Eye Field Differentiation and Generation of Corneal Organoids from Human Induced Pluripotent Stem Cells

Abstract

Purpose : To generate and differentiate human iPSCs into eye field precursors and to establish enriched cultures of corneal epithelium.

Methods : Human dermal fibroblasts were reprogrammed into iPSCs by lentiviral method using the Yamanaka factors. Stably reprogrammed iPSC colonies were characterized for stemness, genomic stability and pluripotency by immunostaining, RT-PCR, FACS, karyotyping and teratoma assay. Direct differentiation of 80% confluent cultures were done using the differentiation medium (DM: DMEM/F12, 4% KOSR, 4% FBS, 1X NEAA, 1X Glutamax, 1X Pen-Strep) for 3 days. Subsequently, the cultures were shifted to retinal differentiation medium (RDM: DM + 2% B27) for a month to induce eye field specification. The distinct eye field primordial (EFP) clusters that emerged at 4 weeks were either continued as adherent cultures or excised for suspension cultures. In adherent cultures, rare EFPs gave rise to whole eye ball like structure with transparent corneal primordia on the surface and neuro-retinal cup on the basal side, at 8 weeks. In suspension cultures, about 30-40% of the EFPs gave rise to distinct retinal primordial (RP) and transparent, bubble-like corneal primordial (CP) structures, at 6 weeks. The delicate CPs are manually dissected out of EFP clusters and cultured separately for further 8-10 weeks in CDM (DM + 1% N2 + 5 μg/mL Insulin + 5 ng/mL FGF + 10 ng/mL EGF) for further tissue maturation or grown on human amniotic membrane (hAM) as explant cultures.

Results : The dermal fibroblast derived hiPSC lines exhibited the properties of pluripotent stem cells by forming teratomas in nude mice. Our optimized eye field differentiation protocol resulted in 40.05±3.89 EFPs per million cells in a 6-well plate (n=3). The EFPs could further mature and gave rise both retinal cups and corneal primordial structures more efficiently in suspension cultures. The isolated and maturing CPs at 8-10 weeks displayed similar anatomical features and marker expression profiles as that of developing corneal tissues and expanded well on hAM. IHC and RT-PCR evaluation confirmed the expression of PAX6, p63, K3/12, K13, K15, K19, VIM, COL1A1, COL4A1, MUC5AC and CD200.

Conclusions : Human iPSCs can be efficiently differentiated into complex three dimensional corneal tissues and offers an alternative tissue source for regenerating different layers of the cornea.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.