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Curr Eye Res. 2016 Apr;41(4):558-68. doi: 10.3109/02713683.2015.1038359. Epub 2015 Apr 16.

Optimized Culture System to Induce Neurite Outgrowth From Retinal Ganglion Cells in Three-Dimensional Retinal Aggregates Differentiated From Mouse and Human Embryonic Stem Cells.

Author information

1
a Laboratory for Retinal Regeneration , RIKEN Center for Developmental Biology , Kobe , Japan .
2
b Department of Ophthalmology and Visual Science , Graduate School of Biomedical Science, Nagasaki University , Kobe , Japan .
3
c Regenerative and Cellular Medicine Office, Sumitomo Dainippon Phama Co., Ltd , Kobe , Japan .
4
d Laboratory for Organogenesis and Neurogenesis , RIKEN Center for Developmental Biology , Kobe , Japan .
5
e Environmental Health Science Laboratory , Sumitomo Chemical Co., Ltd. , Osaka , Japan , and.
6
f Laboratory for in vitro Histogenesis , RIKEN Center for Developmental Biology , Kobe , Japan.

Abstract

PURPOSE:

To establish a practical research tool for studying the pathogenesis of retinal ganglion cell (RGC) diseases, we optimized culture procedures to induce neurite outgrowth from three-dimensional self-organizing optic vesicles (3D-retinas) differentiated in vitro from mouse and human embryonic stem cells (ESCs).

MATERIALS AND METHODS:

The developing 3D-retinas isolated at various time points were placed on Matrigel-coated plates and cultured in media on the basis of the 3D-retinal culture or the retinal organotypic culture protocol. The number, length, and morphology of the neurites in each culture condition were compared.

RESULTS:

First, we confirmed that Venus-positive cells were double-labeled with a RGC marker, Brn3a, in the 3D-retina differentiated from Fstl4::Venus mouse ESCs, indicating specific RGC-subtype differentiation. Second, Venus-positive neurites grown from these RGC subsets were positive for beta-III tubulin and SMI312 by immunohistochemistry. Enhanced neurite outgrowth was observed in the B27-supplemented Neurobasal-A medium on Matrigel-coated plates from the optic vesicles isolated after 14 days of differentiation from mouse ESCs. For the differentiated RGCs from human ESCs, we obtained neurite extension of >4 mm by modifying Matrigel coating and the culture medium from the mouse RGC culture.

CONCLUSION:

We successfully optimized the culture conditions to enhance lengthy and high-frequency neurite outgrowth in mouse and human models. The procedure would be useful for not only developmental studies of RGCs, including maintenance and projection, but also clinical, pathological, and pharmacological studies of human RGC diseases.

KEYWORDS:

Culture procedure; neuritogenesis; pluripotent stem cells; retinal ganglion cell; three-dimensional optic vesicle

PMID:
25880804
DOI:
10.3109/02713683.2015.1038359
[Indexed for MEDLINE]

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