An organoid screening framework to engineer intestinal epithelial composition

Barrier epithelia are essential to organismal homeostasis, and changes in their cellular composition are observed in human diseases. Within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Nevertheless, we lack suitable approaches to identify biological targets and small molecules to modulate them. Here, we develop an extendable framework utilizing a physiologically-inspired organoid model to identify unknown, druggable regulators of intestinal stem cell differentiation that translate to intended manipulations in vivo. Specifically, we miniaturize and adapt an organoid model of Paneth cell differentiation to enable multiplexed phenotypic screening on the scale of thousands of samples, and employ longitudinal single-cell RNA-sequencing to understand hit biology. Strikingly, we identify that inhibitors of the nuclear exporter XPO1 modulate stem cell fate commitment, significantly increasing the abundance of Paneth cells independent of known differentiation cues. Our framework elucidates small molecules which modulate tissue stem cell biology and their underlying targets without the need for a priori knowledge of in vivo pathway biology. Overall design: Single-cell RNA-sequencing experiments on Seq-Well S^3 platform of differentiating intestinal organoids (ENR+CD +/- KPT-330). We performed a longitudinal comparison between untreated and KPT-330-treated or-ganoids over a 6-day differentiation. We collected 17 samples at following timepoints: 6 hours (0.25 days), and 1, 2, 3, 4, or 6 days. Each sample consists of single cells from >1,000 organoids from pre-differentiation ENR+CV organoids and both ENR+CD and ENR+CD + KPT-330 (160 nM) conditions. For time points beyond 2 days, media was refreshed every other day. The resulting dataset consists of 19,877 cells. Unique molecular identifier (UMI), percent mitochondrial, and detected gene distributions are similar across samples, within acceptable quality bounds (genes > 500, UMI < 30,000, percent mitochondrial < 35). ENR+CV, ENR+CD media formulations and organoid culture protocols are per Mead, et al. BMC Biology 2018.