Abstract
Purpose:
To examine whether human adipose-derived stem cells (ASCs) could support the growth of human limbal stem/progenitor cells (LSCs).
Methods:
Three different culture methods were tested, including culturing the LSCs directly on the feeder cells (regular method), a 3-dimensional (D) culture system (sandwich method) and a 3-D culture system coated with fibrin (fibrin method). Freshly isolated limbal epithelial cells (LECs) in single cell suspension or cell sheets were co-cultured with the feeder cells for 2 weeks. Cultured LECs were examined for their cell morphology, proliferation rate, p63alpha-bright population and expressions of putative LSC markers, ABCG2, ΔNp63, N-cadherin and keratin (K) 14, and the differentiation marker K12. LECs cultured on 3T3-J2 feeder cells using the regular method were used as a control.
Results:
Single LECs cultured on ASCs had limited proliferation and displayed differentiated morphology in all three culture methods. When LECs were cultured as cell sheets, the sandwich method supported the highest proliferation rate for ASCs, which was 4.0-fold higher than the control (p<0.01). Compared to the control, cell sheets cultured with ASCs expressed a similar mRNA level of ABCG2, ΔNp63 and N-cadherin in all three culture methods, a significantly lower level of K14 in sandwich and fibrin methods (decreased by 54% and 72%, respectively, p<0.05) and a significantly lower level of K12 in the regular, sandwich and fibrin methods (decreased by 65%, 85% and 90%, respectively, p<0.05). LECs cultured with ASCs were compact and small in size, and contained comparable percentages of p63alpha-bright progenitor cells to the control in all three culture methods (regular, 5%; sandwich, 5%; fibrin, 3%; 3T3-J2 control, 7%). The absolute number of p63alpha-bright cells produced in the ASC sandwich method was 7-folds higher than that in the control (p=0.051).
Conclusions:
ASCs support the growth of LSCs in the form of cell sheets but not single cell suspension. The sandwich method appears to be a more favorable culture method to expand LSCs on ASCs than the regular method.
Keywords: 482 cornea: epithelium •
721 stem cells