Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

Djida Ghoubay-Benallaoua,Elisabeth Dupin,Céline De Sousa,Marie-Claire Schanne-Klein,Gaël Latour,Raphaël Martos,Vincent Borderie,Alexander V Ljubimov

doi:10.1371/journal.pone.0188398

Abstract

Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green’s medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green’s media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

Highlights

The cornea is a transparent window essential for vision, which forms the central part of the ocular surface [1]
The isolated limbal cells (ILC-scraping+ and ILCscraping-) were plated in three different culture conditions to compare their capacity for formation of spheres and adherent colonies: uncoated wells with Green’s medium and 3T3 feeders, Essential 8 medium (E8) medium, and E8 medium supplemented with Epithelial Growth Factor (EGF)
In feeder-free E8 medium (E8) and E8 medium supplemented with EGF (E8+), ILC-scraping+ (Fig 1A and 1B) formed spheres that eventually floated free in the medium, and isolated adherent cells, but no colonies

Summary

Introduction

The cornea is a transparent window essential for vision, which forms the central part of the ocular surface [1]. The stromal and endothelial layers are derived from the cranial neural crest cells that migrate along the optic vesicles and home to the anterior eye region [5, 6, 7, 8, 9, 10]. Epithelial and stromal limbal stem cells, usually referred to as limbal stem cells (LSC) for epithelial cells and stromal stem cells (SSC) for stromal cells, are required to maintain corneal transparency [11]. Both stem cell types are located in the limbal niche [12]. Stromal wound healing is a complex process involving cell death at the site of injury, migration of quiescent keratocytes followed by cell proliferation, differentiation and extracellular matrix synthesis and remodeling [16]

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