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Purpose: It is established by laboratory and clinical evidence that corneal stem cells are localised at limbus but are not isolated as yet due to the lack of stem cell markers. Recently we had discovered a novel anatomical structure at limbus, termed as the Limbal epithelial crypt (LEC), which have the potential of a stem cell niche as determined from previous anatomical and immunohistological studies. We aim to characterise the differential gene expression of the LEC by comparing with other regions of the ocular surface epithelium by 30,000 genes human oligonucleotide microarray analysis with the view to find putative stem cell marker. Methods: On the frozen cryostat sections of the corneoscleral epithelium from cadaver donor tissue, Laser Capture Microdissection (LCM) by PALM® Microbeam systems was done. From the LMD tissue the RNA was extracted, amplified and hybridised to human oligonucleotide array chip. Raw data was obtained with Genepix Pro 6 software and uploaded on BASE where filtration and normalisation was done. Further analysis was done on TMev software by SAM and T Test. Results: Microarray analysis was performed on BASE and with Significance Analysis of Microarray (SAM) on TMev. The analysis showed significant gene expression indicative of stem cells such as specific and differentiation markers.Transcription factors, cell adhesion molecules, genes involved in cell metabolism and respiration showed up regulation in the LEC. Conclusions: The microarray analysis data supports our hypothesis that LEC harbours stem cells along with neighbouring niche cells and hence represents a Limbal Stem Cell niche.This is being confirmed with real time PCR. |
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