Patient-Specific iPSC-Derived Cornea Organoids for Investigating Aniridia-Associated Corneal Disorders

Summary

We describe a protocol for generating cornea organoids from iPSCs derived from aniridia patients, creating a patient-specific model to study corneal disorders. These organoids mimic key structural and functional features, providing a reliable in vitro platform for investigating disease mechanisms, testing potential treatments, and advancing personalized regenerative approaches for anterior eye diseases.

Abstract

Aniridia is a rare congenital eye disorder marked by the partial or complete absence of the iris, and it is often associated with corneal opacification and limbal stem cell deficiency. The underlying genetic mutations, primarily affecting the PAX6 gene, disrupt both the development and function of the cornea. The most common ocular features besides iris defects are nystagmus, foveal hypoplasia, cataract, glaucoma and aniridia-associated keratopathy (AAK). Although in vivo models offer valuable insights into the pathology of aniridia, patient-derived in vitro models are crucial for investigating disease mechanisms and evaluating potential therapies. In this study, we present an in vitro AAK model using patient-derived induced pluripotent stem cells (iPSCs). Our protocol involves the stepwise differentiation of iPSCs into corneal epithelial-like cells within self-assembled three-dimensional organoids, which mimic the native corneal microenvironment. Through the optimization of differentiation culture conditions, we successfully generated corneal organoids that harbor distinct corneal cell populations, recapitulating the key structural and functional attributes of the human cornea.

Introduction

The cornea is an avascular, transparent tissue forming the outermost layer of the eye, consisting mainly of three layers: an epithelial outer layer, a stromal layer containing a collagen-rich extracellular matrix populated by keratocytes, and an endothelial cell layer¹. The cornea plays an essential role in vision by protecting internal ocular structures and refracting light.

Aniridia is a rare, congenital panocular disorder primarily caused by heterozygous mutations in the paired box gene 6 (PAX6), a master regulator of ocular development, and is characterized by partial or complete absence of the iris². In addition to iris hypoplasia or absence, most patients develop progressive corneal abnormalities collectively referred to as aniridia-associated keratopathy (AAK), which includes epithelial defects, limbal stem cell dysfunction, stromal alterations, and progressive corneal opacification, ultimately leading to visual impairment. AAK affects approximately 78%–90% of aniridia patients, with increasing severity over time³. We aimed to establish a detailed and reproducible protocol to generate cornea organoids from aniridia patient-derived induced pluripotent stem cells (iPSCs) to model disease-relevant corneal alterations in vitro. Although animal models and ex vivo studies have provided valuable insights into aniridia pathogenesis, species-specific differences and limited access to patient tissue restrict their translational relevance.

Human iPSC-derived organoids have emerged as powerful in vitro systems for modelling human development and disease^4,5. Corneal organoids, in particular, enable the generation of three-dimensional tissue-like structures that more closely resemble native corneal architecture, including stratified epithelial organization, stromal – epithelial interactions, and extracellular matrix deposition, compared to conventional two-dimensional monolayer cultures, which lack spatial organization, physiological cell – cell interactions, and microenvironmental complexity^4,5. Several differentiation strategies, including SEAM-based and ocular progenitor-driven approaches, have been reported to generate corneal-like tissues from iPSCs⁶. These approaches typically involve an initial phase of ocular lineage induction, followed by progressive tissue specification and selection from heterogeneous ocular progenitor populations. Creating cornea organoids provides the opportunity to study different cell types that are present in corneal tissue^7,8. Moreover, aniridia patients’ iPSC-derived cornea organoids enable the study of the genetic background of the disease and offer a unique opportunity to study aniridia.

In the present protocol, both wild-type and aniridia-derived iPSC lines are subjected to an identical, multi-stage differentiation strategy involving sequential exposure to differentiation (DM), retinal differentiation (RDM), and corneal differentiation media (CDM) over a prolonged, weeks-long culture period. A critical experimental step is the transition from adherent culture to suspension conditions, during which selected corneal-like regions are maintained to promote epithelial self-organization.

While this approach supports reproducible generation of corneal-like epithelial structures suitable for comparative disease modelling, it does not fully recapitulate complex in vivo features such as neural innervation or complete limbal niche organization, which should be considered when assessing the method’s applicability.

In this context, we describe a standardized protocol for generating cornea organoids from patient-derived iPSCs carrying PAX6 mutations associated with aniridia. Importantly, the differentiation strategy itself is not modified between wild-type and aniridia-derived lines; instead, disease-specific phenotypes emerge from the patient-specific genetic background within an otherwise uniform experimental framework.

Overall, this method provides a robust and reproducible platform for studying corneal development and aniridia-associated disease mechanisms and may serve as a foundation for future mechanistic and translational studies.

Protocol

All procedures were carried out in accordance with the ethics committee's guidelines. The study has been registered in the Human Pluripotent Stem Cell Registry (hPSCreg) database, and the ethical approvals have been documented under the France Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation (MESRI; IE-2018-967)⁹. The iPSCs are designated with the codes AAKIPSi001-A (AAK1), AAKIPSi002-A (AAK2), AAKIPSi003-A (AAK3), and WT (from health donors). Furthermore, the ethics committee at Hacettepe University has approved the use of this research, as indicated by the reference number 16969557-1213. The human cell lines used in this study required national regulatory authorization at the time of acquisition. The approval was granted under a consortium framework in which one of the authors was involved. While the present manuscript is not directly related to that consortium project, the biological materials utilized were obtained and authorized within that ethical framework. Therefore, the approval from this university was appropriately cited to ensure full regulatory transparency. All details related to the materials, reagents, and equipment used in this protocol are listed in the Table of Materials. All experimental procedures were carried out within a laminar flow cabinet (BSL-2) to maintain a sterile environment. This setup ensures that all materials and equipment used in the study are adequately sterilized, thereby minimizing the risk of contamination and ensuring the integrity of the experimental results.

1. Generation of human induced pluripotent stem cell (hiPSC) derived cornea organoids

1. Maintenance and initial culture of aniridia-patient-derived human induced pluripotent stem cells (AAK1, AAK2, AAK3)¹⁰ under feeder-free conditioned media (Figure 1)
   1. Prepare the following in advance: Coat one well in a 6-well plate with 1 mL of an ice-cold extracellular matrix (ECM) solution at a concentration of 0.1 mg/mL. This coating procedure must be handled on ice, not to solidify the ECM, and must be allowed to incubate at 37 °C for a minimum duration of 30 min, adhering to the manufacturer’s instructions to ensure proper substrate preparation.
   2. Thaw a cryopreserved aliquot of 1 x 10⁶ hiPSC (WT, AAK1, AAK2, AAK3). Do not fully thaw the frozen cells; instead, allow the frozen cell suspension in the cryovial to thaw until approximately half of the volume remains frozen.
   3. Immediately transfer the partially thawed suspension into 1 mL of complete maintenance medium at room temperature (RT) containing 10 µM Rho-associated protein kinase (ROCK) inhibitor, pre-aliquoted in a 15 mL tube. Do not use any pipette during transfer.
   4. Centrifuge at 300 x g for 5 min and discard the supernatant.
   5. Then resuspend the cells in 1 mL of complete maintenance medium containing 10 µM ROCK inhibitor.
   6. Determine the cell density with trypan blue exclusion. Count cells using a hemocytometer after viability staining with trypan blue. Briefly, mix 20 µL of cell suspension 1:1 with 20 µL of 0.4% trypan blue, followed by the addition of 60 µL of PBS, resulting in a final 1:5 dilution of the original cell suspension. From this mixture, load 10 µL into the hemocytometer chamber. Count viable (trypan blue–negative) cells in four large squares. Calculate the average number of cells per square and multiply by 5 (cell suspension dilution factor) and 10,000 to determine the final cell concentration (cells/mL).
      ​Cells/mL = (Average cell count per square) x 5 x 10⁴
   7. Seed the cells onto a pre-coated ECM well in a 6-well plate, ensuring a cell density of 2 x 10⁴ cells/cm² per well.
   8. Observe the cell morphology and attachment using phase-contrast microscopy at 10x magnification. Then, incubate the 6-well plates at 37 °C with 5% CO₂ to ensure optimal growth conditions.
      NOTE: ROCK inhibitor must only be used overnight while passaging or thawing the cells. During daily medium changes, add the medium gently along the wall of the well. Avoid warming complete maintenance medium in a water bath; instead, equilibrate the medium at room temperature for 15 min before use.
2. Spontaneous differentiation phase (Day 0 – 2)
   1. Prepare the differentiation media (DM) by mixing the following: 8% KnockOut Serum Replacement (KSR), 1x non-essential amino acids (NEAA), 1x Glutamax, 1x Penicillin-Streptomycin, and Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12).
   2. Once hiPSC cultures reach approximately 70%–80% confluence, to induce spontaneous differentiation⁸, gently aspirate and completely discard the medium, then replace it daily with 1 mL of fresh DM per well until Day 3, when the retinal differentiation phase is initiated according to experimental timeline.
3. Retinal differentiation phase (Day 3 – 47)
   ​NOTE: Retinal differentiation medium is used for extended periods to allow the formation of heterogeneous ocular progenitor populations.
   1. Prepare the retinal differentiation media (RDM) by mixing the following: 8% KSR, 1x NEAA, 1x Glutamax, 2% B27, 1x Penicillin-Streptomycin, and DMEM/F-12.
   2. After 2 days of spontaneous differentiation, refresh the culture medium daily by carefully aspirating the DM medium and replacing it with 1 mL of fresh RDM medium per well. Continue this for a total of 30 days.
      NOTE: A significant increase in cell density is expected during this 30-day period. It is essential to make microscopic observations of cell density in different areas of each well. To prevent cell lifting, washing must be avoided, and care must be taken during medium changes. Add the media through the wall of the well. Equilibrate the medium at RT for 15 min before use.
   3. By Day 32, cells reach an apparent state of confluence and form multilayered 2.5D aggregates. At this stage, initiate the transition to suspension culture by gently scraping the entire well using a 1000 µL pipette tip. During this process, fragment the cell layer into small clusters.
      ​NOTE: Although all cells are released from the culture surface, multilayered 2.5D aggregates are preferentially collected and transferred to suspension culture (Supplementary Figure 1), as their enrichment promotes faster self-assembly and the formation of larger organoids compared with single cells or monolayer fragments.
   4. Immediately after scraping, collect the resulting cell clusters and transfer them into low-attachment 6-well culture plates containing RDM to establish suspension culture conditions.
   5. Maintain cells in suspension culture for an additional 15 days, with daily medium replacement using 2 mL of fresh RDM per well. During the first 2 days of suspension culture, avoid medium change to prevent loss of small cell clusters. During suspension culture, cells gradually self-organize and give rise to 3D, transparent, corneal-like structures, monitor regularly by phase-contrast microscopy at 4x and 10x magnification.
4. Corneal differentiation phase (Day 48 – 90)
   1. Prepare the corneal differentiation media (CDM) by mixing the following: 8% KOSR, 1x NEAA, 1x Glutamax, 1x N2-MAX, 1x Insulin-Transferrin-Selenium, 5 ng/mL FGF2, 10 ng/mL EGF, and DMEM/F-12.
   2. After the initial 15-days (from Day 32 – Day 47) of suspension culture in low-attachment plates (37 °C, 5% CO₂), replace RDM with CDM while maintaining the aggregates under suspension conditions at Day 47. Refresh CDM every other day with 2 mL per well, ensuring organoids are always fully submerged in the culture medium. During this period, transparent, bubble-like corneal primordial (CP) structures progressively emerge.
   3. Continue suspension culture in CDM until Day 90, allowing further maturation and spatial segregation of transparent CP regions from more opaque tissue compartments.
   4. At Day 90, mechanically separate transparent CP regions from more opaque tissue compartments using sterilized forceps and tweezers when CP-specific downstream analyses are intended.
      ​NOTE: Mechanical isolation of CP regions is performed when analyses specifically require CP-enriched structures. If other tissue compartments or overall organoid architecture are of interest, organoids may be maintained intact to preserve structural heterogeneity.
   5. Continue the suspension culture in CDM for either isolated CP structures or intact organoids, depending on the experimental objective.

2. Characterization of corneal organoids

1. Morphological and structural evaluation of organoids
   1. Monitor cornea organoids regularly using brightfield microscopy at 4x and 10x magnification. Evaluate size, transparency, and overall morphology at defined time points and document structural changes by image acquisition.
2. Immunofluorescence-based characterization
   1. Cornea organoid fixation
      1. Prepare 4% PFA and 30% sucrose solutions before fixation. Take the cornea organoid from culture with an autoclaved scooper. Transfer the organoid into the 4% PFA in a 1.5 mL tube and incubate for 20 min at room temperature.
      2. After incubation, discard the PFA from the tube carefully without damaging the cornea organoid. Add 1 mL of 30% sucrose solution to the tube and place it at 4 °C. After 24 h, prepare and label 5 mL tubes and liquid nitrogen for OCT fixation.
      3. Discard the sucrose solution from the tube carefully. Transfer the organoid into the sterile scooper. With a 20 µL pipette, remove the excess sucrose solution from the organoid carefully.
      4. Pour OCT into the 5 mL tube cap, and place the organoid into the OCT. Use another sterile scooper to transfer the organoid into the OCT-containing cap.
      5. Add OCT to cover the surface of the cornea organoid. Pop the balloons in the OCT with a needle to obtain clear sections of the organoid.
      6. Close the cap of the 5 mL tubes and use tweezers to dip into the liquid nitrogen. Store the samples in -20 °C until sectioning.
      7. Section them at 5 µm thickness using a cryostat and also store them at -20 °C till staining. Maintain consistent section thickness and tissue orientation to ensure reproducible staining.
   2. IF staining
      1. To prepare cornea organoid sections for IF staining, wash the slides 3x with PBS-T (0.3% Triton X-100) at RT for 10 min.
      2. Clean the slide with tissue paper and make a border with PAP pen. Add an appropriate amount of blocking solution to completely cover the tissue section (typically 100 – 200 µL per slide, depending on the PAP pen boundary) and incubate it at RT for 1 h.
      3. Aspirate the blocking solution and make a border with the PAP pen. Add the 150 μl of primary Ab (PAX6, p63, OCT3/4, NANOG, Na⁺/K⁺-ATPase, AQP1, CK3, CK5, CK14, CK19, N-cad, KERA, COLIV, the dilution rates are listed in the Table of Materials) solutions onto the slide and incubate it at RT for 1 h.
      4. Wash 2x with PBS-T (0.3% Triton X-100) at RT for 10 min. Make the border again with a PAP pen.
      5. Add the 150 μl of secondary Ab solution (Anti Rabbit-488, Anti Mouse-594, the dilution rates are listed in the Table of Materials) onto the slide and incubate it at RT under dark conditions for 2 h. Wash 2x with PBS-T (0.3% Triton-X) at RT for 10 min. Clean the slides and mount with DAPI solution.
      6. For undifferentiated iPSCs, perform blocking and permeabilization after fixation and proceed with staining using the same protocol. Visualize stained samples using fluorescence or confocal microscopy at 4x and 10x magnification.

Results

Stemness of human induced pluripotent stem cells (hiPSCs)

To assess the stemness of human iPSCs used in the experiments, we performed brightfield imaging, immunostaining, and qRT-PCR analysis. We evaluated the expression of key pluripotency markers, including OCT3/4, SOX2, and NANOG, which are core transcription factors required for the maintenance of pluripotency and self-renewal in human pluripotent stem cells¹¹.

B...

Discussion

Comparative analysis between control organoids and those derived from aniridia patients reveals defects in epithelial stratification, abnormal extracellular matrix deposition, and dysregulated expression of the PAX6 gene⁹.

The growth rate of iPSCs derived from different aniridia patient samples, specifically AAK1, AAK2, and AAK3 lines, exhibits variability, potentially reflecting the underlying genetic diversity, caused primarily by mutations in the PAX6

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL serological pipettes	ZA Biotekno	Labselect SP-003-10
15 mL Conical Tubes	Labselect	CT-002-15A
2 mL serological pipettes	Labselect	Labselect SP-003-2
5 mL serological pipettes	Cleanteks	Biofil GSP010005
50 mL Conical Tubes	Golden Gate	CT-002-50
50 mL serological pipettes	BIOFILL	GSP020050
Anti Mouse 594	Jackson Immuno	715-585-150	Dilution Range: 1:100 - 1:800
Anti Rabbit 488	Jackson Immuno	711-545-152	Dilution Range: 1:100 - 1:800
Autoclave VAPOUR-Line lite	VWR chemicals	VAPOUR-Lineeco 25
B27 Supplement	GIBCO	12587010	with vitamin A
Benchtop Centrifuge	Eppendorf	5702
Benchtop microscope DMI1	Olympus	CKX41
Biological Safety Cabinets Class II	Thermo	Safe 2020
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A8806
CellBanker 1	Worthington Industries	LABS-20K
Coverslips 24 mm x 50 mm	ISOLAB	280
Cryomatrix, OCT	Fisher Healthcare	30226281
Cryovial	BioSigma	N403522
DAPI	NEOFROXX	1322
DAPI	NEOFROXX	1539	0.5 ug/mL
DMEM/F12 Media	GIBCO	31330-038
Dulbecco's Phosphate-bufferd Saline (DPBS)	Gibco	14190144	1X
EGF	GIBCO	PHG0311
Ethanol 100%	Sigma	64-17-5
Ethanol 70%	Sigma	64-17-5	Ethanol dilutions
Extracellular Matrix (ECM)	Corning	354277	LDEV-free
FGF2	GIBCO	PHG0024
GlutaMax	GIBCO	35050061
GoTaq Master Mix	Promega	M7123
Incubator	Memmert INCO 153 med	44043
Insulin-Selenium-Transferrin	GIBCO	41400045
KnockOut Serum (KOSR)	GIBCO	10828028
Microtome RM2235	Leica	RM2235
Mounting medium	abcam	ab104135
mTSER Media	STEMCELL	85850
Mutiwell Culture Plates (6-well)	CORNING	3516
N2 Supplement	R&D	AR003
Non-essential amino acids (NEAA)	LONZA	BE13-114E
P20, P200 and P1000 pipettes	Gilson	F167350
Paraffin	VWR chemicals	10048502
Paraformaldehyde	Sigma-Aldrich	F8775
Primer Antibody Aquaporin mAb	abcam	ab9566	Dilution Range: 1:50-1:200
Primer Antibody CK14	Santa Cruz Biotechnology	sc-53253	Dilution Range: 1:50-1:500
Primer Antibody CK19	ProteinTech	14965-1-AP	Dilution Range: 1:150-1:600
Primer Antibody CK3	NOVUS	NBP2-91997 0.1 ML	Dilution Range: 1:50-1:200
Primer Antibody CK5	Thermo	MA517057	Dilution Range: 1:200-1:1000
Primer Antibody Collagen Type IV	abcam	ab6586	Dilution Range: 1:100-1:500
Primer Antibody Keratocan	Bioss	bs-11054R	Dilution Range: 1:50-1:200
Primer Antibody Na/K - ATPase	Santa Cruz Biotechnology	sc-21712	Dilution Range: 1:50-1:500
Primer Antibody N-Cadherin	abcam	ab76011	Dilution Range: 1:50-1:500
Primer Antibody p63	SANTACRUZ	sc-25268	Dilution Range: 1:50-1:500
Primer Antibody Pax6	Novus	NBP2-44576	Dilution Range: 1:50-1:200
RNA isolation kit	Macherey–Nagel	740984.50
Rock Inihibitor (Y-27632)	TOCRIS	1254
Slides	StarFrost	MBB-0302-55A	Adhesive, ground
Stainless steel forceps no:5	Sigma	F6521-1EA
Stainless Stell Scalpel Sterile N 21	Swann-Morton	1033060
The OneScript Plus cDNA Synthesis Kit	Applied Biological Materials	G236
Triton-X100	NEOFROXX	8500
TRIzol reagent	Thermo	15596026
Trypan blue	GIBCO	15250061
Ultra low attachment (ULA) 6-well  plate	Corning	3471
Water bath	Nüve	Nb9