Long-term engraftment of mature human photoreceptors following subretinal transplantation of naïve hPSC-derived retinal organoids

Abstract

Purpose : Retinal organoid (RO) differentiation from conventional, primed human pluripotent stem cells (hPSCs) is optimized in only a few ‘permissive’ lineage-primed hPSC lines. We recently established that primed hPSC can be chemically-reverted to tankyrase/PARP inhibitor-regulated naïve epiblast-like hPSC (N-hPSC) with an enhanced multi-lineage potency devoid of interline variability.Here, we tested whether N-hPSC-derived RO possess improved engraftment and in vivo development of mature photoreceptors.

Methods : Isogenic primed vs N-hPSC were differentiated with a 3D retinal protocol. Horseshoe-shaped neural domains matured into retinal cups (RC) with laminated layers. 6 primed hPSC lines were naive-reverted and differentiated into ROs. Conventional hPSC incapable of HS development differentiated into RCs following naive reversion with efficiencies comparable to “permissive” hPSC lines. Naïve vs primed 13 week-old RCs were dissected into neural retinal sheets and transplanted into the subretinal space of NOG-SCID mice. Human photoreceptor specification and engraftment was evaluated at 3-10 months post-transplant.

Results : Eye field-specific (2 weeks) gene expression, and photoreceptor progenitor markers (8-20 weeks) were detected in greater quantities in naïve RCs (e.g., CRX⁺RCV⁺), as were HuC/D⁺ ganglion/amacrine cells, PROX1⁺ horizontal cells, and MITF⁺ pigmented epithelium. N-hPSC-derived RO displayed improved maturation of rhodopsin⁺ photoreceptors with proper histo-architecture. Eyes transplanted with N-hPSC-derived retinal sheets were sectioned and evaluated by confocal microscopy, and revealed extensive engraftment of mature human (HNA+) cells in tubulation-like structures (Fig. 1). Engrafted human retinal tissue tubes at 10 months developed a full repertoire of mature rod and cone photoreceptors (e.g., rhodopsin⁺, L/M opsin⁺, recoverin⁺) (Fig. 2), astrocytes (GFAP⁺vimentin^-), and Mueller cells (GFAP⁺vimentin⁺).

Conclusions : Naïve reversion of conventional hPSCs abolished the interline variability of RO development, dramatically augmented retinal fate specification, and potentiated long-term survival of transplanted photoreceptors for at least 10 months. Tankyrase inhibitor-regulated N-hiPSC represent a new class of human stem cells with high epigenetic plasticity, improved multi-lineage functionality, and high impact for ocular regenerative medicine.

This is a 2020 ARVO Annual Meeting abstract.

 

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Fig. 2