Culture Systems of Dissociated Mouse and Human Pluripotent Stem Cell–Derived Retinal Ganglion Cells Purified by Two-Step Immunopanning

Abstract

Purpose: We aimed to establish purification and culture systems for retinal ganglion cells (RGCs) differentiated from mouse and human pluripotent stem cells (PSC) for in vitro and regenerative medicine studies. Methods: We used a two-step immunopanning method to purify RGCs from mouse and human PSC-derived three-dimensional (3D) retinal organoids. To assess the method, we purified RGCs from 3D retinal organoids derived from embryonic stem cells (ESCs) generated from Thy1-EGFP transgenic (TG) mice. In addition, 3D retinal organoids differentiated from human induced PSCs (iPSCs) were cultured for up to differentiation day (DD) 120, and RGCs were purified by immunopanning. RGC marker expressions were confirmed by immunostaining and reverse transcription-quantitative PCR. The purified RGCs were cultured, and neurite outgrowth was measured and analyzed using an IncuCyte Zoom system. Results: Mouse RGCs purified from Thy1-EGFP TG mouse retinas and the ESC-derived 3D retinas could be maintained for approximately 2 to 3 weeks, expressing the markers BRN3B and SMI-312. Purified RGCs from human iPSC-derived retinal organoids expressed RGC markers and could be maintained for up to 4 weeks. The RGCs collected at DD 90 to 110 extended longer neurites than those collected at younger stages. Conclusions: We successfully purified RGCs from mouse and human PSC-derived 3D retinal organoids cultured for approximately 120 days. RGCs from older retinal organoids would be useful for neurite tracking. This method would be effective not only for studying the pathology of human RGC diseases but also for therapeutic drug studies and RGC transplantation.