Different cell sizes in human limbal and central corneal basal epithelia measured by confocal microscopy and flow cytometry.
OBJECTIVE
In the epidermis, the highest clonogenicity, a feature of stem cells (SCs), is found in the smallest keratinocyte. In the limbal-corneal (LC) epithelium the SCs are exclusively localized in the basal epithelial layer of the limbal domain. The current study was conducted to determine whether this spatial SC arrangement is reflected in differences in the cell size between limbal and corneal cells.


METHODS
In vivo confocal microscopy was used to scan and measure the size of the cells of the central cornea and the superior limbus in five normal subjects, from the superficial to the basal cell layer. Limbal and corneal pure epithelial sheets were isolated by dispase digestion from human tissues and dissociated into single cells by trypsin digestion. The forward (FSC; a relative measure of cell size) and side (SSC; a relative measure of cytoplasmic complexity) light-scattering properties of these cells were determined by flow cytometry.


RESULTS
Confocal microscopy showed that diameters of the basal cells of the limbal and corneal zones were 10.1 +/- 0.8 and 17.1 +/- 0.8 micro m, respectively. The corresponding values for the superficial layers were 19.9 +/- 1.6 and 36.6 +/- 1.6 microm, respectively (P < 0.0001). The mean FSC and SSC of the limbal cells amounted to 65.7% +/- 8.7% and of the corneal cells, 74.4% +/- 4.6%. Furthermore, only 1.40% +/- 0.83% and 0.69% +/- 0.37% of the corneal cells had FSC and SSC equal to the lowest 15% of FSC and SCC of the limbal cells, respectively, indicating that the limbus contained a substantial proportion of very low FSC and SSC cells for which there was no corneal counterpart.


CONCLUSIONS
The data collectively demonstrate that the smallest cells are located in the limbal basal epithelium. This feature may help isolate corneal SCs located in the limbus.
Romano
Andre C
AC
TissueTech, Inc., and Ocular Surface Research and Education Foundation, 7000 SW 97 Avenue, Miami, Florida 33173, USA.
Espana
Edgar M
EM
Yoo
Sonia H
SH
Budak
Murat T
MT
Wolosin
J Mario
JM
Tseng
Scheffer C G
SC
D000328:Adult; D001485:Basement Membrane / Q000166:cytology; D002469:Cell Separation / Q000379:methods; D048429:Cell Size; D003315:Cornea / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D018613:Microscopy, Confocal; D013234:Stem Cells / Q000166:cytology*