Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Jan 26, 2020; 12(1): 35-54
Published online Jan 26, 2020. doi: 10.4252/wjsc.v12.i1.35
Sphere-forming corneal cells repopulate dystrophic keratoconic stroma: Implications for potential therapy
Himanshu Wadhwa, Salim Ismail, Jennifer J McGhee, Bert Van der Werf, Trevor Sherwin
Himanshu Wadhwa, Salim Ismail, Jennifer J McGhee, Trevor Sherwin, Department of Ophthalmology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland 1023, New Zealand
Bert Van der Werf, Department of Epidemiology and Biostatistics, School of Population Health, Faculty of Medical and Health Sciences, The University of Auckland, Auckland 1023, New Zealand
Author contributions: Wadhwa H and Ismail S participated in the experimental design, data acquisition analysis and manuscript writing; McGhee JJ participated in experimental design and cell and tissue preparation; Van der Werf B participated in data analysis and manuscript writing; Sherwin T participated in study design, experimental design and manuscript writing; All authors read, edited and approved the final manuscript.
Supported by Save Sight Society of New Zealand, No. 37116543; New Zealand Wound Care Society, No. 3713325; John Hamel MacGregor Trust.
Institutional review board statement: This study was conducted under ethical approval by the Northern X Regional Ethics Committee and since reviewed by the Northern A Health and Disability Ethics Committee (New Zealand). Ethics reference: NTX/07/08/080/AM06
Conflict-of-interest statement: Prof. Sherwin reports grants from Save Sight Society of New Zealand, grants from Auckland Medical Research Foundation, grants from New Zealand Wound Care Society, and grants from John Hamel MacGregor Trust during the conduct of the study.
Data sharing statement: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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 Non Commercial (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: See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Trevor Sherwin, PhD, Professor, Department of Ophthalmology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1023, New Zealand. t.sherwin@auckland.ac.nz
Received: June 25, 2019
Peer-review started: June 29, 2019
First decision: July 31, 2019
Revised: September 11, 2019
Accepted: November 13, 2019
Article in press: November 13, 2019
Published online: January 26, 2020
Processing time: 187 Days and 18 Hours
Abstract
BACKGROUND

Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss. Cells extracted from peripheral corneas can form stem cell-enriched spheres, which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue.

AIM

To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue.

METHODS

Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells. These spheres were implanted into incisions created in full thickness and onto the surface of 10 µm thin sections of keratoconic and normal stromal tissues in vitro. Tissue sections were used to maximise use of limited keratoconic tissue available for research. Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d (D14) from implantation. Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment.

RESULTS

Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10 µm thin en face tissue sections. No observable differences were seen in sphere migration, proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14. Spheres stained positively with Calcein-AM up to D14. Cell migration increased from day 0 to D14, occurring radially in three dimensions from the sphere and in alignment with tissue edges. Cell proliferation marker, EdU, was detected at day 10. Implanted spheres stained positively for putative stem cell markers ∆Np63α and ABCB5, while ABCG2, ABCB5, ∆Np63 and p63α were detectable by droplet digital PCR up to D14. Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin (myofibroblast marker) in some migrated cells. Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers, indicating an appropriate response to repopulating diseased tissue.

CONCLUSION

Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.

Keywords: Keratoconus; Cell culture; Immunohistochemistry; Quantitative PCR; Digital PCR; Spheroid; Holoclone; Neurosphere; Regeneration

Core tip: Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss. Cells extracted from peripheral corneas can form stem cell-enriched spheres, which we introduced into normal and keratoconic corneal stroma. This study shows for the first time that implanted stem cell-enriched spheres are capable of repopulating the dystrophic corneal stromal surface in a directed manner. Spheres may therefore be used to replace or supplement diseased cells in keratoconic patients, thereby serving as an adjunct to current treatments.