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.

Robust and Precise Wounding and Analysis of Engineered Contractile Tissues.

Fibrous tissue gap closure is a critically important process initiated in response to traumatic injury. Recent three-dimensional (3D) bioengineered models capture cellular details of this process, including wound retraction and closure, but have high failure rates, are labor-intensive, and require considerable expertise to develop and implement with tools that are typically not available in standard wet laboratories. Here, we develop a simple and effective 3D-printed wounding platform to reliably create and puncture arrays of prestressed tissues and monitor subsequent wound dynamics. We demonstrate the ability to create a range of wound sizes in a contractile collagen/fibroblast tissue, within 125 µm of the desired target location, with high degrees of circularity. Wounds exhibit an initial expansion due to tissue prestress, and sufficiently small wounds close completely within 24 h, while larger wounds initially closed much more rapidly, but did not complete the closure process. Simulating the dynamics of tissue retraction with a viscoplastic finite element model indicates a temporary elevation of circumferential stresses around the wound edge. Finally, to determine whether active wounding and retraction of the tissue significantly affect closure rates, we compared active puncture of prestressed tissue with passive removal of a structure that prevents closure, and found that active wounding and retraction substantially accelerated wound closure when compared with the passive case. Taken together, our findings support the role of active tissue mechanics in wound closure arising from an initial retraction of the tissue. More broadly, these findings demonstrate the utility of the platform and methodology developed here in further understanding the mechanobiological basis for wound closure. Impact Statement In vitro models to study wound formation and closure in prestressed tissue are typically challenging to implement. This work provides an easily accessible approach to produce and analyze wounds in arrays of contractile tissues that recapitulate critical features of wound retraction and closure in animal models. The specific modeling and experiments results presented here suggest that mechanobiology effects arising from wound retraction in viscoplastic extracellular matrices could play an important role in driving wound closure.