Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Oct 26, 2020; 12(10): 1171-1183
Published online Oct 26, 2020. doi: 10.4252/wjsc.v12.i10.1171
Enrichment of retinal ganglion and Müller glia progenitors from retinal organoids derived from human induced pluripotent stem cells - possibilities and current limitations
Kristine Karla Freude, Sarkis Saruhanian, Alanna McCauley, Colton Paterson, Madeleine Odette, Annika Oostenink, Poul Hyttel, Mark Gillies, Henriette Haukedal, Miriam Kolko
Kristine Karla Freude, Alanna McCauley, Colton Paterson, Madeleine Odette, Annika Oostenink, Poul Hyttel, Henriette Haukedal, Department of Veterinary and Animal Sciences, Group of Stem Cell Models for Studies of Neurodegenerative Diseases, Section for Pathobiological Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg 1870, Denmark
Sarkis Saruhanian, Miriam Kolko, Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen 2100, Denmark
Mark Gillies, Save Sight Institute, South Block, Sydney Eye Hospital, Sydney 2000, Australia
Miriam Kolko, Department of Ophthalmology, Copenhagen University Hospital, Rigshospitalet-Glostrup, Glostrup 2600, Denmark
Author contributions: Freude KK and Kolko M designed and coordinated the study; Saruhanian S, McCauley A, Paterson C, Odette M, Oostenink A, Freude KK, and Hyttel P performed the experiments, acquired and analyzed the data; Saruhanian S, McCauley A, Paterson C, Odette M, Hyttel P, Haukedal H, and Freude KK interpreted the data; Gillies M provided intellectual input into the experimental design; Freude KK, Haukedal H and Kolko M wrote the manuscript; all authors approved the final version of the manuscript.
Supported by Innovation Fund Denmark, No. 4108-00008B; The Bagenkop Nielsens Ø jen-Fond, No. 115227; Hø rslev-Fonden, No. 116967; Beckett Fonden, No. 116936; and Velux Foundation, No. 1179261001/2.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board at the Department of Drug Design and Pharmacology at the University of Copenhagen.
Conflict-of-interest statement: The authors declare they do not have conflicts of interest regarding the publication of this article.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The ARRIVE Guidelines have been adopted.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Kristine Karla Freude, BSc, DPhil, MSc, PhD, Associate Professor, Department of Veterinary and Animal Sciences, Group of Stem Cell Models for Studies of Neurodegenerative Diseases, Section for Pathobiological Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegårdsvej 7, Frederiksberg 1870, Denmark. kkf@sund.ku.dk
Received: June 11, 2020
Peer-review started: June 11, 2020
First decision: June 20, 2020
Revised: July 3, 2020
Accepted: August 16, 2020
Article in press: August 16, 2020
Published online: October 26, 2020
Processing time: 137 Days and 2.3 Hours
ARTICLE HIGHLIGHTS
Research background

Degenerative eye diseases, such as glaucoma, are characterized by a progressive loss of retinal ganglion cells (RGCs), neurons within the eye required for image and non-image transformation. RGCs have been shown to interact with, and be dependent upon, Müller glia. Both cell types are central in the pathology of glaucoma.

Research motivation

The development of in vitro differentiation protocols for generating retinal cells and tissues is crucial to identify disease-relevant cellular changes and to test potential intervention strategies in a human context.

Research objectives

In this study we investigated the potential of retinal organoids to produce RGC and Müller glia progenitors and the efficiency with which these cell types can be isolated and expanded as enriched monolayer cultures.

Research methods

Retinal organoids were differentiated from human induced pluripotent stem cells (hiPSCs), then magnetic-activated cell sorting applied to isolate RGC and Müller glia progenitors. Gene and protein expression were assessed in both retinal organoids and monolayers of RGC and Müller glia progenitors via qPCR and immunocytochemistry respectively. Furthermore, retinal organoid ultrastructure was examined via transmission electron microscopy.

Research results

Retinal organoids were grown successfully for at least 56 d and the presence of RGC and Müller glia progenitors validated via optimized whole mount immuno-cytochemistry. Furthermore, pure populations of RGC and Müller glia progenitors could be isolated using magnetic-activated cell sorting and successfully propagated in enriched monolayers for up to 2 wk.

Research conclusions

Retinal organoids generated from hiPSCs recapitulate eye development in terms of their cellular heterogeneity and tissue architecture. Key cell types, such as RGC and Müller glia progenitors, can be isolated from the retinal organoids and expanded for subsequent cell-specific phenotype characterizations in disease-relevant contexts.

Research perspectives

In vitro generation of the relevant cell types affected by degenerative eye diseases is important in order to study specific cell type disease vulnerability and selective cellular interactions. The generation of retinal organoids with subsequent isolation of RGC and especially Müller glia progenitors is an important step for in vitro disease modeling.