Niche function of amniotic membrane stromal cells in corneal epithelial stem cell expansion

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 contain devitalized stromal and/or epithelial cells. In this study, we intend to evaluate the efficacy of the fresh denuded AM with live stromal cells (LAM) for ex vivo expansion of LESCs.

Methods: Epithelium of fresh human AM was removed after brief dispase II digestion. AM stromal cells remained alive, or devitalize through repeated freeze-thaw. Limbal epithelial cell sheets or single cells isolated from rabbit limbal tissues were cultured on the basement membrane side of the live AM (LAM) or dead AM (DAM) in SHEM medium for different durations. Some epthelia were labeled with BrdU before culturing and then traced for 7 days. The outgrowth of the epithelial sheets, epithelial colony-forming-efficiency (CFE) of expanded cells on 3T3 feeder layers 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 Western blot analysis.

Results: Limbal 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 denuded AM with devitalized stromal cells, i.e, DAM. There were more BrdU label retaining cells on LAM. 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 with low seeding density of 100/cm2 on the LAM could generate stratified epihtelium and be easily separated as an intact cell sheet, while cells on DAM could not.

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: 482 cornea: epithelium • 721 stem cells