Reconstruction of a human hemicornea through natural scaffolds compatible with the growth of corneal epithelial stem cells and stromal keratocytes

[摘要] Purpose: To reconstruct a humanhemicornea in vitro by means of limbal stem cells cultured onto humankeratoplasty lenticules (HKLs) and to obtain a natural corneal graftfor clinical applications. Methods: Limbal stem cells were seededonto HKLs with or without the presence of feeder layers of lethallyirradiated 3T3-J2 cells and compared with the current “gold standard”scaffold, i.e., the fibrin glue. The effects of the scaffold on thepreservation of stemness and/or induction of differentiation pathwayswere investigated through analysis of a variety of markers, includingp63 and ΔNp63α for stemness, 14-3-3σ for early differentiation,keratins 3, 14, 12, and 19 to determine cell phenotype, and α6, β1, andβ4 integrins to evaluate interactions with the stroma. Integrity of thestroma was assessed through analysis of keratan sulfate, CD-34 andaldehyde dehydrogenase 3A1 (ALDH3A1) (for keratocytes), visual systemhomeobox 1 (VSX1), and alpha-smooth muscle actin (α-SMA) (forfibroblasts and myofibroblasts). The structural properties of thereconstructed “hemicornea” were investigated through scanning electronmicroscopy. To evaluate the preservation of the stemness potential,cells were trypsinized from each scaffold and clonogenic/proliferativecharacteristics analyzed. Results: Limbal stem cells expanded ontoHKLs gave rise to a stratified squamous keratinized epitheliummorphologically similar to that of normal corneas. The resultingcorneal epithelium was characterized by basal expression of p63 andΔNp63α, while expression of 14-3-3σ, keratin 3, and keratin 12 wasfound in the upper cell layers. The basal cuboidal epithelial cellswere anchored to the basement membrane and expressed keratin 14 and α6,β1, and β4 integrins. In the stroma of HKLs, keratocytes maintained thebiosynthetic and phenotypic appearances typical of resting/quiescentcells and expressed keratan sulfate, CD-34, and ALDH3A1. Fibroblastictransformation was observed with the appearance of VSX1 and α-SMA.Scanning electron microscopy analysis showed that HKLs maintained theirnative conformation with collagen fibrils interconnected to the networkand parallel to the corneal surface. HKLs did not alter theclonogenic/proliferative capacity of limbal stem cells. No differenceswere seen when HKL was compared to fibrin glue, one of the scaffoldscurrently used for limbal stem cell transplantation. Conclusions: Our findings demonstratethat HKL could be a suitable scaffold for corneal epithelial stem cellsas they were shown to proliferate, express differentiation markers, andbind to the underlying stroma with no alterations in clonogenicpotential. HKLs have some advantages over currently used scaffolds,such as the possibility to allow cell growth with no feeder layers, tobe freeze dried, and to preserve the integrity and viability of stromalkeratocytes. The development of a tissue-engineered “hemicornea” mightoffer new therapeutic perspectives to patients affected by total limbalstem cell deficiency with stromal scarring.

[学科分类] 生物化学/生物物理