Robust differentiation of human enteroendocrine cells from intestinal stem cells

Enteroendocrine (EE) cells are the most abundant hormone-producing cells in humans and are critical regulators of energy homeostasis and gastrointestinal function. Challenges in converting human intestinal stem cells (ISCs) into functional EE cells, ex vivo, have limited progress in elucidating their role in disease pathogenesis and in harnessing their therapeutic potential. To address this, we employed small molecule targeting of the endocannabinoid receptor signaling pathway, JNK, and FOXO1, known to mediate endodermal development and/or hormone production, together with directed differentiation of human ISCs from the duodenum and rectum. We observed marked induction of EE cell differentiation and gut-derived expression and secretion of SST, 5HT, GIP, CCK, GLP-1 and PYY upon treatment with various combinations of three small molecules: rimonabant, SP600125 and AS1842856. Robust differentiation strategies capable of driving human EE cell differentiation is a critical step towards understanding these essential cells and the development of cell-based therapeutics.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. For all experiments, n = 3 was chosen as the minimal replicate number based on prior studies showing significance with similar sample sizes. We determined this to be sufficient due to relatively low variability between samples in the same group, allowing for clear delineation of differences between groups and statistical significance. The only exception to this is Supplementary Figure 4c, as the G14 group only had an n = 2 due to the loss of a sample. However, repeats of this study yielded similar results and the n = 2 still allowed for statistical significance, so we included it in this study.
Data was not excluded from analysis.
All replication attempts from the same organoid line were successful and showed results similar to those in the paper. Due to the variability seen between human samples, each experiment also was performed on at least three separate human organoid lines. These findings also showed similar results to the results shown in the paper, as evidenced by Figures 2d, 3d, 4c, 5b, 5d, and 5f, 5h, 8d, 8g, and 8i which show data from multiple organoid lines.
All organoid samples were analyzed equally and lines were used for experiments based on availability at the time. Therefore, no randomization was required.
No commonly misidentified cell lines were used.