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Development. 2019 Jan 9;146(1). pii: dev171686. doi: 10.1242/dev.171686.

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

Author information

1
Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA.
2
Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53706, USA.
3
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA.
4
McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA.
5
Aragon Institute for Health Research (IIS Aragón), Lozano Blesa University Hospital, Zaragoza 50009, Spain.
6
Department of Ophthalmology, Lozano Blesa University Hospital, Zaragoza 50009, Spain.
7
Murdoch Children's Research Institute, The Royal Children's Hospital, Parkville, Victoria 3052, Australia.
8
Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia.
9
Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
10
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.
11
Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA dgamm@wisc.edu.
12
Department of Ophthamology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53705, USA.

Abstract

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.

KEYWORDS:

Cell culture; Differentiation; Human pluripotent stem cells; Organoids; Photoreceptors; Retina

PMID:
30567931
DOI:
10.1242/dev.171686

Conflict of interest statement

Competing interestsD.M.G. and M.J.P. have an ownership interest in Opsis Therapeutics LLC, which has licensed the technology to generate optic vesicles from pluripotent stem cell sources reported in this publication. D.M.G. also declared intellectual rights through the Wisconsin Alumni Research Foundation and a consultant role with FUJIFILM Cellular Dynamics International. All other authors indicated no potential conflicts of interest.

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