Transcriptional Profiling of Enriched Populations of Stem Cells Versus Transient Amplifying Cells

Abstract

The basal layer of limbal and central corneal epithelium is enriched in stem cells and transient amplifying cells, respectively. This physical separation of stem and transient amplifying cells makes the limbal/corneal epithelium an exceptionally suitable system for isolating basal cells enriched in these two proliferative populations. Prior attempts to isolate epithelial stem cells used methods such as proteolytic tissue dissociation and cell sorting that could potentially alter their gene expression profile. Using laser capture microdissection, we were able to isolate resting limbal and corneal basal cells from frozen sections with minimal tissue processing, thereby improving the yield and quality of RNA. Analyses of RNA isolated from 300 limbal and corneal basal cells from eight mice revealed a set of ϳ100 genes that are differentially expressed in limbal cells versus corneal epithelial basal cells. Semiquantitative reverse transcription-PCR confirmed the up-regulation of three limbal and three corneal genes. LacZ identification of epiregulin from epiregulin-null mice and immunohistochemical staining of wild type mice confirmed that epiregulin, one of the limbal epithelium-enriched genes, was associated with the limbal epithelial basal cells. Within the limbal and corneal basal cells, we detected previously unknown genes that were differentially expressed in these two regions that contribute further to our understanding of the unique heterogeneity of these two closely related basal cell populations. Our findings indicate that we can obtain accurate gene expression profiles of the stem cell-enriched limbal basal cell population in their "natural" quiescent state.The ability to identify, purify, and characterize epithelial stem cells is a critically important issue in epithelial stem cell biology as it will further our understanding on how stem cells are regulated. Of the various epithelial tissues studied, the limbal/corneal epithelium has yielded a great deal of information about stem cells (1). Based on a combination of (i) keratin expression data (2); (ii) in vivo and in vitro cell kinetic data (3-7); (iii) centripetal migration studies (8 -13); and (iv) corneal regeneration and reconstitution studies (14 -20), it is well accepted that the corneal epithelial stem cells are preferentially located in the limbal epithelial basal layer (for reviews, see Refs. 1 and 21-27). Furthermore, since the limbal stem cells can be millimeters away from their central corneal progeny transient amplifying (TA) 2 cells (1,2,4,7,24,28), the limbal/corneal system is ideally suited for isolating relatively pure populations of epithelial stem and TA cells for subsequent analyses.A lack of stem cell-specific markers has been an impediment for the isolation and subsequent biological and biochemical characterization of epithelial stem cells. Recently, transcriptional profiling of enriched populations of putative epithelial stem cells has been used to search for epithelial stem cellspecific markers. Methods used to isolate the putativ...

Cofactors of LIM Domains Associate with Estrogen Receptor α to Regulate the Expression of Noncoding RNA H19 and Corneal Epithelial Progenitor Cell Function

“…4). There is a statistically significant overlap of genes differentially regulated by DN-CLIM at P3 and the limbal epithelially enriched genes determined by the aforementioned study (33). Interestingly, all of the limbal epithelially enriched genes that are affected by DN-CLIM in the mouse cornea are down-regulated, further supporting a role for CLIM as transcriptional activators of a gene program regulating corneal progenitor cells (Fig.…”

“…We took advantage of previously published data comparing gene expression in central corneal and limbal cell populations captured by laser microdissection in the mouse (33) (Fig. 4).…”

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

“…The spatial separation of stem and transient amplifying cells in the limbal and central cornea allows an easy way to produce a stem cell enriched (limbal) and stem cell free (corneal) cell population. In recent years, several research groups, including ourselves, compared subsets of mRNAs and proteins expressed in the limbal and central cornea by the currently available high throughput nucleic acid profiling and proteomic techniques (81)(82)(83)(84)(85)(86)(87). Several of these molecules have been described as potential stem cell markers (Table 1).…”

Molecular profile of organ culture-stored corneal epithelium: Lgr5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells

“…Several groups have reported the gene expression profile of fresh limbal and corneal epithelial cells which has significantly contributed to the understanding of cellular pathways and phenotype (1,2). Surprisingly, the molecular profiling of long-term organ culture media-stored corneal epithelium has not been reported in the literature.…”