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.

De novo apical domain formation inside the Drosophila adult midgut epithelium

In the adult Drosophila midgut, basal intestinal stem cells give rise to enteroblasts that integrate into the epithelium as they differentiate into enterocytes. Integrating enteroblasts must generate a new apical domain and break through the septate junctions between neighboring enterocytes, while maintaining barrier function. We observe that enteroblasts form an apical membrane initiation site when they reach the septate junction between the enterocytes. Cadherin clears from the apical surface and an apical space appears above the enteroblast. New septate junctions then form laterally with the enterocytes and the AMIS develops into pre-apical compartment before it has a free apical surface in contact with the gut lumen. Finally, the enterocyte septate junction dissolves and the pre-enterocyte reaches the gut lumen with a fully-formed brush border. The process of enteroblast integration resembles lumen formation in mammalian epithelial cysts, highlighting the similarities between the fly midgut and mammalian epithelia.

Drosophila renal stem cells enhance fitness by delayed remodeling of adult Malpighian tubules

Drosophila renal stem cells (RSCs) contradict the common expectation that stem cells maintain tissue homeostasis. RSCs are abundant, quiescent and confined to the peri-ureter region of the kidney-like Malpighian tubules (MTs). Although derived during pupation like intestinal stem cells, RSCs initially remodel the larval MTs only near the intestinal junction. However, following adult injury to the ureter by xanthine stones, RSCs remodel the damaged region in a similar manner. Thus, RSCs represent stem cells encoding a developmental redesign. The remodeled tubules have a larger luminal diameter and shorter brush border, changes linked to enhanced stone resistance. However, RSC-mediated modifications also raise salt sensitivity and reduce fecundity. Our results suggest that RSCs arose by arresting developmental progenitors to preserve larval physiology until a time in adulthood when it becomes advantageous to complete development by RSC activation.One-Sentence SummaryActivated Drosophila renal stem cells rebuild the adult Malphigian tubules using a less efficient but more stone-resistant design.

MLL1 is required for maintenance of intestinal stem cells

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.