Abstract
Purpose :
Human stem cell derived in vitro models of cornea tissue can aid our understanding of the process of wound healing and may be useful for pharmacological screening, in a cost effective manner, while reducing the reliance on animal experiments. Previous reports of 3D corneal colonies demonstrate corneal features, but require a dissection step that may reduce scalability. Here, we report the generation of 3D eye organoids from human embryonic stem cells (hESCs), with cornea characteristics independent of dissection
Methods :
H9 ESCs were seeded at 3000 cells/well in a 96 ultra-low bind well plate in mTeSR media with 2.5% matrigel and 2μM Thiazovivin (THIA). Differentiation into eye organoids were stimulated at day 2 by changing to differentiation media (DM): GMEM with 10% knockOut Serum Replacement, 1mM Sodium pyruvate, 1mM non-essential amino acids, 2mM L-glutamate, 1% penicillin-streptomycin solution and 55μM β-mercaptoethanol. Corneal specification was enhanced at day 32-46 by changing to DM:CnT-PR (1:1) with 10ng/mL keratinocyte growth factor (KGF) and 2μM THIA, before changing to maintenance media; DMEM:F12, 2% B-27 supplement, 10ng/ml KGF and 2μM THIA at day 48. Corneal features were characterized by immunohistochemistry (IHC).
Results :
By day 20, ≈20% (N=3x96) of the organoids formed transparent blebs interpreted as pre-cornea formation. At day 28, organoids show pigmented regions (RPE or iris pigment cells). By day 48, organoids show transparent, opaque and pigmented regions, Figure 1. IHC revealed an organized outer layer consisting of tightly packed cells (epithelium) on top of a thin acellular structure reminiscent of Bowman’s membrane (BM). The epithelial layer was positive for corneal markers: TP63α, PAX6, ZO-1, Keratin 15 and Keratin 12 (Figure 2). Cells found beneath the putative BM were vimentin positive and resemble keratocytes of the corneal stroma (Figure 2).
Conclusions :
We report a method for culturing 3D eye organoids from hESCs exhibiting features resembling the three outer layers of the cornea; Epithelium, BM and stroma. These organoids may be useful to study wound healing and identification of therapeutic compounds.
This is a 2021 ARVO Annual Meeting abstract.