Purchase this article with an account.
S. M. Sharma, T. Fuchsluger, R. Dana, U. V. Jurkunas; Ex vivo Expansion of Limbal and Oral Epithelium Co-Cultured With Human Dermal Fibroblast Feeder Layer. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1939.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate whether stem cell-like properties are maintained by co-culture of human limbal and oral epithelial cells with non-transformed human dermal fibroblast feeder layer.
Human limbal and oral epithelial cells were expanded ex vivo by co-culturing with murine 3T3 fibroblasts, human dermal fibroblasts (DF), humans bone marrow cells (BM), and with no feeder layer (NF), using the cell suspension technique and/or explant methods. Cellular morphology was monitored with phase contrast microscopy and stem cell characteristics were assessed by using quantitative real time PCR and immunohistochemistry. For the explants, cell outgrowth area was marked and measured.
Limbal epithelial cells grown with dermal feeder had a higher expression of ABCG2 and p63 mRNA as compared to the cells grown on NF (p = 0.008 and p= 0.007, respectively) and BM feeder layer (p= 2.42E-07 and p= 4.14E-08, respectively). Similarly, higher expression of ABCG2 and p63 was detected in oral cells co-cultured with DF as compared to cells grown on NF (p= 0.01 and p= 0.001, respectively) or with BM (p= 0.04 and p= 0.03, respectively). Interestingly, the phenotype of cells co-cultured with DF was comparable to cells co-cultured with murine 3T3 feeder layer; they expressed putative stem cell marker, integrin-β1, and differentiated cell marker, cytokeratin-3. Quantitative analysis of the area of outgrowth on limbal cells cultivated via the explant method on DF showed exponential growth starting from day 2 to day 15, which was superior to cells grown with NF.
Our findings suggest that use of dermal fibroblasts is conducive to epithelial cell growth, differentiation, and expression of stem cell markers. Because dermal fibroblasts could be readily obtained from a small dermal biopsy, use of such autologous feeder cells minimizes the need for animal-derived products and enhances ex vivo cell expansion suitability for human transplantation.
This PDF is available to Subscribers Only