title: Corneal stromal cell plasticity: in vitro regulation of cell phenotype through cell-cell interactions in a three-dimensional model.
abstract: In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims at recapturing this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined: epithelial explant; transwell; and the use of conditioned media. The aim was to determine whether the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta-1 (TGF-β1) was then used to determine whether the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial-stromal cell interactions. Two different nondestructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial-stromal co-culturing with TGF-β1, and wortmannin media supplementation, respectively, affect stromal cell behavior and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology, and protein expression were investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a nonactivated keratocyte cell lineage in terms of behavior and biological properties; and then back again via TGF-β1 media supplementation. It was then revealed that epithelial-stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal-epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury by which such interactions are vital.
authors.affiliation: Institute for Science and Technology in Medicine, School of Medicine, Keele University , Stoke-on-Trent, United Kingdom .
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Wilson
authors.forename: Ying
authors.initials: Y
authors.lastname: Yang
authors.forename: Alicia J
authors.initials: AJ
authors.lastname: El Haj
mesh_terms: D000328:Adult; D000730:Androstadienes / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D003319:Corneal Stroma / Q000166:cytology*; D055119:Elastic Modulus / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D053558:Keratin-3 / Q000378:metabolism; D008954:Models, Biological; D010641:Phenotype; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D054457:Tissue Scaffolds / Q000737:chemistry; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D015415:Biomarkers; D053558:Keratin-3; D053773:Transforming Growth Factor beta1; D014746:Vimentin; D000077191:Wortmannin
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