Abstract
Purpose :
Derivation of retinal tissue in a dish creates new opportunities for cell replacement
therapies of blindness and addresses the need to preserve retinal architecture to restore vision.
Here we report efficient derivation of 3-dimensional (3D) retinal tissue from human embryonic
stem cell (hESC) lines ESI053, ESI049, ESI017 (ES Cell International lines, Crook et al., 2007, Cell
Stem Cell., and also WA01 (H1) and WA09 (H9).
Methods :
3D retinal tissue was derived from five hESC cell lines using a feeder-free system and a
protocol modified from Singh et al., 2015, Stem Cells & Devel. Human embryonic stem cell
lines were karyotyped and fingerprinting analysis was done to assign molecular genetic identity
to each line. Retinal organoids were allowed to differentiate for 8 weeks before fixing with 4%
paraformaldehyde, processing for frozen immunohistochemical analysis and cutting 12 micron-
thick sections. Immunohistochemistry was done to visualize the expression of retinal markers of
several key retinal lineages essential for cell therapies. Cell division in hESC-3D retinal tissue
was evaluated using Ki67 antibody.
Results :
Karyotype of all hESC lines were normal. Fingerprinting signature of each hESC line
was developed for further identity testing for cell therapy applications. Immunohistochemical
profiling of 8-week old retinal organoids derived from all hESC
lines revealed strong expression of retinal progenitor markers
OTX2, CRX, PAX6, BLIMP1, NEUROD1, photoreceptor
markers (RCVRN, RXR Gamma), amacrine markers (CALB2,
CALR) and ganglion marker (BRN3B). Retinal tissue derived
from all hESC lines appeared to be similar morphologically
(shown: 8-week retinal tissue, WA09 line), demonstrated initial
stages of lamination (with amacrine and ganglion markers facing
the basal side) and differentiated with approximately the same developmental dynamics in a dish.
Long-term growth (up to several months) of retinal organoids from several lines demonstrated
progressive growth and preservation of translucent color of the rim, containing developing neural
retina.
Conclusions :
These results enable testing of hESC-3D retinal tissue from ESI lines (for which cGMP-grade hESC stocks available) in vivo in animal models with retinal degeneration for developing cell therapies to repair retina and ameliorate vision loss.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.