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Besides invention of iPSC technology, recent progress of stem cell-based organoids is founded on long-standing 3D-reaggregate approaches from embryonic tissues. In particular, histotypic in vitro reconstruction of avian retinal spheroids was most prolific. For instance, a complete reconstitution of all retinal layers was possible, which was supported by Wnt signalling and factors from the retinal pigmented epithelium (RPE); similar in vitro findings are still missing for mammals. Using an established model of reaggregates from dispersed retinal cells of the neonatal Gerbil [1], we show here that in contrast to supernatant from RPE (RPECM), supplementation with Wnt3a induced a correct inside-out polarity of retinal layers. XAP1+ precursors of photoreceptors (PRs) were correctly found on the external face of the sphere, but general cell differentiation remained limited. If Wnt3a was present for 4 days in vitro (div) only, the correctly polarized tissue further differentiated, e.g., more calretinin+ amacrine cells (CR+ ACs) sent out processes into an ipl-like layer and rhodopsin expression of PRs became detectable. Finally, if Wnt3a during 4 div was followed by RPECM treatment, all retinal layers with most cell types were arranged in correct order, as shown by markers including CR, Pax6, AChE, PKCá, CRALBP, and Cern901. LiCl experiments showed that the canonical Wnt/β-catenin pathway was involved. We conclude that Wnt3a conveyed a correct inside-out laminar polarity but kept cells in an undifferentiated state, while RPECM strongly promoted retinal differentiation. Both together supported a nearly complete retinal tissue reconstruction from fully dispersed cells, as never achieved before for cells from any mammalian retina.

international journal of stem cell research and therapy

Reconstructing Mammalian Retinal Tissue: Wnt3a Regulates Laminar Polarity in Retinal Spheroids from Neonatal Mongolian Rats, while RPE Promotes Cell Differentiation