Amniotic Membrane With Live Stromal Cells Facilitates Preservation And Expansion Of Corneal Epithelial Stem Cells

Abstract

Purpose: : Amniotic membrane (AM) has been broadly used as carrier for ex vivo expansion of limbal epithelial stem cells (LESCs), whereby AM works as a surrogate niche for stem cells. Previous studies used cryopreserved AM which contains devitalized stromal and/or epithelial cells. In this study, we intend to evaluate the efficacy of the fresh denuded AM with live stromal cells for ex vivo expansion of LESCs.

Methods: : Epithelium of fresh AM was removed after brief dispase II digestion. AM stromal cells remained alive, or devitalized through repeated freeze-thaw. Limbal epithelial cell sheets isolated from rabbit limbal explants were cultured on the basement membrane side of the live AM (LAM) or dead AM (DAM) for up to 7 days. The outgrowth of the epithelial sheets, epithelial colony-forming-efficiency (CFE) on 3T3 feeder layers, and BrdU labeling with or without tracing were performed. The whole-mount and section immunofluorescence staining for cytokeratin K12 and K14, p63, Ki67, and BrdU were conducted. The expression of K12, K14, p63, Ki67 was also determined by real time PCR and Western blot analysis. Outgrowth epithelial sheets generated from LAM or DAM were transplanted onto the corneal surface of a limbal stem cell deficiency rabbit model and followed up for 2 months.

Results: : Epithelial cells on LAM showed more homogeneous, compact, and smaller in cell size. The CFE of cells expanded on LAM was significantly higher than that on DAM. There were more BrdU label retaining cells on LAM, while short term BrdU labeling did not show significant difference. K14, p63, and Ki67 expression was higher in epithelial cells expanded on LAM. K12 was negative in the basal epithelium on LAM, while was full-thickness positive in the epithelium on DAM. Furthermore, the epithelial cells on the LAM could be easily separated as an intact cell sheet and successfully reconstructed ocular surface in limbal stem cell deficiency rabbit model, while cells on DAM could not generate intact cell sheet.

Conclusions: : AM with live stromal cells facilitates ex vivo expansion and stemness preservation of limbal epithelial stem cells. AM stromal cells may act as niche cells in ex vivo corneal epithelial tissue engineering.

Keywords: cornea: epithelium • differentiation • proliferation