Human Limbal Progenitor Cells Expanded on Intact Amniotic Membrane Ex Vivo

Abstract

Background: The transplantation of human limbal epithelium on amniotic membrane as a substrate is a new treatment for limbal stem cell deficiency. Limbal epithelial stem cells are characterized by a slow cell cycle and the lack of K3 keratin and connexin 43 (Cx43), a gap junction protein. We investigated Cx43 expression, gap junction intercellular communication (GJIC), and proliferative activity of limbal epithelium expanded on amniotic membrane. Methods: Connexin 43 expression and bromodeoxyuridine (BrdU) incorporation were determined by immunohistology. The GJIC was investigated by a scrapeloading dye transfer assay. Expression of Cx43 and K3 keratin as well as BrdU-retaining nuclei were also analyzed after xenotransplantation in nude mice. Results: Limbal epithelium showed mean±SD 12.4% ±14.5% positive units of Cx43 expression and a low BrdU labeling index of 2.4% ± 0.9% (n = 5), of which the latter was due to slow cycling, as proved by its increase to 62.0%±9.5% after continuous BrdU labeling for 5 days. Most of the expanded epithelium did not show GJIC (83%), significantly more than that grown on plastic (6%; PϽ.002). Basal cells of the stratified epithelium after xenotransplantation did not express Cx43 and K3 keratin, but their nuclei retained BrdU. Conclusion: These results support the hypothesis that intact amniotic membrane preferentially preserves and expands Cx43-negative, keratin K3-negative, and GJICdeficient limbal epithelium, a phenotype resembling that of stem cell-containing limbal basal epithelial cells in vivo. Clinical Relevance: Intact amniotic membrane is a suitable substrate for bioengineering limbal epithelia for ocular surface reconstruction.

Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells

“…It is generally accepted that outgrowth is slower on intact AM than on epithelium-denuded membrane [8,15]. We speculate that the AM cells directly hinder growth, and impair attachment by forming a barrier between the membrane and the limbal cells.…”

Amniotic membrane transplantation in surgical management of ocular surface squamous neoplasias: long-term results

“…15 The promotion of epithelization is implemented by the basement membrane of AM that serves as a substrate for normal migration, differentiation, and adhesion of epithelial cells, and prevents epithelial apoptosis. 16 Moreover, the epithelium of AM stimulates healing of the ocular surface by producing several growth factors, such as epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor, and basic fibroblast growth factor. 17 The anti-inflammatory activity of AM might be explained by the presence of receptor antagonist of inflammation mediators including interleukin-1 and regulation of the production of activin.…”

Factors Influencing Outcomes in Cultivated Limbal Epithelial Transplantation for Chronic Cicatricial Ocular Surface Disorders

“…There are experimental evidences showing that the AM is a natural substrate ideal for restoring the stromal niche of the limbal epithelial cells and the limbal epithelial cells cultivated on intact AM epithelium retain stem cell characteristics. 8,20,29,30 On the other hand, Koizumi et al reported that the cultivated limbal epithelial cells showed faster outgrowth with better stratification in morphology compared with those with intact amniotic epithelial cells. 31 They concluded that the denuded AM is the best carrier for corneal epithelial sheet preparation.…”