Corneal Epithelium Cell Marker Acquisition by Human Adipose Tissue Mesenchymal Stem Cells (hAT-MSC)

Abstract

Purpose: : transplantation of autologous corneal stem cells is not possible in cases of bilateral limbal stem cell deficiency (LSCD). To restore ocular surface in these patients it is then mandatory to find an extraocular source of autologous stem cells i.e. mesenchymal stem cells (MSC). The aim of this study was to investigate if human adipose tissue-MSC (hAT-MSC) can acquire specific markers of corneal epithelium.

Methods: : hAT-MSCs were isolated from human lipoaspirates, expanded up to 3-4 passages. Their MSC phenotype and multipotent capacity (by differentiation towards osteoblast, adipocytes, and chondrocytes) was demonstrated. Corneal epithelium cell phenotype was induced by incubating hAT-MSC with conditioned growth culture media on plastic and collagen IV substrates for 1, 8, 15, and 22 days. Corneal epithelial and MSC specific cell marker expression was analyzed by flow cytometry (CD73, CD90, CD105, CD45, CD34, CD14, CD19, HLA-DR), immunofluorescence (CK3, CK12), and real time-PCR (CK3, CK12) with TaqMan probes. The human corneal epithelium cell line HCE was used as positive control.

Results: : hAT-MSC incubation with conditioned growth media induced a significant increase in the expression of the two specific corneal epithelium cell markers CK3 and CK12. This induction was time-dependent, peaking at 15 days of culture. hAT-MSCs grown in the same conditions but cultured with only basal media did not show corneal epithelial cell marker enhancement. CK3 was preferentially induced by Collagen IV substrate and CK12 by plastic.

Conclusions: : hAT-MSCs were successfully enhanced to acquire corneal epithelium markers. This suggests that autologous adipose tissue could be a potential source of stem cells for ocular surface reconstruction in cases of bilateral LSCD.

Keywords: cornea: epithelium • differentiation • regeneration