4. 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.
5. Centrifuge at 100 x g for 3 min at room temperature to collect the cells.
6. Aspirate the supernatant and gently resuspend the cells with 1 mL of hiPSC seeding medium using a P1000 pipette.
7. Mix 10 µL of cell suspension with 10 µL of trypan blue solution for cell counting. Transfer 10 µL 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.
8. Seed the cells in the ultra low-attachment round bottom microwell plate with a density of 2.7 × 10⁵ cells/well to yield 500-1,000 cells per EB.
9. Add 2 mL of hiPSC seeding medium to each well and mix the solution gently to distribute cells evenly.
10. Culture the cells at 37 °C with 5% CO₂ for 24 h.

3. Vascular differentiation (Day 1 - 5)

1. On Day 1, prepare the mesoderm induction medium (MIM) as described in Table 3 and Table 4 and prewarm it at 37 °C for 20 min. The MIM medium must be made freshly.
2. Use a P200 pipette to carefully aspirate the medium from the microwell plate without disturbing the EBs at the bottom.
3. Add 2.5 mL of MIM slowly using a P200 pipette. Culture the EBs in MIM at 37 °C with 5% CO₂ for 2 days.
4. 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 °C with 5% CO₂ for another 2 days.

4. Hydrogel embedding (Day 5)

1. Prepare the collagen I mixture as described in Table 6. Place a 50 mL tube on ice and add collagen I solution, H₂O, 10× DMEM/F12 medium, sodium bicarbonate 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.
2. 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.
3. Place a 12-well plate on ice for 2 min.
4. 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.
5. Put the plate in the 37 °C incubator for 2 h to solidify the first layer of hydrogel.
6. Keep the remaining collagen I-basement membrane matrix mixture on ice for later use.
7. 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.
8. Dropwise, add 1.5 mL of collagen I-basement membrane mixture to the aggregates and gently mix them using wide-bore P200 pipette tips.
9. Transfer the plate with the first layer of hydrogel from the incubator (step 4.5) to the biosafety cabinet.
10. 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.
11. Incubate the plate at 37 °C with 5% CO₂ for another 2 h.
12. Prepare the vascular differentiation medium 2 (VDM2; Table 8) and prewarm the medium in a 37 °C water bath for 20 min prior to use.
13. Add 2 mL of VDM2 to each well and culture the cells at 37 °C with 5% CO₂.
14. 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 µL/well). Change the medium every 2-3 days.

5. 3D Immunofluorescent assay

1. 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).
2. Add 2 mL of 4% paraformaldehyde (PFA) to the sample and incubate at 4 °C overnight with paraffin film sealing.
3. Remove the PFA solution and wash the sample with 2 mL PBS four times (15 min each time).
4. Add 2 mL of blocking buffer (Table 9) and incubate at room temperature for 2 h.
5. 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 °C for 2 days with gentle shaking.
   NOTE: The primary antibody (CD31, PDGFRβ, and ERG1) is recommended to be diluted in the blocking buffer at a ratio of 1:400 (see Table of Materials for more information).
6. Remove the primary antibody solution and wash with 2 mL PBST for 1 h six times.
7. 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 °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.
8. Remove the secondary antibody solution and wash with 2 mL TBST six times (1 h each time). Add DAPI (1 µg/mL) in the wash buffer used for the last wash step if nuclei staining is needed.
9. 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.
10. Use wipes to absorb the remaining solution without touching the gel or the organoids.
11. 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.
12. Transfer the organoids with the clearing reagent carefully to the center of the glass-bottom dish using a spatula.
13. Cover the organoids or gel piece with a 24 mm × 24 mm coverslip.
14. Push the coverslip down gently until it lays flat, and sandwich the samples with the glass bottom.
15. Remove the redundant clearing reagent in the dish and seal the coverslip with nail polish.
16. For 3D imaging, use a confocal microscope with a 10× objective. Use tile scan and Z-stack scan to obtain whole-mount images of organoids.

Results

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 ...

Discussion

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

Materials

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