Single-Cell Transcript Profiles Reveal Multilineage Priming in Early Progenitors Derived from Lgr5+ Intestinal Stem Cells

• Single-cell analyses of intestinal Lgr5+ cells identify two discrete populations
• Both pools express stem-cell genes, but only one activates terminal cell markers
• Multilineage-primed Lgr5+ cells express features of bipotential progenitors
• A suite of informatics tools reveals that these progenitors originate in Lgr5+ ISCs

SummaryLgr5+ intestinal stem cells (ISCs) drive epithelial self-renewal, and their immediate progeny—intestinal bipotential progenitors—produce absorptive and secretory lineages via lateral inhibition. To define features of early transit from the ISC compartment, we used a microfluidics approach to measure selected stem- and lineage-specific transcripts in single Lgr5+ cells. We identified two distinct cell populations, one that expresses known ISC markers and a second, abundant population that simultaneously expresses markers of stem and mature absorptive and secretory cells. Single-molecule mRNA in situ hybridization and immunofluorescence verified expression of lineage-restricted genes in a subset of Lgr5+ cells in vivo. Transcriptional network analysis revealed that one group of Lgr5+ cells arises from the other and displays characteristics expected of bipotential progenitors, including activation of Notch ligand and cell-cycle-inhibitor genes. These findings define the earliest steps in ISC differentiation and reveal multilineage gene priming as a fundamental property of the process.

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