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Johannes Menzel-Severing, Matthias Zenkel, Angelika Mößner, Friedrich E. Kruse, Ursula Schlötzer-Schrehardt; TGFβ/BMP Pathway Specific Expression Profiling of Limbal Epithelial Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5245.
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Previous works have shown that laser capture microdissection (LCM) is a suitable technique to select limbal epithelial cells for molecular characterization. Here, we used LCM to determine the expression profile of transforming growth factor beta/bone morphogenetic protein (TGFβ/BMP) signaling pathway related genes in limbal and central corneal epithelial cells.
The PALM MicroBeam system (Carl Zeiss, Germany) was used to isolate clusters of limbal basal epithelial cells with high nuclear:cytoplasmic ratio from serial cryosections of human donor corneas. Basal epithelial cells from central cornea were obtained using the same technique for direct comparison of gene expression. Linear amplification of isolated RNA was achieved using the MessageAmp II aRNA Kit (Ambion, USA). Successful amplification and RNA quality were monitored on a 2100 Bioanalyzer (Agilent Technologies, USA). Gene expression analysis was performed using pathway-specific RT2 Profiler PCR Arrays (SABiosciences, USA).
Using LCM, we were able to obtain RNA of sufficient quality and quantity to determine gene expression from well-defined subpopulations of ocular surface epithelial cells. Several components of the canonical TGFβ pathway showed stable expression in both corneal and limbal basal epithelium; this list includes TGFβ-receptors and SMAD signaling effectors. Ligands such as TGFβ1 and BMP6 were differentially expressed in basal epithelial cells isolated from central cornea, as compared to limbal cell clusters. Up-regulation in corneal basal epithelial cells was also found for TGFβ target genes such as TGFBI (keratoepithelin, a well-described extracellular matrix constituent of the corneal stroma) as well as adhesion molecules and transcription factors.
TGFβ and BMP signaling has been suggested to play a role in regulating cell cycle progression and differentiation in a number of stem cell populations, including that of the corneal surface. These data obtained from two distinct, homogeneous populations of corneal epithelial cells add to our understanding of the pathway-specific regulatory cytokine network. Further studies are warranted to elucidate potential involvement of TGFβ/BMP pathway components in regulating cellular cross talk within the limbal epithelial stem cell niche.
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