Abstract
Purpose :
Human induced pluripotent stem cells (hiPSC) derived-3D retinal organoids are an invaluable tool to study retinal development and to recapitulate clinical phenotypes, overcoming the limitations of animal models. However, the maturation of photoreceptors (PR) requires extensive differentiation culture times (6+ months) needed to investigate the disease-phenotype and to test therapeutic strategies. The goal of our study is to accelerate the maturation of PR in retinal organoids by incorporating small molecule in culture conditions.
Methods :
Retinal organoids were differentiated from hiPSC and hESC (embryonic stem cell) lines using the embryoid-body approach. 3D-retinal organoids were intermittently treated with 1nM and 0.5 nM 9-cis Retinoic acid (RA) on Days (D) 30-60 and D90-120 of differentiation, respectively. Light and dark culture conditions parameters were included during organoid differentiation. Organoid treated with all-trans retinoic acid (ATRA) from D30-120 days of differentiation were utilized as control. Expression and localization of PR markers including opsins and phototransduction, were analyzed by immunohistochemistry (IHC) and gene expression (qRT-PCR) at D120, 150 and 180. All data are represented as Mean±SEM (N=3 independent differentiation experiments).
Results :
Retinal organoids treated with 9-cis RA showed accelerated and significantly increased expression of recoverin, cone arrestin, visual arrestin, GUCY2D, GUCA1A, GUCA1B, rhodopsin, short, medium, and long wavelength opsin markers at D120 and D150 for hESC and hiPSC, respectively. Control organoids treated with ATRA showed the initial expression at D180. The degree of 9-cis RA molecule effects was cell-line dependent. Dark culture conditions did not have any effect on marker expressions to regular light exposure.
Conclusions :
Intermittent doses of 9-cis RA into media promotes early time-point PR maturation in 3D retinal organoids with the degree of effect being cell line-dependent. Our modified differentiation strategy has the potential to expedite the understanding of modeling inherited retinal degenerations for intervening therapies and to generate mature PR for cell-based transplantation therapies.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.