September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Culture of retinal ganglion cells (RGC) purified from 3D retinal organoids differentiated from human induced pluripotent stem (iPS) cells
Author Affiliations & Notes
  • Wataru Kobayashi
    Laboratory for Retinal Regeneration RIKEN Center for Developmental Biology, Kobe, Japan
    Department of Ophthalmology, Tohoku University Graduate School of Medical Science, Sendai, Japan
  • Akishi Onishi
    Laboratory for Retinal Regeneration RIKEN Center for Developmental Biology, Kobe, Japan
  • Yuji Takihara
    Department of Ophthalmology, Faculty of Medical Science, University of Fukui, Fukui, Japan
  • Masaru Inatani
    Department of Ophthalmology, Faculty of Medical Science, University of Fukui, Fukui, Japan
  • Toru Nakazawa
    Department of Ophthalmology, Tohoku University Graduate School of Medical Science, Sendai, Japan
  • Masayo Takahashi
    Laboratory for Retinal Regeneration RIKEN Center for Developmental Biology, Kobe, Japan
  • Footnotes
    Commercial Relationships   Wataru Kobayashi, None; Akishi Onishi, None; Yuji Takihara, None; Masaru Inatani, None; Toru Nakazawa, None; Masayo Takahashi, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 6074. doi:
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      Wataru Kobayashi, Akishi Onishi, Yuji Takihara, Masaru Inatani, Toru Nakazawa, Masayo Takahashi; Culture of retinal ganglion cells (RGC) purified from 3D retinal organoids differentiated from human induced pluripotent stem (iPS) cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6074.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We have previously reported neurite outgrowth from retinal organoids differentiated from human induced pluripotent stem cells (iPSCs). To enable more precise analyses of retinal ganglion cell (RGC) neurons, we established the purification method of the RGCs.

Methods : 3D retinas differentiated from 201B7 human iPSCs were maintained for 50–100 days. The RGCs were purified using a two-step immunopanning method with antimacrophage antibodies and anti-Thy 1.1 antibodies. The RGCs were plated on dishes coated with poly-D-lysine and laminin, and maintained in serum-free medium with supplements.

Results : RAX and CHX10 were expressed at differentiation day (DD) 22, and BRN3B-positive cells were localized at the inner half of the DD35 optic vesicles. RGCs purified from human 3D retinal organoids by immunopanning attached in the first 24 hours, and began to project neurites. Three days after immunopanning, the neurites had reached three times the length of the cell body, and connected to other RGCs. The purified RGCs could be maintained for about one month, with sustained neurites growth. Most of the RGCs were BRN3B-positive, and the neurites could be visualized by the pan-axonal antibody SMI-312. However, some of these did not grow neurites.

Conclusions : We purified RGCs from human 3D retinal organoids for long-term culture. This method may be useful for studying the pathogenesis of RGC diseases, such as glaucoma, and in drug screening.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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