Differentiation Status of Limbal Epithelial Cells Cultured on Intact and Denuded Amniotic Membrane Before and After Air-Lifting

Abstract

The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of colla-gen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5 th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture.

Investigation of K14/K5 as a Stem Cell Marker in the Limbal Region of the Bovine Cornea

“…However, there still remains no decisive stem cell marker. Therefore, to identify corneal epithelial stem cells a combination of various cell makers including both positive and negative putative corneal epithelial stem cell makers must be used [15], [16], [17], [18].…”

Comparative Analysis of the Basement Membrane Composition of the Human Limbus Epithelium and Amniotic Membrane Epithelium

“…46 Exposure of cultivated limbal epithelial cells to the amniotic BM promotes the formation of a well-differentiated, stratified, firmly adherent corneal epithelial equivalent for tissue engineering strategies. 20,21,43 However, the absence of limbusspecific environmental niche factors in HAM, as shown in this study, may not enable the preservation of a stem cell phenotype and may contribute to the reported depletion of stem cells in the culture. 22 It has to be considered that the amniotic BM may be modulated by the expanded limbal epithelial cells and alter its composition during the cultivation period.…”

“…39 Although neither preparation has been shown to be superior, it seems that HAM with intact epithelial cells supports the proliferative capacity and the preservation of an undifferentiated phenotype, 16,36,[40][41][42] whereas HAM with the BM denuded of epithelial cells supports stratification, differentiation, and attachment of expanded limbal cells. 20,21,43 These observations suggest that direct exposure of cultivated cells to amniotic BM components provides a microenvironmental cue to promote differentiation of cultivated cells. However, other studies have provided evidence that removing amniotic epithelial cells did not affect the expression of putative stem cell markers, such as deltaN-p63alpha and ABCG2.…”

“…It rather supports previous reports showing that denuded amniotic BM provides signals to support growth, stratification, and differentiation of expanded limbal cells but does not support stemness. 20,21,43 Further differences between amniotic and corneolimbal BMs related to the presence of endostatin and collagen type XVIII in the corneal BM and their absence in HAM. This observation is consistent with previous findings that neither native nor cryopreserved HAM inhibited angiogenesis but promoted capillary tube formation by endothelial cells in vitro, 45 despite its antiangiogenic effect after transplantation in vivo.…”

Reconstruction of Auto-Tissue-Engineered Lamellar Cornea by Dynamic Culture for Transplantation: A Rabbit Model

“…Using this process, 2–6 layers of stratified epithelium could be formed on different scaffolds [15]–[22], adhesive molecules and cell-cell junctions have been verified by immunofluorescence staining [17] and transmission electron microscopy [17]–[19]. The classical static culture method could promote epithelium growth and produce a favorable morphological outcome on amniotic membrane [23], compressed collagen [18], silk fibroin [24], corneal stromal lamella discs [25], and so on.…”