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Arman S. Zaman, Patrick J. Pham, Hua Mei, Thuy Truong, Martin N. Nakatsu, Sophie X. Deng; Optimizing the Detection of Limbal Epithelial Stem/Progenitor Cells Using In Vivo Laser Scanning Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3522.
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© ARVO (1962-2015); The Authors (2016-present)
To optimize the detection of regions within the normal limbus that harbor a high density of undifferentiated corneal epithelial stem/progenitor cells using in vivo laser scanning confocal microcopy (LSCM).
Human sclerocorneal tissues were obtained from healthy donors and divided into four to six regions. Images of each limbal region were taken using the Heidelberg Retina Tomograph III at three locations: anterior, middle and posterior limbus. The limbal epithelial layer was then isolated from each region and cultured on growth-arrested NIH 3T3 feeder layers. Colony forming efficiency (CFE) was assessed after 14 days. Total RNA was extracted from colonies and reverse transcribed to cDNA. Quantitative real-time PCR (qRT-PCR) was performed to detect the mRNA expression levels of putative stem cell and differentiation markers.
The limbal palisades of Vogt (POV) as well as regions with high cell density (HCD) but lacking the POV were detected using LSCM. The average CFE of the regions with the POV was similar to that in the HCD regions (P=0.13). The cultured corneal epithelial stem/progenitor cells from the HCD regions had higher expression levels of putative stem cell markers ABCG2 and ΔNp63α (P<0.05) and a lower expression of the differentiation marker keratin 12 (P<0.05) compared to the POV regions.
These findings demonstrate that LSCM can be used to evaluate the microstructure of the corneal limbus. Furthermore, we found that the HCD regions that lack the POV also harbor limbal stem/progenitor cells. Thus, the POV may not be the only location where limbal stem/progenitor cells reside and other HCD regions may be promising target niches for limbal stem cell transplant.
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