Human umbilical cord lining cells as novel feeder layer for ex vivo cultivation of limbal epithelial cells

Abstract

PURPOSE. To determine the effectiveness of human umbilical cord-derived mucin-expressing cord lining epithelial cells (CLEC-muc) as feeder cells in a coculture system for the cultivation of human limbal stem cells. METHODS. Human CLEC-muc were cultured in PTTe-1 medium and treated with mitomycin C to arrest their growth to make the feeder layer. Single-cell suspension of limbal cells was prepared from corneal rim collected from the Singapore Eye Bank. Limbal cells were cultured in a coculture system with CLEC-muc as well as 3T3 cells as feeder layer. We compared the colony-forming efficiency and cell morphology of the limbal cells cultured in the two different feeder layers. We also compared the expression level of several putative limbal stem cell markers, such as HES1, ABCG2, ΔNP63, and BMI1, in the cultured limbal cells by immunostaining and quantitative (q)RT-PCR. Expression of cytokeratins CK14, CK15, CK19, CK3, and CK4 was further compared. RESULTS. Human limbal epithelial cells cultured in both types of feeder layers showed comparable cell morphology and colony-forming efficiency. These cells exhibited a similar expression pattern of HES1, ABCG2, ΔNP63, BMI1, CK14, CK15, CK19, and CK3 as detected by immunostaining and PCR. CONCLUSIONS. Human CLEC-muc may be a suitable alternative to conventional mouse 3T3 feeder cells, which may reduce the risk of zoonotic infection.