Influence of Substrate on Gene Expression Associated with Stemness in Human Limbal Epithelial Cell Graft

Abstract

Purpose: Protocols for generation of grafts from human limbal epithelial cells (HLECs) differ to some extent between clinics. Initiation of the cultures is performed using the explant culture technique or a suspension of dissociated cells, and a commonly used substrate is the human amniotic membrane (HAM). Outcome after transplantation is linked to various factors, including to content of cells presenting qualities associated with stemness. In contrast to HAM, cell culture coated plastic inserts (PIs) provide a readily accessible and standardized substrate. We here expanded HLECs on HAM and on PIs and examined the generated grafts for selected markers associated with stemness.

Methods: Limbal tissues were obtained after removal of the central tissue for transplant purposes. The corneo-limbal ring was divided in samples and placed epithelial side down on either HAM or on PIs in 6 well plates, and cultivated in complex medium (DMEM/F12 supplemented with 5% FBS, DMSO, human EGF, insulin, transferrin, selenium, hydrocortisone, cholera toxin, gentamicin and amphotericin B) for 3 weeks. Samples were processed for analysis using Flow Cytometry and qRT-PCR.

Results: After 3 weeks, we have previously reported expression of genes associated with stemness such as ABCG2, P63, OCT4, SOX2, KRT3, and OCLN to be similar in cultures maintained on the two types of substrate. Using FACS sorting, we here detected that expression of P63 was increased in the side population obtained using PIs compared with HAM. In contrast, the expression of ABCG2 was increased in the side population obtained when using HAM as a substrate.

Conclusions: Our findings show that genes related to stemness may be similarly expressed in grafts engineered using different substrates. However, we further demonstrate that the expression of such genes in the side population may differ considerably in grafts engineered on different substrates.

Keywords: 482 cornea: epithelium • 721 stem cells • 654 proliferation