Cultivation of Human Induced Pluripotent Stem Cells into 2D Eye-Like Organoids on Purified Keratan Sulphate

Abstract

Purpose : Previously, we showed how human induced pluripotent stem cells (hiPSCs) could be expanded into Self-formed, Ectodermal, Autonomous, Multi-zones (SEAMs), which are small, circular cellular colonies in which cells in four distinct concentric zones represent different component tissues of the eye (Hayashi et al., Nature 2016;531:376-80). Glycosaminoglycans (GAGs) are important components of the stem cell niche in a number of tissues (Hayes et al. Stem Cells Dev 2016;25:836-47) and here we test the hypothesis that keratan sulphate (KS), the most abundant GAG in cornea, affects SEAM formation.

Methods : Porcine eyes from freshly slaughtered animals were obtained and GAGs extracted from ten excised corneas by guanidine hydrochloride treatment. Extracts were then subjected to density gradient centrifugation and GAGs separated by HPLC with ion exchange and size-exclusion columns. Purified high- and low-sulphated KS GAGs (identified by antibodies 5D4 and 1B4, respectively) at concentrations of 0 µg/cm² (control group), 0.25 µg/cm², 0.5 µg/cm² and 1.0 µg/cm², in conjunction with laminin 511 at 0.5 µg/cm², were used to coat cell culture plates on which hiPSCs were generated into SEAMs and their growth patterns studied.

Results : Both high- and low-sulphated KS GAGs were able to support SEAM growth at all coating concentrations used. Notably, at day 10 of culture, hiPSCs grown on the highest concentration of 5D4-positive, high-sulphated KS (i.e.1.0 µg/cm²) consistently generated more than 90 SEAM colonies/dish, above the SEAM formation rate for all other experimental conditions tested. Immunofluorescence microscopy revealed the highest positive signal for the transcription factor protein, Oct4 occurred after KS coating, with qPCR analysis indicating elevated Oct4 gene expression levels. Finally, SEAMs grown for 4-weeks on wells that included 5D4-positive KS in the coating showed high gene expression levels for PAX6, a master regulator of eye development, and p63, an indicative corneal epithelial stem cell marker, with immunohistochemistry revealing increased numbers of p63/PAX6-positive hiPSC-derived corneal epithelial-like cells in zone 3 of the SEAM.

Conclusions : When used in combination with laminin 511 as a substrate for hiPSC growth, KS influences SEM formation, with a possible enhancement of the hiPSC/SEAM-derived corneal epithelial cell population.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.