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Wataru Kobayashi, Akishi Onishi, Sunao Sugita, Kyoko Iseki, Yuji Takihara, Masaru Inatani, Toru Nakazawa, Masayo Takahashi; Differential purification of retinal ganglion cells from human induced pluripotent stem cell derived 3D retinal organoids.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4914.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion (RG) cells are traditionally difficult to study from both tissue explant and human induced pluripotent stem (iPS) cell derived 3D retinal organoids. Therefore, a method to efficiently purify RG cells from large 3D retinal organoid differentiation may help to overcome current limitations. In this study, we compared cell populations and sorting quality between the two-step immunopanning method and the MACS (Magnetic-Activated Cell Sorting) system.
Human 3D retinal organoids differentiated from 201B7 iPS cells were maintained for 50 to 75 days. In the two-step immunopanning method, dissociated RG cells were purified by anti-macrophage and anti-Thy 1.1 antibodies. In the MACS system, the dissociated RG cells were purified by magnetic microbeads conjugated to anti-Thy1 antibodies. The purified iPS cell derived RG cells (iPS-RG) cells were then plated on dishes coated with poly-D-lysine and laminin, and were maintained in serum-free medium with neurotrophic factors. The growth and morphology of the neurites were visualized by immunocytochemistry with BRN3B and SMI-312 antibodies.
The iPS-RG cells purified by both immunopanning and MACS methods attached on to dishes in the first 24 hours and extended neurites. Our iPS-RG cells from either method could be cultured for about three weeks. One to three days after plating, the RG cells purified by MACS grow faster than those purified by immunopanning. Conversely, there were more cells with glia-like morphology retained in MACS-purified cell cultures. Interestingly, neurites of iPS-RG cells were similar between assays, yet the length of the neurites from MACS purified iPS-RG cells was longer.
We validated the neurite growth of human iPS-RG cells purified with two distinct methods and cultured over an extended period. The purity of iPS-RG cells by MACS system was lower than the two-step immunopanning method, but the glia-like cells may positively influence on neuritogenesis of purified iPS-RG cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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