Wataru Kobayashi1,2, Akishi Onishi1, Hung-Ya Tu1, Yuji Takihara3, Michiru Matsumura1, Kazuko Tsujimoto1, Masaru Inatani3, Toru Nakazawa2,4,5, Masayo Takahashi1. 1. Laboratory for Retinal Regeneration, Center for Developmental Biology, RIKEN, Kobe, Hyogo, Japan. 2. Department of Ophthalmology, Tohoku University Graduate School of Medical Science, Sendai, Miyagi, Japan. 3. Department of Ophthalmology, Faculty of Medical Science, University of Fukui, Fukui, Japan. 4. Department of Advanced Ophthalmic Medicine, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan. 5. Department of Retinal Disease Control, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
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.
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.
Authors: Elizabeth E Capowski; Kayvan Samimi; Steven J Mayerl; M Joseph Phillips; Isabel Pinilla; Sara E Howden; Jishnu Saha; Alex D Jansen; Kimberly L Edwards; Lindsey D Jager; Katherine Barlow; Rasa Valiauga; Zachary Erlichman; Anna Hagstrom; Divya Sinha; Valentin M Sluch; Xitiz Chamling; Donald J Zack; Melissa C Skala; David M Gamm Journal: Development Date: 2019-01-09 Impact factor: 6.868
Authors: Samuel W Lukowski; Camden Y Lo; Alexei A Sharov; Quan Nguyen; Lyujie Fang; Sandy Sc Hung; Ling Zhu; Ting Zhang; Ulrike Grünert; Tu Nguyen; Anne Senabouth; Jafar S Jabbari; Emily Welby; Jane C Sowden; Hayley S Waugh; Adrienne Mackey; Graeme Pollock; Trevor D Lamb; Peng-Yuan Wang; Alex W Hewitt; Mark C Gillies; Joseph E Powell; Raymond Cb Wong Journal: EMBO J Date: 2019-08-22 Impact factor: 11.598
Authors: Michael Yamakawa; Samuel M Santosa; Neeraj Chawla; Evguenia Ivakhnitskaia; Matthew Del Pino; Sebastian Giakas; Arnold Nadel; Sneha Bontu; Arjun Tambe; Kai Guo; Kyu-Yeon Han; Maria Soledad Cortina; Charles Yu; Mark I Rosenblatt; Jin-Hong Chang; Dimitri T Azar Journal: Biochim Biophys Acta Gen Subj Date: 2020-03-12 Impact factor: 3.770
Authors: Sarah K Ohlemacher; Kirstin B Langer; Clarisse M Fligor; Elyse M Feder; Michael C Edler; Jason S Meyer Journal: Adv Exp Med Biol Date: 2019 Impact factor: 2.622
Authors: Roxanne Hsiang-Chi Liou; Thomas L Edwards; Keith R Martin; Raymond Ching-Bong Wong Journal: Int J Mol Sci Date: 2020-06-16 Impact factor: 5.923