Single-Cell mRNA Sequencing Reveals Rare Intestinal Cell Types

Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating sub-populations of cells are based on mRNA or protein expression of only few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumor cells is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbor stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone producing intestinal cells. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. Reassuringly, RaceID confirmed the existence of known enteroendocrine lineages, and moreover, discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID by this proof-of-principle experiment we then apply the algorithm to ex vivo isolated LGR5 positive cells and their direct progeny and demonstrate homogeneity of the stem cell pool. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs. Overall design: Small intestinal crypts were isolated from a single wild-type C57BL/6 mouse, a Reg4-dsRed-knock-in mouse and an Lgr5-GFP-DTR mouse. The crypts were propagated and expanded in culture as organoids. For each experiment, multiple organoids were harvested and dissociated into single cells. Each experiment was done twice, using different passage of the same organoid culture. We also included a pool-and-split control for 96 Reg4-dsRed positive intetsinal cells and a control library with 5 mouse embryonic stem cells (wells 1-5), 5 mouse embryonic fibroblasts (wells 6-10), 75 random organoid cells (wells 11-85), 5 wells without primer and without template (wells 86 and 93-96), and five wells with primer and without template (wells 87-92). We also sequenced two 96 well plates of Lgr5-EGFP positive single cells isolated ex vivo, and Lgr5 progeny collected after five days of lineage tracing. Label induction was performed using an Lgr5-Cre reporter mouse expressing YFP from Rosa26 promoter with a loxP flanked transcriptional road block in between. Five 96 well plates of YFP positive were sequenced. Sample number four also contains also unrelated samples (single cell barcode 49-96), which should be discarded.

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Grün D, Lyubimova A, Kester L, Wiebrands K, Basak O, Sasaki N, Clevers H, van Oudenaarden A