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:
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.
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
Research background

Keratoconus is a disease in which the front part of the eye, the cornea, becomes cone-shaped resulting in impaired vision. It is not clear why this disease occurs and why it progresses, although current treatments can help to improve vision. Reports in the literature of cross linking treatments that removed some of the native cells and strengthened the matrix, only halted or slowed the disease process for relatively short periods. On the contrary, transplanting healthy tissue containing healthy cells and matrix reduced recurrence rates. From this, we hypothesised that introducing healthy cells may be able to stop progression of the disease process. Stem cells possess many reparative and regenerative characteristics. Stem cell-enriched spheres cultured from healthy human corneal donors have been shown to be able to elicit healing responses and also can be implanted into normal corneal tissue to repopulate it. However, this regenerative ability of spheres has not previously been studied in diseased corneal tissue.

Research motivation

This study aimed to analyse how stem cell spheres behave in keratoconic tissue. It was not known whether stem cell spheres could survive or how they would behave when implanted into diseased corneal tissue. The eventual goal is to be able to use stem cell spheres for implantation and direct them to regenerate or repair diseased cornea with minimal invasiveness to donors and recipients.

Research objectives

Our research objectives were to implant stem cell spheres into keratoconic tissue and observe cell survival, proliferation, migration and differentiation. This data will inform the use of stem cell spheres for implantation into diseased tissue as a therapeutic tool.

Research methods

Spheres were implanted into full-thickness keratoconic tissues and also onto 10 µm thin slices of keratoconic stromal tissues. Similar implants were done in non-keratoconic tissues for comparison. Spheres were stained with the live cell stain Calcein-AM and imaged between days 0 and 14. Sphere implanted tissues were also analysed using indirect immunohistochemistry and droplet digital PCR.

Research results

Our results showed that spheres were able to survive to 14 d after being implanted into keratoconic and non-keratoconic tissues, both into full-thickness tissues as well as onto 10 µm tissue slices. There were no significant differences observed between how spheres migrated on keratoconic tissue compared to non-keratoconic tissue. Cells migrated from spheres radially and aligned with tissue edges. Cells were observed to increase in number with time by direct observation and by detection of cell proliferation markers. Putative stem cell markers were still detected 14 d post implantation but with lower levels of expression in the spheres implanted on keratoconic tissue compared to those implanted on normal tissue. Stromal cell markers increased while epithelial cell markers reduced indicating that spheres exhibit a response appropriate to the stimulus of stromal tissue. Future work will determine whether the cells will ultimately differentiate into keratocytes or how sphere-derived cells would progress in vivo.

Research conclusions

This study provided a novel insight into the implantation of healthy cells aimed at reducing disease progression in degenerative diseases like keratoconus. It has shown early insights into how spheres behave when implanted into diseased keratoconic corneal tissues. If healthy cells derived from implanted stem cell spheres can influence the diseased milieu into a healthier scenario, stem cell sphere implantation could be used to supplement corneal cross-linking procedures and delay the deterioration of vision for patients with keratoconus.

Research perspectives

This study informs the use of stem cell-enriched spheres as therapeutic agents in ocular tissue matrices. Future research would aim to study these interactions and how best to progress towards being able to use stem cells as a therapeutic adjunct to current treatments.