RT info:eu-repo/semantics/article
T1 Comparison of functional limbal epithelial stem cell isolation methods
A1 López Paniagua, Marina
A1 Nieto Miguel, Teresa
A1 De La Mata Sampedro, Ana
A1 Dziasko, Marc
A1 Galindo de la Rosa, Sara
A1 Rey, Esther
A1 Herreras Cantalapiedra, José María
A1 Corrales, Rosa María
A1 Daniels, Julie T
A1 Calonge, Margarita
K1 Cell culture; Cell suspensions; Explants; Limbal stem cells; Ocular surface.
AB The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either of the isolation techniques from the donor limbal tissue, then the cell suspension is probably the best option as long as the cells are expanded following our culture conditions.
PB ELSEVIER
SN 0014-4835
YR 2016
FD 2016
LK https://uvadoc.uva.es/handle/10324/64734
UL https://uvadoc.uva.es/handle/10324/64734
LA eng
NO Experimental Eye Research. 2016;146:83-94.
NO Producción Científica
DS UVaDOC
RD 05-jun-2024