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Ziming Luo, Kun-Che Chang, Bogdan Tanasa, Marius Wernig, Jeffrey L Goldberg; Rapid protocol for induced retinal ganglion cell differentiation from human stem cells. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3315.
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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 developed a rapid protocol for directly induced RGC differentiation from human stem cells.
We directly induced RGC-like cells (iRGCs) from hiPSCs and hESCs by overexpression of NGN2. Cells gained their RGC fate in less than two weeks in the full-Sato medium (RGC culture medium) supplied by Notch inhibitor, DAPT. Immunostaining and qRT-PCR were performed to confirm their RGC characteristics. Calcium imaging stimulated by GABA agonist muscimol was used to evaluate their electrophysiologic maturities. Then, we utilized single-cell RNA sequencing (scRNA-seq) to further delineate the iRGC differentiation and compare their transcriptomic profiles to fetal and organoid RGCs. All experiments were conducted at least three times independently. Data were analyzed by ANOVA and post-hoc t-test with Tukey correction, with a P-value of <0.05 considered statistically significant. All use of animals conformed to the ARVO statement for the Use of Animals in Research, and was approved by the IACUC and the Institutional Biosafety Committee of Stanford University.
Within one week of induction, neuronal morphology including neurite growth was observed. Progeny expressed RGC markers, including BRN3a, ISL1, HuD, and NEFL after 8 days of differentiation. qRT-PCR confirmed the reduction of pluripotent genes (POU5F1 and NANOG) and the upregulation of neuronal genes (PAX6, BRN3a, and ISL1). iRGCs demonstrated muscimol-induced calcium influx similar to immature primary mouse RGCs. Unbiased clustering in scRNA-seq data showed that iRGCs distributed closely but not overlapping with day-59 and day-82 fetal human RGCs, indicating high similarity. Compared with day-45 retinal organoid-derived RGCs, iRGCs largely overlapped in most clusters. However, for some marker genes including BRN3a, Brn3b, and NEFL, iRGCs demonstrated expression patterns more similar to fetal RGCs than to retinal organoid RGCs.
In the present study, we describe a new, effective method that generates a homogeneous population of iRGCs after over-expression of a single transcription factor in human stem cells. These iRGCs are highly similar to two-month-old fetal RGCs, although they develop in less than two weeks. The simplicity of this system may benefit the translational studies on human RGCs.
This is a 2021 ARVO Annual Meeting abstract.
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