Abstract
Purpose :
Congenital aniridia is caused by heterozygous mutations on the PAX6 gene leading to reduced amount of PAX6 protein (haploinsufficiency), abnormal eye development and aniridia-associated keratopathy (AAK). It is generally accepted that this progressive corneal opacification is due to limbal stem cell (LSC) deficiency, leading to disrupted corneal epithelial renewal. However, other earlier features like inflammation and loss of innervation could indirectly cause limbal niche dysfunction and late LSC loss. In this study, we produced induced pluripotent stem cells (iPSC) from AAK patients and addressed the question of whether PAX6 haploinsufficiency affects LSC lineage commitment.
Methods :
The study was conducted with AAK-iPSCs derived from three AAK patients carrying different mutations and from two healthy controls. The established iPSC lines were characterized according to the general standards of PSCs, maintained in a feeder-free culture system, and thereafter differentiated to LSCs with our previously established method. Characterization of the cells at days 7, 10-11 and 24-26 was carried out using qPCR. Additionally, phase contrast microscopy, immunofluorescent labeling of PAX6, ABCG2, and p63/p40, as well as flow cytometry analysis of ABCG2 and ABCB5 were performed at days 10-11 and 24-26. Functionality of the differentiated cells was assessed at d24 by a wound healing assay.
Results :
During LSC lineage commitment, characterization of the AAK lines showed lowered PAX6 expression as compared to the controls. Still, the AAK-iPSCs successfully differentiated towards LSC lineage and showed similar wound healing capacity as healthy controls. However, extensive batch-related variation in the LSC marker expression and wound healing efficacy was detected both in the control and AAK lines. Interestingly, preliminary examination of PAX6 expression during early lineage commitment at d7 revealed an expression peak only in the control iPSC lines.
Conclusions :
The AAK-iPSC can be differentiated towards LSCs, supporting the presence of LSCs in young aniridia patients before cell loss later in life. Whether the observed lack of PAX6 expression peak during early LSC commitment with AAK-iPSC reflects developmental regulation of PAX6 remains to be studied further. Moreover, these AAK-iPSC will be useful to test whether they influence e.g. corneal immune cells or neurons, and reciprocally, before LSC loss.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.