Abstract
Purpose :
Induced pluripotent stem cells (iPSCs) derived human retinal organoids (hRetOrg) have emerged as a powerful system for studying retinal diseases while formation of a fovea has not been yet achieved in current methods. We hypothesize that foveated-hRetOrg can be generated if in vivo developmental molecular programs underlying fovea formation, namely Retinoic Acid (RA) signaling, are recapitulated in this in vitro system. We therefore settled to longitudinally characterize RA signaling in hRetOrg during retinogenesis period.
Methods :
We established a hRetOrg culture system generated from a human control iPSC line, IMR(90)-4, based on Zhong et al. 2014 and Cowan et al. 2020 methods. We collected hRetOrg from the beginning of retinogenesis at week (W) 5 up to fully differentiated stages at W21 for gene expression analysis by qRT-PCR, in situ hybridization and immunohistochemistry. We also employed electroporation technique to perform localized transfections in defined hRetOrg domains using customized electrodes and chambers.
Results :
We confirmed recapitulation of in vivo retina developmental key points in our hRetOrg cultures, such as formation of eye-field at day (D) 7, optic vesicle –like configurations at D16 and optic cup-like structures at D25. Around W5, neural retinal cells spontaneously began to differentiate following in vivo birth dating orders. Immunohistochemistry analysis of W21 hRetOrg showed Rhodopsin, SW, and M/LW cone expressions in outer segment-like structures confirming full differentiation of photoreceptors in our cultures. Regarding RA signaling analysis, we found that RA synthesizing enzymes ALDH1A1 (dorsally expressed in vivo) and ALDH1A3 (ventrally expressed in vivo) are expressed in non-overlapping domains with slightly different dynamics. Expression of RA degradative enzymes is more complex and highly variable among organoids. Additionally, we also developed an efficient gene transfection method of hRetOrg via electroporation for precise spatiotemporal regulation of expression of any gene of interest.
Conclusions :
This study thoroughly characterizes RA signaling in current hRetOrg cultures during retinogenesis period, constituting a reference where upon genes of interest can be manipulated in order to recapitulate in vivo patterns of foveal molecular mechanisms, which ultimately can lead to the formation of foveated-hRetOrgs.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.