Aniridia-Associated Keratopathy Stems From Limbal Stem Cell Niche Dysfunction

keratopathy bullous condition in eye of elderly patient before penetrating keratoplasty
An investigation reviews the connections between abnormalities in the limbal niche microenvironment and aniridia-associated keratopathy.

Ocular surface alterations in aniridia-associated keratopathy (AAK) stem from the primary dysfunction and gradual breakdown of the limbal stem cell niche due to PAX6-related effects, according to research results published in The Ocular Surface

Congenital aniridia is a rare ocular condition, caused by a haploinsufficiency of the Pax6 transcription factor resulting in heterozygous mutations in the PAX6 gene locus. Insufficient Pax6 has been associated with abnormal ocular structure development, manifested clinically as iris hypoplasia, corneal opacification, cataract, glaucoma, nystagmus, and foveal and optic nerve hypoplasia. 

Patients living with aniridia are at high risk of childhood glaucoma (median age of onset, 8 years), and a large proportion of these patients — between 78% and 100% — develop AAK, a severe corneal phenotype resulting in slowly progressing limbal stem cell deficiency leading to corneal epithelial erosions, corneal vascularization, progressive conjunctivalization, pannus formation, and, ultimately, corneal opacification, and vision loss. 

Currently, the pathophysiology of AAK and the mechanisms that cause PAX6 mutations to result in AAK are controversial, the study explains.

Researchers reported on structural and molecular alterations present in the human limbal niche of a 16-year-old girl with congenital aniridia, AAK, and secondary glaucoma with a “blind and painful” left eye requiring enucleation due to uncontrolled intraocular pressure IOP and lens luxation. 

In situ morphological and immunohistochemical data were supplemented by mRNA expression studies undertaken on limbal epithelial cells cultivated from limbal biopsies both of this patient and of other patients with aniridia. 

The primary patient presented with painful buphthalmia due to secondary aniridia-related glaucoma, congenital aniridia, complete loss of corneal transparency with corneal vascularization, LESC in the superior half, dry eye syndrome, and a subluxated cataractous lens in the left eye. In the right eye, corneal vascularization involved 360 ˚ of the limbus. 

AAK diagnosis was made based on classical clinical features: irregular corneal epithelium, recurrent erosions, and an opaque, vascularized cornea. Both eyes had AAK graded stage 2 and stage 3. Genetic analysis identified a “large, heterozygous PAX6 gene deletion” that encompassed exons 11 to 15 and exon 9 in the neighboring ELP4 gene. 

The left eye underwent 3 glaucoma surgeries (2 cyclophotocoagulations and 1 trabeculotomy) and was enucleated in 2017 due to painful secondary glaucoma, vitreous hemorrhage, and crystalline lens luxation. 

Three small limbal biopsies were taken from the superior, nasal, and temporal regions in order to isolate and cultivate limbal epithelial progenitor cells for molecular analysis. The biopsies underwent histopathology, electron microscopy, and immunohistochemistry. 

Corneoscleral tissue samples were also obtained from 5 normal human donor eyes (mean age, 38±9.5 years) and were used as controls. 

Light microscopic analysis showed a rudimentary iris stump forming peripheral anterior synechiae, a maldeveloped closed chamber angle with no signs of regular outflow structures, a pupillary membrane, and an atrophic ciliary body. Central corneal findings showed an irregularly thickened and thinned corneal epithelium consisting of 3 to 15 cell layers. 

Morphological differences were distinct, and seen between superior and inferior limbal regions that reflected different LESC disease progression. Within the superior region, palisade structures were “largely absent;” in the inferior region, investigators found flattened but identifiable palisade structures. 

Results of Fontana-Masson staining demonstrated intense argentaffin reaction for melanin pigment that was contained within limbal melanocytes, and adjacent LESC in normal control eyes. This was completely absent in the inferior limbus of the eye with aniridia. 

In the superior-temporal limbal quadrant, residual palisade structures contained no distinct LESC clusters; rather they had a uniform population of less differentiated cells that continued into the basal layer of the corneal epithelium. 

Immunolabeling for Pax6 was seen in the inferior-temporal quadrant in most corneal and limbal epithelial cells in AAK. Cytoplasmic localization of Pax6 was not noted in the control eyes. Ocular surface epithelia in AAK were “completely negative” for corneal epithelial differentiation markers keratin 12 (K12) and K3, but positive for conjunctival differentiation markers K13 and K19. 

Abnormal K10 and K13 expression in AAK epithelial cells was correlated with cytoplasmic localization of Pax6. Consecutive imaging of conjunctival, limbal, and corneal epithelial demonstrated differential expression patterns suggest “transdifferentiation of corneal epithelial cells rather than mere conjunctivalization of the cornea.” 

Desmosome protein expression — including desmocollin-2 and desmoglein-1 — was only seen in the superficial epithelial cells of the cornea and limbus in AAK. 

K15, K14, and P-cadherin, all established limbal stem/progenitor cell markers, were expressed in limbal basal epithelial cells in both AAK and control samples. However, in AAK, positively labeled progenitor-like cells were not organized into limbal clusters, rather forming a “continuous multilayered band of undifferentiated basal/suprabasal cells” that extended from the limbus to the corneal surface. 

These findings, among others, indicate that undifferentiated cells that exhibit a limbal epithelial cell/progenitor cell phenotype are well preserved in AAK, but not organized in a typical fashion. 

In the patient with AAK, the limbal epithelium was infiltrated heavily with CD18-positive immune cells from the stroma, as well as interspersed with Melan A-positive melanocytes, which have been identified as an important “niche cell” population. In AAK, these cells were increased markedly, and displaced from their basal position, as well as expressed reduced Pax6 levels in their nuclei. 

The extracellular matrix of the limbal microenvironment also demonstrated substantial alterations, including immunolocalization of classical basement membrane components like collagen type IV and perlecan. Conversely, subepithelial matrix components like tenascin-C, versican, vitronectin, fibulin-2, and fibrillin-2, had “markedly increased staining intensities” had increased staining intensities in AAK but weak and patchy staining patterns in controls. 

Transcript levels of PAX6 were not significantly different between patients with aniridia and the 5 healthy donors. However, transcript levels of KRT12 (K12), KRT3 (K3), DSG1, and ALDH1A1 were significantly reduced in AAK vs controls, indicative of a correlation to reduced PAX6 dosage. mRNA expression levels of KRT13 (K13), KRT10 (K10), KRT17 (K17), KRT15 (K15), and ABCG2 were not significantly different between groups, nor were transcript levels of evaluated extracellular matrix genes. This indicated that differences seen on the protein level are not caused by transcriptional dysregulation in limbal epithelial cells. 

Study limitations include the use of a single case description and prolonged glaucoma medication use. 

“Genetic and environmental factors (oxidative stress, inflammation) seem to interact in the pathogenesis of AAK, with reduced nuclear Pax6 dosage affecting both normal limbal epithelial differentiation and melanocyte pigmentation and function,” according to the researchers. “[These findings] are fully compatible with previous data from animal models.” 

“They further support the concept that AAK is caused by a primary and progredient dysfunction of the limbal stem cell niche, preventing homing and survival of LESC,” the study says. “As a consequence of these findings, any therapeutic approach in aniridia patients should also consider strategies for niche restoration to halt the decline of LESC function.” 


Schlötzer-Schrehardt U, Latta L, Gießl A, et al. Dysfunction of the limbal epithelial stem cell niche in aniridia-associated keratopathy. Ocul Surf. Published online June 6, 2021. doi:10.1016/j.jtos.2021.06.002