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Single-Cell mRNA Sequencing Enables ID of Rare Intestinal Cells

NEW YORK (GenomeWeb) – Using a single-cell messenger RNA sequencing approach, a team of researchers from the Netherlands has uncovered rare intestinal cell types.

Hubrecht Institute's Alexander van Oudenaarden and his colleagues sequenced the transcriptomes of hundreds of cells from mouse intestinal organoids and developed a computational method to identify rare cell types within such a complex population. Using their approach, they identified Reg4 as a novel marker of enteroendocrine cells, a rare population of hormone-producing intestinal cells, as they reported today in Nature.

"We believe that single-cell mRNA sequencing in combination with the RaceID algorithm," which they developed, "is a powerful tool to unravel heterogeneity of rare cell types in both healthy and diseased organs," van Oudenaarden and his colleagues wrote in their paper.

The researchers sequenced 238 randomly selected cells from intestinal organoids, small epithelial structures that harbor all major intestinal cell types, to generate more than 3,000 transcripts from each. More than 3,700 genes, they noted, had more than five transcripts in each cell.

By clustering these single-cell transcriptomes, the researchers found six major cell types. But to find rare cells, they focused on outliers that couldn't be explained by either technical or biological gene expression noise. They then grouped these outliers themselves based on transcriptome correlation.

Both differential gene analysis and the RaceID algorithm that the researchers developed confirmed the presence of rare cells in these groups, including goblet, tuft, Paneth, and enteroendocrine cells. RaceID further uncovered three secretory precursor clusters that co-expressed Neurog3 with Krt7, Pax4, or Ang4.

As a whole, the researchers noted that enteroendocrine cells secrete some 10 different hormones, though each cell only secretes a subset of those hormones. The researchers profiled the heterogeneity of and identified markers for enteroendocrine cells, finding that the top scoring genes included the proteinase Pappa2 and Reg4, a relatively unknown gene. Using FISH in a mouse intestine, the researchers confirmed Reg4 was an enteroendocrine cell marker.

They then noted that Reg4-expressing cells could be divvied by RaceID into three groups, including known cell types as well as novel ones that they confirmed in vivo.

The researchers then applied their RaceID approach to examine stem cells marked by Lgfr5 expression, as it's unclear whether intestinal stem cells are heterogeneous or not.

Using organoids with an Lgfr5-green fluorescent protein reporter gene, they purified stem cells and used RaceID to classify them. This approach classified the cells into a single homogenous cluster, though with a few outliers. A complementary approach likewise found them to be largely homogenous.

Together, the researchers noted that this indicates that Lgfr5-positive cells are homogenous, though they are mixed with rare populations of Paneth and enteroendocrine cells.

"[W]e demonstrated here the ability of RaceID to correctly classify different cell types in a complex mixture and reveal heterogeneity," they said.