期刊
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
卷 59, 期 2, 页码 776-787出版社
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.17-22406
关键词
pluripotent stem cell; retinal ganglion cell; 3D retinal organoid; immunopanning; neurite outgrowth
资金
- Research Center Network for Realization of Regenerative Medicine (MEXT)
- Program for Intractable Disease Research Utilizing Disease-Specific iPS Cells (Japan Science and Technology Agency [JST])
- Program for Intractable Disease Research Utilizing Disease-Specific iPS Cells (Japan Agency for Medical Research and Development [AMED])
- JSPS KAKENHI [24687010]
- Kato Memorial Bioscience Foundation
- Grants-in-Aid for Scientific Research [24687010, 17K16994] Funding Source: KAKEN
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据