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Kun-Che Chang, Ziming Luo, Xin Xia, Cara Knasel, Jeffrey Louis Goldberg; Retinal organoid differentiation and transplantatio. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2504.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cell (RGC) replacement therapy could provide an approach to vision restoration in glaucoma and other optic neuropathies. Here we studied regulatory mechanisms of RGC differentiation from human embryonic stem cells (hESCs) in three-dimensional retinal organoid culture, and cell transplantation in vivo.
We utilized the H9-Brn3b-tdTomato-Thy1.2 hESC cell line for retinal organoid differentiation. We co-treated organoids with Sox4 and GDF-15. RGC markers Brn3a, HuD, Islet1 and RBPMS, and photoreceptor progenitor marker Recoverin were detected in retinal organoids by immunofluorescence (IF) staining. IF and qRT-PCR were performed to evaluate developmental stages. Calcium imaging stimulated by GABA agonist muscimol was used to evaluate electrophysiologic maturity in developing RGCs. Organoid-derived RGCs were injected intravitreally in the adult mouse eye, and co-stained with human nuclei markers. All experiments were conducted at least three times independently. Data were analyzed by ANOVA and post-hoc t-test with Tukey correction, with Pvalue of <0.05 considered statistically significant. All research was conducted in compliance with IACUC approval and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
In our retinal organoid culture, tdTomato signal indicating Brn3b expression was initially detected in the central zone at day 26,. IF and qRT-PCR results showed that organoids at similar number of days post-tdTomato detection expressed retinal cell markers in a similar pattern. Brn3b-positive RGCs were HuD- and Islet1-positive, but RBPMS-negative in early stages. Co-treatment with Sox4and GDF-15 promoted organoid-to-RGC differentiation. From day 35, 10 days after tdTomato expression, we detected Recoverinexpression by IF, intially on the basal side of retinal organoids and later on the apical side. Organoid-derived RGCs showed a muscimol-induced calcium influx similar to immature primary mouse RGCs. Finally, organoid-derived RGCs survived and extended neurites in the ganglion cell layer in mouse retina 7 days after transplant in vivo.
RGCs differentiate from human stem cell-derived retinal organoid cultures, but only to an immature electrophysiologic state. Further understanding of the maturation process in retinal organoids may enhance RGC transplant and ultimately provide a strategy for therapeutics in glaucoma and other optic neuropathies.
This is a 2020 ARVO Annual Meeting abstract.
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