Abstract
Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied. Here, we established a protocol for the efficient isolation and cultivation of pure populations of human limbal melanocytes, which could be expanded at high yield by using recombinant laminin (LN)-511-E8 as culture substrate. Co-cultivation of limbal melanocytes with limbal epithelial stem/progenitor cells on fibrin hydrogels pre-incubated with LN-511-E8 resulted in multilayered stratified epithelial constructs within ten days. By reproducing physiological cell–cell and cell–matrix interactions of the native niche environment, these biomimetic co-culture systems provide a promising experimental model for investigating the functional roles of melanocytes in the limbal stem cell niche and their suitability for developing advanced epithelial grafts for ocular surface surface reconstruction.
Highlights
Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied
We have recently reported that the LN chains α2, α3, α5, β1, β2, β3, γ1, γ2 and γ3 are strongly expressed in the limbal basement membrane, and that the α5-containing isoforms LN-511 and LN-521 enabled efficient expansion of limbal epithelial stem and progenitor cells (LEPCs) in both 2D and 3D cultures[18]
We have previously reported that fibrin-based hydrogels incorporating the limbus-specific LN-511 isoform and its biologically active C-terminal domain (E8 fragment) resulted in multilayered stratified corneal epithelial constructs after 14 days in culture[18]
Summary
Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied. Using recombinant LN-511-E8 fragments, we obtained high yields of pure LMs, which could be successfully applied for tissue engineering of fibrin-based corneal epithelial constructs Such biomimetic 3D co-culture systems of LMs and LEPCs may represent powerful tools for studying stem cell-niche cell interactions and for ocular surface reconstruction
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