A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction

Abstract

The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.

P-Cadherin Is Expressed by Epithelial Progenitor Cells and Melanocytes in the Human Corneal Limbus

“…After seeding of both cell types on DHL scaffolds and three weeks of cultivation, stratification (2–3 layers) of the epithelium, and darkly pigmented cells interspersed between the epithelial cells (supposed to be melanocytes) were confirmed by H&E staining (the dotted line represents the BM; Figure 5 E(ii)). We also observed a repopulation of the limbal stroma ( Figure 5 E(ii)) similar to earlier observations [ 29 ].…”

“…The repopulation potential of sorted CD90 − CD117 − P-cad + LEPC was tested by seeding these cells on DHLscaffolds; we also used CD90 − CD117 + P-cad + LM as they share a common locality with LEPC in vivo. Hematoxylin and Eosin (H&E) staining of the DHL specimen showed a regular arrangement of collagen fibrils, connective tissue protrusions (black arrows), invaginations (white arrows), and vascular gaps (dashed circles) in the ECM ( Figure 5 E(i)) [ 29 ]. After seeding of both cell types on DHL scaffolds and three weeks of cultivation, stratification (2–3 layers) of the epithelium, and darkly pigmented cells interspersed between the epithelial cells (supposed to be melanocytes) were confirmed by H&E staining (the dotted line represents the BM; Figure 5 E(ii)).…”

“…For routine histology, the scaffolds were fixed in 4% paraformaldehyde for 30 min and embedded in paraffin. The 5 µm thick sections were cut and stained with hematoxylin (Haematoxylin Gill III, Surgipath, Leica, Germany) and eosin Y (Surgipath, Leica, Germany) as described previously [ 29 ].…”

“…Decellularized human limbal scaffolds (DHL) were prepared as described previously [ 29 ]. Recellularization of decellularized scaffolds was also carried out as described previously [ 29 ].…”

“…Decellularized human limbal scaffolds (DHL) were prepared as described previously [ 29 ]. Recellularization of decellularized scaffolds was also carried out as described previously [ 29 ]. Briefly, for DHL-LEPC/LM scaffolds, the cultured LEPC (P1) and LM (P1) were seeded together in a ratio of 3:1 on the decellularized corneal surface and cultured in CCM (0.4 mM Ca 2+ ).…”