This experiment aims to investigate the potential ability of mesenchymal stem cells (MSCs) to produce beneficial factors for corneal recovery when the cells were seeded on a xenogeneic acellular corneal matrix (ACM) in vitro.
MSCs and corneal limbal stem cells (LSCs) from the corneal limbus region of rats were isolated and cultured. Batch 1 from each type of cell was seeded on a Beagle cornea ACM at a density of 3×103 cells/mm2 for 7 days. The proliferation activity of the cells was quantitatively determined at 1, 3, 5, and 7 days with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Keratin3/12 (CK3/12) growth factors, including vascular endothelial growth factor (VEGF), pigment epithelium-derived factor, epidermal growth factor, and transforming growth factor-beta1, integrin subunits such as α5β1, and α6β1, and elements of the extracellular matrix (ECM) such as fibronectin and laminin were investigated with immunofluorescence staining, reverse transcriptional polymerase chain reaction, and western blot assay, respectively.
The basic expression of growth factors in MSCs was much higher (n=6, p<0.05) than that in the LSCs, including VEGF, epidermal growth factor, and transforming growth factor-beta1. After being seeded on the ACM, those factors in MSCs expressed continuously at a high level, but the seeded corneal epithelium cells presented a downregulated trend in these factors. The expression of VEGF in seeded MSCs decreased, which was similar to the trend for the seeded LSCs (n=6, p<0.05). The expression of keratin3, a sign of mature epithelium cells, was also present in the MSCs after being seeded for 7 days. The expression of pigment epithelium-derived factor by the seeded and normal culture MSCs was equal, while the expression of this factor was not detected in either the seeded or the normal cultured LSCs. There were no significant differences between the integrin subunits (α5, α6, β1) and the extracellular matrix, including fibronectin and laminin, generated by normal cultured or seeded MSCs and LSCs.
Under the ACM microenvironment, the MSCs presented beneficial factors for corneal recovery comparable to those presented by corneal LSCs. This indicates that MSCs, when combined with an ACM, may compose a competent corneal substitute for healing corneal wounds.