Single-molecule transcript counting of stem-cell markers in the mouse intestine

Tissue stem cells are long lived cells that can give rise to all the different cell types in a tissue. These cells are hard to study as they generally make up a small minority and are often morphologically indistinguishable from their differentiated progenies. A key goal in stem cell research is to identify ‘stem cell markers’, the small set of genes that are specifically expressed only in the tissue stem cells and not in their differentiated progenies.

HFSP Long-Term Fellow Shalev Itzkovitz and colleagues
authored on Fri, 16 December 2011

Identification of the locations and molecular identities of tissue stem cells has been impeded by lack of sensitive methods to study gene expression in single cells in intact tissue. Traditional methods such as immunohistochemistry and RNA in-situ hybridization are very limited in their sensitivity and specificity. We developed a method that enables the integer number of transcripts of any gene in single cells to be counted in intact mouse tissue We applied this technique to study the co-expression of a large panel of putative intestinal stem cell markers, revealing the precise location and molecular identities of stem cells in the mouse intestine.

Figure:  Three-color single molecule fluorescence in-situ hybridization of intestinal stem cell markers facilitates a quantitative mapping of their co-expression in intact mouse tissue.

The mouse small intestine constitutes one of the best studied model systems for adult stem cell biology. While it is widely accepted that the intestinal stem cells reside at the bottom of intestinal crypts – deep structures that protrude into the underlying connective tissue, the precise location and molecular identity of crypt stem cells remains highly debatable. Different genes were suggested to specifically mark crypt stem cells that reside either at the very bottom of the crypts or rather higher up along the crypt axis, but comprehensively studying their co-expression has not been possible with existing techniques. To address this challenge we applied a method that enables the detection of single mRNA molecules as bright spots under a fluorescence microscope. The method is based on the specific accumulation of short fluorescently labeled oligo-nucleotide sequences that are designed to be complementary to successive regions of the gene of interest. We used this method to simultaneously measure different genes and wrote software to automatically detect the spots and assign them to individual cells. By performing these measurements on hundreds of intestinal crypts for a comprehensive panel that includes all previously suggested stem cell markers, we created a spatial expression map revealing the precise location and co-expression pattern of the proposed stem cell markers.

We found that most of the previously suggested stem cell markers are in fact much broader in their expression range than previously realized, and that they are expressed not only in the stem cells but also in the differentiated progenies. Strikingly, all of the markers were co-expressed in a small group of cells residing at the very bottom of the crypts, which specifically expressed a single gene – Lgr5. Thus, it seems that all the genes in our panel, including those that were assumed to mark mutually exclusive stem cells, mark a single common stem cell population, the Lgr5-cells residing at the crypt bases. Our single molecule transcript counting technique can be generically applied to study the molecular identity of tissue stem cells and to quantitatively explore gene expression signatures at the single cell level in other tissues and in tumors.



Single molecule transcript counting of stem cell markers in the mouse intestine, Itzkovitz S, Lyubimova A, Blat I, Maynard M, van Es J, Lees J, Jacks T, Clevers H, van Oudenaarden A, Nature Cell Biology, 2011, doi: 10.1038/ncb2384

Pubmed link