The main area of interest is on adult stem cells - specifically limbal epithelial stem cells, which play an important role in corneal epithelial homeostasis and hence corneal transparency. We have earlier established a two parameter concept for identifying potential stem cells, and developed a simple method for ex-vivo expansion of the limbal/buccal epithelial stem cells for corneal surface reconstruction in limbal stem cell deficient patients in compliance with good manufacturing practice. With the objective of improving stem cell expansion and transplantation outcome, the current focus is to understand the mechanism involved in the maintenance of stemness at
(i) molecular level - the role of specific genes, regulatory elements and associated signaling pathways and (ii) cellular level - the microenvironment or niche in the limbal stroma.
Limbal niche plays an important role in the maintenance of stemness. We propose that limbal niche cells are the mesenchymal stem cells (MSC) in the limbal stroma. The location of these MSCs in situ was identified by the presence of unique clusters of cells in the anterior limbal stroma, which were double positive for CD90 and CD105 subjacent to the basal limbal epithelium, extending between rete pegs, palisades of Vogt. They were absent in posterior limbal stroma and throughout the corneal stroma indicating that these unique clusters of cells are the specific cells present in niche which can be considered as niche cells . Functional characterization of these cultured stromal cells revealed that limbal stromal cells are better feeder layer than corneal stromal cells as it supported higher colony forming efficiency and holoclones formation indicating their role in maintenance of stemness. Studies are now being carried out to understand the secretory profile of these limbal stromal MSCs as well the extracellular matrix.
Representative confocal images of limbal and corneal cryosections (radial) double immunostained for CD90 and CD105. (Nuclear counterstaining with propidium iodide (blue)). (A) CD90 (green) and CD105 (red) were found in several unique clusters of cells in the anterior limbal stroma subjacent to the basal limbal epithelium. (B) Such cells were absent in the corneal stroma and this was confirmed by observing sequential images of the cornea.
It is estimated that SCs account to 0.1-10% of total limbal epithelial cells and till date, there is no specific method for their isolation. Due to the low SC content in the population analyzed, no single specific positive marker has been identified for limbal epithelial stem cells (LESCs). We have now established a method to enrich the SC content up to 80% (by isolating the limbal basal epithelial cells followed by Laser Capture Micro dissection (LCM) of cells with high N/C ratio, which we propose to be a better population to understand the molecular signature of these LESCs. Further we have demonstrated that the ?Np63a isoform is expressed at mRNA levels only in the enriched SC population and not in the differentiated cells indicating a probable role of this isoform in maintenance of stemness. The current focus is to understand the molecular mechanisms that are associated with the maintenance of stemness by transcriptome and miRNA profiling of these enriched population of stem cells.
Semi quantitative RT PCR analysis for ?Np63 isoforms (a and ?). with GAPDH- . ?Np63a expression found only in small limbal basal cells with high N/C ratio and not in large cells with low N/C ratio; and ? isoforms were expressed in limbal basal cells with high and low N/C ratio. ?Np63 isoforms were not expressed in central corneal epithelial cells.
Enrichment of human limbal epithelial stem cells to understand the stem cell related properties by whole genome analysis and p63 isoform expression profile, Funding Agency: Department of Biotechnology (2011-2014).
To analyse the limbal epithelial stem cell niche in limbal stem cell deficient patients. Funding Agency: AMRF-Aurolab Research Grant (2011-2014)
Effect of VEGF levels in Tenon fibroblast on the outcome of glaucoma surgery. Funding Agency:AMRF-Aurolab Research Grant (2012-2014).
Factors Responsible for the Generation of Epithelial Sheet Rich in Stem Cells under ex-vivo conditions from the Limbal and Buccal Biopsy Champalimaud. Funding Agency: AMRF (2008-09).
Developing Xenobiotic-free Culture Conditions to Generate Stem-Cell Rich Epithelium for Corneal Surface Reconstruction. Funding Agency: ALCON Anterior Segment Research Grant (2008-2011).
Antigenic Mimicry between Leptospiral and Human Lens Proteins. Funding Agency: ALCON Anterior Segment Research Grant (2008-2011).
Cytokine Profile in Aqueous Humor of Parasitic Granuloma. Funding Agency: ALCON Anterior Segment Research Grant (2008-2011).
Translational Research to Generate Corneal/Buccal Epithelial Stem Cells with GMP Compliance for Corneal Surface and Socket Reconstruction. Funding Agency: Defence Research Development Organisation (DRDO) (2011-2013).
Evaluation of surface free energy of Aravind Aqueous Drainage Implant (AADI) in comparison with Baerveldt implant. Funding Agency: AMRF-Aurolab Research Grant (2012-2013).