Abstract
Purpose :
To assess the effects of RB1 loss-of-function on retinal development in vitro using retinal organoids (ROs) derived from a healthy and RB1-null human induced pluripotent stem cell (hiPSC) lines.
Methods :
Adipose-derived mesenchymal cells from familial retinoblastoma patient were reprogrammed into iPSCs using episomal Yamanaka factors (RB1+/-). Isogenic mutant lines were generated by CRISPR-Cas9 knockout of RB1 in a healthy iPSC line (RB1-/-). The control and mutant iPSCs were characterized and differentiated into retinal lineage. Proliferating retinal progenitors in ROs were BrdU-pulsed. Terminal differentiation potential was assessed over time using RT-PCR and immunohistochemistry.
Results :
Upon retinal differentiation, the patient specific RB1+/- and CRISPR-edited RB1-/- mutant iPSC lines formed eye fields in 2D adherent cultures at 3-4 weeks of differentiation [~35 eye-fields/million cells; n=4]. When assessed at different time-points of maturation, RB1 haploinsufficiency or its total absence resulted in precocious differentiation and depletion of SOX2+ retinal stem cells (RSCs) and early emergence of OTX2+, CRX+ multipotent retinal precursors (MRPs) at day-40 (D40) of differentiation. We also observed an altered spatial positioning and disorderly arrangement of OTX2+, CRX+ lineage committed MRPs, suggesting retinal lamination defects. Complete loss resulted in extended proliferation and expansion of OTX2+/BAF60C+ precursors and CRX+/BrdU+ precursors. These proliferating MRPs fail to exit cell cycle and do not terminally differentiate into ARR3+ mature cone photoreceptors. This triggered massive apoptosis around D55, resulting in gradual disintegration of RB1-/- ROs at later stages.
Conclusions :
The RB1-/- RO model confirms that RB1 loss does not affect eye field formation and retinal lineage commitment, but causes precocious differentiation and early depletion of RSCs. The MRPs continued to proliferate, lost their spatial cues, and failed to terminally differentiate into mature retinal neurons. This suggests that the conflicting signals in RB1-/- MRPs trigger apoptosis or render them prone to accumulate additional mutations, leading to cancerous transformation. However, RB1 is critical for the regulation of RSC differentiation; and for spatial organization, survival and terminal differentiation of MRPs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.