Improved generation and long-term engraftment of retinal organoids from Tankyrase/PARP-Inhibitor-Regulated Naïve human pluripotent stem cells (TIRN-hPSC)

Abstract

Purpose : Generation of retinal organoids (RO) from conventional, primed hPSCs is inefficient and optimized in only a few ‘permissive’ lines. We recently established naïve epiblast-like TIRN-hPSC with reduced interline variability of differentiation for improved vascular progenitor engraftment. Here, we tested whether TIRN-hPSC-derived ROs also possess improved in vivo development to mature photoreceptors.

Methods : Isogenic primed vs TIRN-hPSC were differentiated with an established 3D RO protocol. Horse-shoe (HS)-shaped domains matured to retinal cups (RC) with laminated layers. Neuroepithelial specification and epigenomic integrity were validated at 4-20 weeks by RT-PCR, immunofluorescence, RNA-Seq, and CpG methylation sequencing. Interline variability of RC differentiation was quantitated to 16 weeks. RC neural sheets were transplanted into the subretinal space of NOG-SCID mice, and human photoreceptor specification and engraftment was evaluated at 6-9 months post-transplant.

Results : Although several conventional hPSC lines failed to differentiate into RCs, all TIRN-reverted hPSC efficiently generated HS domains and well-differentiated RC organoids with efficiencies comparable to “permissive” hPSC lines. Eye field-specific transcripts and photoreceptor progenitor markers (2-20 weeks) were detected in greater quantities in TIRN RCs (e.g., CRX⁺RCV⁺, HuC/D⁺ ganglion/amacrine cells, PROX1⁺ horizontal cells, MITF⁺ pigmented epithelium). TIRN-derived RO displayed improved maturation of rhodopsin⁺ photoreceptors with proper histo-architecture. Confocal microscopic evaluation of NOG eyes transplanted with TIRN-derived RC sheets demonstrated long-term engraftment (10 months) of mature human cells in tabulation-like structures with development of a full repertoire of mature rod/cone photoreceptors (e.g., rhodopsin⁺, L/M opsin⁺, recoverin⁺), astrocytes (GFAP⁺vimentin^-), and Mueller cells (GFAP⁺vimentin⁺). Transcriptomic and epigenetic studies revealed that chemical PARP inhibition of the TIRN method mediated the improved epigenetic plasticity to retinal lineages.

Conclusions : TIRN reversion of conventional hPSCs abolished interline variability of RO development across genetic background, augmented retinal fate specification, and potentiated long-term survival of transplanted photoreceptors for at least 10 months.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.