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A. J. Shortt, G. A. Secker, S. J. Tuft, J. T. Daniels; The Effect of Insulin Like Growth Factor-1 on Colony Forming Efficiency, Colony Size and Cell Morphology of Cultured Human Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1786.
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Insulin like growth factor (IGF) plays an important role in embryonic and post natal growth. This study evaluated the effect insulin like growth factor-1 (IGF-1) on ex-vivo human limbal epithelial stem cells (LESC) in culture
Primary human limbal epithelial cells were isolated into a suspension, seeded onto a layer of mitomycin C growth arrested mouse 3T3 cells and cultured in a serum containing corneal epithelial culture medium (CECM). The effect of adding 2ng/ml, 10ng/ml and 20ng/ml of recombinant human IGF-1 to the CECM was compared with a control group cultured in CECM without IGF-1 supplementation. Colony forming efficiency, holocone colony area, cell morphology and mean holoclone cell size were compared between groups. In each group three replicates were performed using cells from an single donor and the experiment was repeated using three different donors. Differences between groups were analysed by ANOVA.
Supplementation of CECM with IGF-1 did not alter the colony forming efficiency between groups (control =4.4±0.69%, 2ng/ml=3.33±1.0%, 10ng/ml =3.56±0.51% and 20ng/ml =4.67±0.33%) (p<0.05). There was a dose dependent increase in holoclone colony area with increasing concentrations of IGF-1 (control =0.11±0.07cm2, 2ng/ml= 0.14±0.06cm2, 10ng/ml =0.16±0.15cm2 and 20ng/ml =0.19±0.05%) however this was not statistically significant. Holoclone cell morphology in the 10ng/ml and 20ng/ml IGF-1groups was markedly different from the control and 2ng/ml groups in that cells were more regular in appearance and there was less variability in cell size. The mean cells diameter in the 10ng/ml and 20ng/ml IGF-1 groups was significantly smaller (12·28±0·12µm and 13.02±0·19µm) than in the control group (18.82±0·92µm) (p<0.05).
The addition of IGF-1 in a standard co-culture system for limbal epithelial cells results in a small but not significant increase in colony size and a significant reduction in the mean cell size. These results indicate that IGF-1 may act upon LESC to increase their proliferation and maintain their stem cell phenotype. The effect of IGF-1 on LESC marker expression remains to be evaluated.
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