Cellular therapy of corneal epithelial defect by adipose mesenchymal stem cell-derived epithelial progenitors

Francisco Bandeira,Melina Setiawan,Jodhbir S Mehta,Tze-Wei Goh,Gary Hin-Fai Yam

doi:10.1186/s13287-019-1533-1

Abstract

BackgroundPersistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE). This study investigated CE reformation using human adipose mesenchymal stem cells (ADSC), which derived epithelial progenitors via mesenchymal-epithelial transition (MET).MethodsSTEMPRO human ADSC were cultured with specific inhibitors antagonizing glycogen synthase kinase-3 and transforming growth factor-β signaling, followed by culture under a defined progenitor cell targeted-epithelial differentiation condition to generate epithelial-like cells (MET-Epi), which were characterized for cell viability, mesenchymal, and epithelial phenotypes using immunofluorescence and flow cytometry. Tissue-engineered (TE) MET-Epi cells on fibrin gel were transplanted to corneal surface of the rat LSCD model caused by alkali injury. Epithelial healing, corneal edema, and haze grading, CE formation were assessed by fluorescein staining, slit lamp bio-microscopy, anterior segment optical coherence tomography, and immunohistochemistry.ResultsCD73high/CD90high/CD105high/CD166high/CD14negative/CD31negative human ADSC underwent MET, giving viable epithelial-like progenitors expressing δNp63, CDH1 (E-cadherin), epidermal growth factor receptor, integrin-β4, and cytokeratin (CK)-5, 9. Under defined epithelial differentiation culture, these progenitors generated MET-Epi cells expressing cell junction proteins ZO1 and occludin. When transplanted onto rat corneal surface with LSCD-induced PED, TE-MET-Epi achieved more efficient epithelial healing, suppressed corneal edema, and opacities, when compared to corneas without treatment or transplanted with TE-ADSC. CE markers (CK3, 12, and CDH1) were expressed on TE-MET-Epi-transplanted corneas but not in other control groups.ConclusionHuman ADSC-derived epithelial-like cells, via MET, recovered the CE from PED associated with LSCD. ADSC can be a viable adult stem cell source for potential autologous epithelial cell-based therapy for corneal surface disorders.

Highlights

Persistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE)
Mesenchymal-epithelial transition of human adipose mesenchymal stem cells (ADSC) to express epithelial phenotype CD73high/CD90high/CD105high/CD166high/CD14negative/ CD31negative ADSC were treated with small molecules inhibiting Glycogen synthase kinase 3 (GSK3) and Transforming growth factor β (TGFβ) signaling together with atRA, as previously reported [19] with minor modifications (Fig. 1)
Epidermal growth factor receptor (EGFR) was detected in 4.5% mesenchymal-epithelial transition (MET) progenitors but in 0.1% ADSC and integrin-β4 (ITGB4) was expressed in 4% MET progenitors but undetected in ADSC. These results demonstrated that small molecule treatment under M1 and M2 cultures on ADSC generated epithelial-like progenitors through MET

Summary

Introduction

Persistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE). The CE is a self-renewing tissue, owing to the epithelial stem cells (limbal stem cells, LSC) present in the limbal basal epithelium at the corneal periphery [2, 3]. This unique population of epithelial progenitors constantly provides new cells for normal epithelial turnover and wound healing. A breach of CE integrity, due to mechanical trauma (like foreign body intrusion, contact lens overuse, and chemical burns); infection, neurotrophic keratopathy, dry eye, systemic and genetic disorders (e.g., thyroid eye diseases, Sjogren’s syndrome, aniridia-related keratopathy caused by Pax mutations, and ectodermal dysplasia caused by P63 mutations), and limbal stem cell deficiency (LSCD); causes persistent epithelial defects (PED), which result in corneal scarring, ulceration, neovascularization, conjunctivalization and, corneal opacification, and visual loss [4]

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