S C Tseng1, F E Kruse, J Merritt, D Q Li. 1. Department of Ophthalmology, Bascom Palmer Eye Institute, William L. McKnight Vision Research Center, Miami, FL 33136, USA.
Abstract
PURPOSE: To compare the supporting mechanism between the serum-free and the fibroblast-cocultured single-cell clonal culture systems. METHODS: Clonal growth, measured by colony forming efficiency (CFE) and size, was compared between rabbit corneal and limbal epithelial cells in a previously-established serum-free MCDB medium supplemented with growth factors, and in a coculture system with a feeder layer of mitomycin C-treated mouse 3T3 fibroblasts grown in the MCDB or DMEM medium plus 20% fetal bovine serum (FBS). RESULTS: Limbal epithelial cells in the serum-free MCDB medium had a significantly lower CFE than corneal epithelial cells (p < 0.001), suggesting that this system promoted more clonal growth of corneal progenitor cells. In contrast, with cocultured 3T3 fibroblasts limbal CFE was significantly increased (p < 0.001), while corneal CFE was not changed, indicating that the 3T3 system promoted more clonal growth of limbal progenitor cells. Addition of 20% FBS in the MCDB medium cocultured with 3T3 fibroblasts significantly promoted both limbal and corneal CFEs (p < 0.001). For both cultures, switching the serum-containing MCDB medium to the serum-containing DMEM medium produced clonal growth only with cocultured fibroblasts. This epithelial growth-promoting activity was not present on the cell surface or in the extracellular matrix, but present in pre-centrifuged and prefiltered 3T3 fibroblast-conditioned media. Both growth-promoting and anti-apoptotic activities were present in fibroblast-derived serum-free conditioned media. In the presence of this anti-apoptotic activity, serum addition promoted clonal growth, and the expression of cornea-type K3 keratin in limbal colonies was negative using AE-5 monoclonal antibody. CONCLUSIONS: Further purification and characterization of this fibroblast-derived anti-apoptotic survival factor will facilitate understanding of the mechanism by which epithelial stem cells are regulated via epithelial-mesenchymal interactions.
PURPOSE: To compare the supporting mechanism between the serum-free and the fibroblast-cocultured single-cell clonal culture systems. METHODS: Clonal growth, measured by colony forming efficiency (CFE) and size, was compared between rabbit corneal and limbal epithelial cells in a previously-established serum-free MCDB medium supplemented with growth factors, and in a coculture system with a feeder layer of mitomycin C-treated mouse 3T3 fibroblasts grown in the MCDB or DMEM medium plus 20% fetal bovine serum (FBS). RESULTS: Limbal epithelial cells in the serum-free MCDB medium had a significantly lower CFE than corneal epithelial cells (p < 0.001), suggesting that this system promoted more clonal growth of corneal progenitor cells. In contrast, with cocultured 3T3 fibroblasts limbal CFE was significantly increased (p < 0.001), while corneal CFE was not changed, indicating that the 3T3 system promoted more clonal growth of limbal progenitor cells. Addition of 20% FBS in the MCDB medium cocultured with 3T3 fibroblasts significantly promoted both limbal and corneal CFEs (p < 0.001). For both cultures, switching the serum-containing MCDB medium to the serum-containing DMEM medium produced clonal growth only with cocultured fibroblasts. This epithelial growth-promoting activity was not present on the cell surface or in the extracellular matrix, but present in pre-centrifuged and prefiltered 3T3 fibroblast-conditioned media. Both growth-promoting and anti-apoptotic activities were present in fibroblast-derived serum-free conditioned media. In the presence of this anti-apoptotic activity, serum addition promoted clonal growth, and the expression of cornea-type K3 keratin in limbal colonies was negative using AE-5 monoclonal antibody. CONCLUSIONS: Further purification and characterization of this fibroblast-derived anti-apoptotic survival factor will facilitate understanding of the mechanism by which epithelial stem cells are regulated via epithelial-mesenchymal interactions.
Authors: De-Quan Li; Zhuo Chen; Xiu Jun Song; Cintia S de Paiva; Hyun-Seung Kim; Stephen C Pflugfelder Journal: Exp Eye Res Date: 2005-04 Impact factor: 3.467