An Optimized Protocol for the Generation of Vascular Organoids from Human Induced Pluripotent Stem Cells

Summary

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

Abstract

Vascular organoids derived from human pluripotent stem cells (hPSCs) have emerged as powerful three-dimensional models for studying vascular development, disease mechanisms, and drug responses. Current vascular organoid protocols enable the generation of self-organizing endothelial–pericyte networks; however, batch-to-batch variability, inconsistent organoid formation, and inadequate pericyte coverage can compromise reproducibility and scalability. This study presents an optimized protocol for generating vascular organoids from human induced pluripotent stem cells (hiPSCs) with improved reproducibility and structural consistency. Key modifications include refined embryoid body formation conditions, optimized growth factor concentrations and timing during mesoderm induction and vascular specification, and improved three-dimensional culture conditions that promote robust pericyte recruitment and endothelial–pericyte interactions. The optimized organoids exhibit highly branched CD31-positive endothelial networks consistently ensheathed by PDGFR-β-positive pericytes, with significantly reduced batch-to-batch variability compared with the original protocol. Detailed, step-by-step procedures are provided for organoid generation, whole-mount immunofluorescence characterization, and quality control assessment. This optimized method enables the scalable production of high-quality vascular organoids suitable for studying vascular cell interactions, modeling disease, and screening compounds, thereby lowering the technical barrier for laboratories seeking to adopt this model system.

Introduction

Three-dimensional (3D) organoids have emerged as powerful tools for modeling human organ development and disease in vitro, offering superior structural biomimicry and pathophysiological relevance compared with conventional two-dimensional cell culture models^1,2. In recent years, human pluripotent stem cell (hPSC)-derived organoids have been successfully established for multiple tissue types, including the intestine³, brain^4,5, kidney⁶, liver^7,8....

Protocol

The use of hPSCs in this study was approved by the Biomedical Ethics Committee of Anhui University (BECAHU_2026-010).

1. Maintenance of human induced pluripotent stem cells

NOTE: Maintain human induced pluripotent stem cells (hiPSCs) under feeder-free conditions. Perform all procedures in a Class II biological safety cabinet using aseptic technique.

1. Preparation of basement membrane matrix-coated culture plates
   1. Remove a vial of basement membrane matrix from −20 °C and thaw it at 4 °C overnight for approximately 12–16 h. Avoid repeated freeze–thaw....

Results

The optimized protocol described here enables the generation of mature vascular organoids from hiPSCs within approximately 15 days using a stepwise differentiation strategy (Figure 1A). Briefly, hiPSC aggregates were generated in low-attachment plates supplemented with 0.25% PVA on an orbital shaker (Day −1). Mesoderm induction was initiated on Day 0 using CHIR99021 (12 µM) and BMP-4 (30 ng/mL) for 3 days, followed by vascular lineage specification with VEGF-A (100 ng/mL) and forskolin (2 µM.......

Discussion

The optimized protocol described herein introduces several key improvements to the vascular organoid differentiation system originally established by Wimmer et al.¹⁸, with the aim of enhancing the homogeneity, reproducibility, and structural integrity of vascular organoid generation. Compared with the original protocol, the optimized method provides significant improvements in the following aspects. First, optimizing aggregate formation conditions substantially improves the homogeneity of embryoid.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm Bottle-Top Filter	Nalgene	FPE204030	For sterile filtration of media and buffers
15 mL Conical Tube	SparkJade	GD0001	Sterile
2 mL Microcentrifuge Tube	Eppendorf	22363204	Sterile
2-Mercaptoethanol (1000×)	Gibco	21985023	Use in fume hood
Alexa Fluor 488 AffiniPure F2 Fragment Donkey Anti-Rabbit IgG (H+L)	Lablead	Y1104	Secondary antibody; dilute 1:300
Alexa Fluor 594 AffiniPure F(ab′)2 Fragment Donkey Anti-Goat IgG (H+L)	Lablead	Y1007	Secondary antibody; dilute 1:300
Anti-CD31 antibody	Cell Signaling	3528s	Primary antibody; dilute 1:100 in blocking buffer
Anti PDGFR β antibody	Zenbio	347269	Primary antibody; dilute 1:100 in blocking buffer
Automated Cell Counter	Life Technologies	Countess™ II	Includes counting chamber slides
B27 Supplement (50×)	Gibco	17504044	For N2B27 medium; thaw at 4ºC, aliquot and store at −20ºC, avoid freeze-thaw
Benchtop Centrifuge	Zonkia	SC-3610	With swinging-bucket rotor
Biological Safety Cabinet (Class II)	Thermo Fisher	NA	NA
BMP4	Proteintech	HZ-1045	Reconstitute in sterile ddH2O to 100 ng/μL, aliquot and store at −20 °C
Bovine Serum Albumin (BSA)	Biosharp	9048-46-8	For blocking buffer
CHIR99021	MCE	HY-10182	Prepare 10 mM stock in DMSO, store at −20ºC for up to 1 year
CO2 Incubator	Fisher Scientific	LS-CO150	37 ºC, 5% CO2, humidified
Confocal Imaging Dish	Biosharp	BS-20-GJM	For imaging after staining
Confocal Microscope	LEICA	NA	Equipped with 10×, 20×, 63× objectives and ZEN software
DMEM	Vivacell	C3113-0500	For Matrigel dilution and collagen I solution
DMEM/F-12, GlutaMAX Supplement	Gibco	10565018	For Matrigel dilution and N2B27 medium
EDTA (0.5 M, pH 8.0)	Invitrogen	AM9260G	Dilute with PBS to 0.02% for passaging
Fetal Bovine Serum (FBS)	Gibco	10270-106	For N2B27 complete differentiation medium (15% final)
FGF-2	Novoprotein	C046	Prepare 1 mg/mL stock in sterile PBS, aliquot and store at −20ºC
Forskolin	R&D Systems	1099	Prepare 10 mM stock in DMSO, store at −20ºC
HEPES (1M)	Gibco	15630080	For collagen I solution
Hoechst	Beyotime	C1071S-4	Prepare 1 mg/mL stock in deionized water, aliquot and store at −20ºC
Inverted Microscope	LEICA	DMIL LED	Routine cell observation
Matrigel (growth factor-reduced)	STEEMA	BM05	Store at −20ºC after aliquoting, avoid freeze-thaw; handle on ice
MEM Non-Essential Amino Acids Solution (NEAA, 100×)	Gibco	11140035	For N2B27 medium
N2 Supplement (100×)	Gibco	17502048	For N2B27 medium; store at −20ºC protected from light
Neurobasal Medium	Gibco	21103049	For N2B27 medium
Nuclease Free Water (DNase/Rnase Free)	Lablead	Doo55WJ	For RNA/cDNA preparation and qPCR
Orbital Shaker	Joan Lab	NA	For washing steps
Paraformaldehyde (4% solution)	Servicebio	G1101	Dilute with PBS to 4% for fixation; use in fume hood
Penicillin-Streptomycin (100×)	Gibco	15140122	For N2B27 medium
PGM1 Medium	Cellapy	A1517001	hiPSC expansion medium; store at 4ºC protected from light, stable for 2 weeks
PGM1 Supplement (25×)	Cellapy	A1517101	For use with PGM1 Basal Medium
Polyvinyl Alcohol (PVA)	Sigma-Aldrich	P8136	Dissolve in PGM1 at 0.25% (w/v) to promote aggregation
PureCol (Bovine Type I Collagen, 3 mg/mL)	Advanced BioMatrix	5005	For collagen I-Matrigel mixture; test batch for polymerization capacity
qPCR Thermal Cycler	Thermo Fisher	StepOnePlus™	With melt curve capability; for real-time PCR detection
Reverse Transcription Kit	Accurate Biology	AG11706	For cDNA synthesis from total RNA; contains RT master mix with reverse transcriptase, buffer, dNTPs, and random primers
RNA Extraction Kit	Yeasen	19221ES50	For total RNA isolation from vascular organoids; includes gDNA elimination columns and RNase-free DNase I
Sodium Bicarbonate (7.5%)	Gibco	25080094	For collagen I solution
Sodium Deoxycholate	Sigma-Aldrich	D6750	Prepare 1% (w/v) solution in deionized water for blocking buffer
Sodium Hydroxide (1.0 N)	Sigma-Aldrich	S2770	For pH adjustment of collagen I solution
Sterile Disposable Pipettes (10 mL)	Bkmamlab	110205007	Individually wrapped, sterile
Sterile Filter Pipette Tips (1000, 200, 20 μL)	Biozym	VT0270/VT0250/VT0220	Sterile, with filter
Sterile Needle (30 G)	NA	NA	For dissecting vascular networks
Sterile Spatula (Round End)	Fisher Scientific	21-401-5	Stainless steel
SYBR Green Master Mix	Accurate Biology	AG11733	2× concentration for qPCR; contains hot-start DNA polymerase, buffer, dNTPs, and SYBR Green dye
Tissue Culture-Treated 12-Well Plate	BD Falcon	353043	For gel embedding and vascular network culture
Tissue Culture-Treated 6-Well Plate	Eppendorf	30720113	Standard TC-treated
Triton X-100	Sigma-Aldrich	T9084	For blocking buffer
Trypan Blue (0.4%)	Thermo Fisher	15250061	For cell counting
Tween 20	Sigma-Aldrich	P7949	For 0.05% PBS-T wash solution
Ultra-Low Attachment 6-Well Plate	Corning	3471	For aggregate formation and differentiation
Ultra-Low Attachment Round-Bottom 96-Well Plate	Beyotime	FULA962-6pcs	For suspension culture of vascular organoids
VEGF-A	Proteintech	HZ-1038	Prepare 1 mg/mL stock in sterile PBS, aliquot and store at −20ºC
Water Bath	Blue pard	HWS-24	37ºC
Y-27632	MCE	HY-10071	Prepare 10 mM stock in sterile PBS or DMSO, store at −20ºC; working concentrations: 5 μM (thawing), 50 μM (aggregate formation)