Nditions, immature colon epithelial cells reside in the bottom on the colonic crypts and express higher levels from the surface marker CD44, whilst differentiated mature cells progressively migrate towards the top rated and progressively shed CD44 CD40LG Inhibitors Related Products expression 14, 15. We focused our analysis on the stem/immature compartment with the colonic epithelium by sorting the EpCAMhigh/CD44+ population (Fig. 1, E ), which, in normal tissues, corresponds towards the bottom in the human colonic crypt 14. To study the much more mature, terminally differentiated cell populations, we analyzed an equal quantity of cells from the EpCAM+/CD44neg/CD66ahigh population, which corresponds to the top of the human colonic crypt (Fig. 1, D, F) 16. In our initially pilot experiments, we tested the method’s feasibility working with properly established reference markers. We analyzed and clustered colon epithelial cells making use of 3 genes encoding for markers linked to either one of several two important cell lineages (i.e. MUC2 for goblet cells and CA1 for enterocytes) or the immature compartment (i.e. LGR5) of the colon epithelium 14, 179. This experiment showed that genes encoding for lineage-specific markers are frequently expressed within a mutually exclusive way, mirroring the expression pattern of corresponding proteins (Supplementary Fig. 5). We then searched for novel gene-expression markers with the diverse cell populations, with a specific concentrate on Erection Inhibitors products putative stem cell markers. We performed a high-throughput screening of 1568 publicly obtainable gene-expression array datasets from human colon epithelia (Supplementary Table 1), applying a bioinformatics strategy developed to determine developmentally regulated genes determined by Boolean implication logic (Supplementary Fig. 6) 20. The search yielded candidate genes whose expression linked with that of other markers previously linked to person colon epithelial cell lineages (Supplementary Fig. 79). Using an iterative method, we screened by SINCE-PCR far more than 230 genes on 8 independent samples of typical human colon epithelium. At each round, genes that have been non-informative (i.e. not differentially expressed in either constructive or unfavorable association with CA1, MUC2 or LGR5) have been removed and replaced with new candidate genes. Thereby, we progressively built a list of 57 TaqMan assays that permitted us to analyze the expression pattern of 53 distinct genes (Supplementary Table 2) with high robustness (Supplementary Fig. ten). This allowed us to visualize and characterize a number of cell populations, making use of both hierarchical clustering (Fig. 1, I) and principal element evaluation (PCA; Fig 1, G ).HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptNat Biotechnol. Author manuscript; accessible in PMC 2012 June 01.Dalerba et al.PageAnalysis on the EpCAMhigh/CD44neg/CD66ahigh population (enriched for “top-of-the-crypt” cells) revealed that this subset, even though transcriptionally heterogeneous, was practically exclusively composed of cells expressing high-levels of genes characteristic of mature enterocytes (e.g. CA1+, CA2+, KRT20+, SLC26A3+, AQP8+, MS4A12+) 14, 213 and led to the discovery of no less than two novel differentially expressed gene expression markers (e.g. CD177, GUCA2B) (Fig. 1, H). To validate the reliability of SINCE-PCR outcomes, we evaluated the distribution of SLC26A3 and CD177 protein expression in tissue sections and we confirmed its preferential expression in the top on the human colonic crypts (Supplementary Fig. 11 and 12). In the present time, it can be.