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Ludovic Zimmerlin, Imran Ahmed Bhutto, Riya Kanherkar, Tea Soon Park, Michael Barbato, Michael Koldobskiy, Ying Liu, Mandeep Singh, Gerard A Lutty, Elias T Zambidis; Improved generation and long-term engraftment of retinal organoids from Tankyrase/PARP-Inhibitor-Regulated Naïve human pluripotent stem cells (TIRN-hPSC). Invest. Ophthalmol. Vis. Sci. 2022;63(7):3725 – F0331.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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