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E. Chankiewitz, M. Zenkel, F. E. Kruse, U. Schlötzer-Schrehardt; Molecular Profiling of Limbal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6077.
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© ARVO (1962-2015); The Authors (2016-present)
The regeneration of the corneal surface is maintained by a pool of stem cells at the limbus. Previous impediments to molecular characterization included the lack of accurate identification and pure isolation of this stem cell population. We screened for genes specifically expressed in limbal stem cells using PCR-based gene expression profiling combined with laser-capture microdissection and immunolabelling.
Serial cryosections obtained from human donor eyes were immunostained with antibodies against putative stem cell and differentiation markers (N-cadherin, desmoglein-3). Small clusters of 5 to 8 immunopositive or -negative cells were microdissected using the P.A.L.M. Microlaser system (Zeiss). Following extraction, total RNA was linearly amplified by the MessageAmpTM II aRNA-Kit. RNA quality and quantity were monitored with the Agilent BioAnalyzer. High quality cDNA was used for real-time PCR analyses with the Human Stem Cell RT2 ProfilerTM PCR Array (Superarray). The expression of 84 stem cell specific genes was screened in limbal stem cells and compared with those in basal epithelial cells of the cornea.
The combination of immunolabelling strategies and laser-capture microdissection enabled the accurate isolation and enrichment of high-quality RNA from small limbal stem cell clusters. Differential gene expression analyses showed an up-regulation of Notch pathway genes (e.g. DTX2, DVL1, NOTCH2, GCN5L2) in limbal stem cell clusters as compared to basal corneal epithelial cells. Moreover transcription factors of the Wnt pathway (e.g. PPARD, BTRC), cell cycle regulators (e.g. CDC2, PARD6A), cell-cell communication regulating genes (e.g. GJB1, GJB2) and cell adhesion molecules (e.g. CDH2, CD44, COL1A1) were higher expressed in the limbal stem cell population.
Accurate gene expression profiling using immunolabelled, microdissected putative stem cell clusters is a powerful method for molecular characterization of small cell populations by enhancing the specificity of expression data. The molecular components and pathways identified contribute to our understanding of stem cell regulation and may be used for improved in vitro techniques for therapeutical purposes.
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