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

Abstract

Purpose: To reconstruct a human hemicornea in vitro by means of limbal stem cells cultured onto human keratoplasty lenticules (HKLs) and to obtain a natural corneal graft for clinical applications.

Methods: Limbal stem cells were seeded onto HKLs with or without the presence of feeder layers of lethally irradiated 3T3-J2 cells and compared with the current "gold standard" scaffold, i.e., the fibrin glue. The effects of the scaffold on the preservation of stemness and/or induction of differentiation pathways were investigated through analysis of a variety of markers, including p63 and DeltaNp63alpha for stemness, 14-3-3sigma for early differentiation, keratins 3, 14, 12, and 19 to determine cell phenotype, and alpha6, beta1, and beta4 integrins to evaluate interactions with the stroma. Integrity of the stroma was assessed through analysis of keratan sulfate, CD-34 and aldehyde dehydrogenase 3A1 (ALDH3A1) (for keratocytes), visual system homeobox 1 (VSX1), and alpha-smooth muscle actin (alpha-SMA) (for fibroblasts and myofibroblasts). The structural properties of the reconstructed "hemicornea" were investigated through scanning electron microscopy. To evaluate the preservation of the stemness potential, cells were trypsinized from each scaffold and clonogenic/proliferative characteristics analyzed.

Results: Limbal stem cells expanded onto HKLs gave rise to a stratified squamous keratinized epithelium morphologically similar to that of normal corneas. The resulting corneal epithelium was characterized by basal expression of p63 and DeltaNp63alpha, while expression of 14-3-3sigma, keratin 3, and keratin 12 was found in the upper cell layers. The basal cuboidal epithelial cells were anchored to the basement membrane and expressed keratin 14 and alpha6, beta1, and beta4 integrins. In the stroma of HKLs, keratocytes maintained the biosynthetic and phenotypic appearances typical of resting/quiescent cells and expressed keratan sulfate, CD-34, and ALDH3A1. Fibroblastic transformation was observed with the appearance of VSX1 and alpha-SMA. Scanning electron microscopy analysis showed that HKLs maintained their native conformation with collagen fibrils interconnected to the network and parallel to the corneal surface. HKLs did not alter the clonogenic/proliferative capacity of limbal stem cells. No differences were seen when HKL was compared to fibrin glue, one of the scaffolds currently used for limbal stem cell transplantation.

Conclusions: Our findings demonstrate that HKL could be a suitable scaffold for corneal epithelial stem cells as they were shown to proliferate, express differentiation markers, and bind to the underlying stroma with no alterations in clonogenic potential. HKLs have some advantages over currently used scaffolds, such as the possibility to allow cell growth with no feeder layers, to be freeze dried, and to preserve the integrity and viability of stromal keratocytes. The development of a tissue-engineered "hemicornea" might offer new therapeutic perspectives to patients affected by total limbal stem cell deficiency with stromal scarring.

Figure 1

Expression of epithelial cell markers in reconstructed hemicorneas. Human corneal epithelial stem cells seeded onto the HKL scaffold form a pluristratified and differentiated epithelium. Cryosections were analyzed through immunofluorescence. The layer of basal cuboidal cells was firmly attached to the underlying extra cellular matrix (ECM) and to the basement membrane through integrins α6 (blue, A), β1 (green, B), and β4 (red, C). The basal expression of p63 isoforms (red, D) and of the stem cell marker ΔNp63α (yellow, E) was always observed, thus suggesting the maintenance of undifferentiated progenitor cells interspersed between differentiated cells. Expression of keratin 14 (green, F) was also observed in the basal layers. Corneal differentiation occurred in all epithelial layers and was evaluated through the analysis of several markers, including 14-3-3σ (pink, G), keratin 19 (orange, H), keratin 3 (yellow, I), and keratin 12 (red, J). Note the presence of the Bowman’s membrane in transmitted-light images (white arrows, D–E). Scale bar=50 μm.

Figure 2

Comparison between HKL and fibrin-glue matrix as scaffolds for cultured human keratinocytes. Human corneal epithelial stem cells were cultured onto HKL (A, C) and fibrin glue (B, D) in submerged conditions for 7 days (A, B) and at the air–liquid interface for 14 further days (C, D). Increased stratification (more than 15 cell layers) and overexpression of p63 isoforms in suprabasal cells were only observed when the fibrin glue was analyzed (D). The growth onto HKL showed a phenotype that was more similar to that of normal human corneal epithelia (A, C). Note that the basal epithelial plane became undulated, yielding an appearance that resembles the palisade of Vogt (bottom inset, C). Scale bar=50 μm.

Figure 3

Keratocytes in the stromal part of the hemicornea were observed after 4',6-diamidino-2-phenylindole (DAPI) staining (A). Expression of specific markers of the corneal stroma was analyzed: keratan sulfate (KS), essential for the maintenance of the correct orientation of collagen fibrils (B); CD-34, a specific marker of keratocytes expressed both in vivo and in vitro (C); ALDH3A1, a marker of quiescent keratocytes in vivo (D); VSX1, found in keratocytes undergoing fibroblastic transformation (E); α-SMA, a muscle protein of F-actin stress fibers, typically found in myofibroblasts (F). Scale bar=50 μm.

Figure 4

Transparency and biomechanical properties of the cornea depend on the structure and organization of corneal stroma. Scanning electron microscope (SEM) images of the HKL showed that the Bowman's membrane structure maintained its native conformation (A: 3500×, scale bar 5μm). Collagen fibers and fibers interconnecting to the network formed collagen bundles, which were regular and parallel to the corneal surface (B: 100×, scale bar 100 μm). These were similar to those observed in normal human corneas.

Figure 5

Cells isolated from each of three scaffolds were serially propagated for three passages. This allowed us to obtain information about the residual clonogenic potential of the cells grown onto the scaffolds and to evaluate the effects of the matrix on the preservation of stemness and induction of differentiation pathways. [HKL + 3T3/J2] = HKLs with 3T3-J2 feeder layer; [HKL - 3T3/J2] = HKLs without 3T3-J2 feeder layer. No difference in the number of clonogenic cells (A) or percentage of aborted colonies (aborted colonies/total colonies ratio; B) was observed when cells isolated from HKLs (in the presence of 3T3-J2 feeder layers) or fibrin were compared. In the absence of a 3T3-J2 feeder layer, reduced number of clonogenic cells and increased percentages of aborted colonies were observed. Despite this, cells were found proliferating for at least three passages in culture, thus suggesting that HKLs might not interfere with the stemness and proliferative potential of corneal stem cells. Error bars indicate SEM (n=3).