Purchase this article with an account.
Amanda Vernon, Stephen J Tuft, Julie T Daniels; Human dermal fibroblasts as potential niche substitutes in clinical limbal epithelial stem cell tissue equivalents. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5173.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Limbal epithelial stem cell (LESC) therapy is used for ocular surface repair. Limbal epithelial cells (LEC) are expanded on a substrate, often amniotic membrane and transplanted, however challenges to this therapy include amnion biological variation. Studies have shown the potential of plastic compressed collagen gels seeded with human limbal fibroblasts (hLF) as alternative substrate. However sourcing autologous hLF from patients may risk inducing LESC deficiency in their healthy eye therefore the potential of human dermal fibroblasts (hDF) was investigated as an alternative
Human LECs were isolated by enzymatic digestion and expanded on 3T3 feeders. A solution of NaOH neutralised rat-tail type I collagen, 10X MEM/fibroblast media containing hDF or hLF (8.8x105 /ml) were incubated (30 min/40°C). Gel solution (2.2ml) was added to each cassette and left to set (30 min/37°C). Plastic compression was achieved using 35g weight, mesh and filter paper (15 min). LECs (6.5 x106/ml) were seeded onto gels and tissue equivalents (TE) were submerged (15 days) in media (CECM) and airlifted (7 days). LEC characteristics were examined by light microscopy and immunostaining (p63α, CK3 and Pax6). Fibroblast RNA was extracted for microarray analysis and qPCR. Cytokine array was performed using conditioned media following serum starving (0.1% BSA)
hDF-populated TE supported a confluent limbal epithelium, with cobblestone morphology. Immunostaining revealed positive expression for p63α, CK3 and Pax6. Quantitative assessment revealed 53% p63α positive cells in hDF-populated TE compared to 35% for hLF control. Microarray analysis of hDF isolated from TE revealed significant upregulation, relative to hLF control, for IL24 (FC, 7.54) and IL13RA2 (FC, 30.2) with qPCR confirming mRNA significantly increased. Cytokine array demonstrated significant upregulation for IL6, CXCL1, with a >2 fold increase for GMCSF
hDFs appear to support LECs capable of proliferation, and maintenance of corneal epithelial markers in a TE. However hDF-populated TE secreted proinflammatory cytokines therefore further validation would be required prior to clinical application as this type of TE might induce inflammation in the recipient. This study suggests not all fibroblasts are the same and it may not always be optimal to use fibroblasts from other tissue sources in TE for LESC therapy
This PDF is available to Subscribers Only