Modeling ion channel function with murine intestinal 3D stem cell cultures

Abstract

Cystic Fibrosis (CF) is a persistent and progressive genetic disease, manifesting as frequent lung, gastrointestinal, and pancreatic inflammation and chronic infection. The majority of patients with CF have a mutation caused by a single amino acid deletion of phenylalanine (F508del) in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein; this causes an unstable protein to form, with subsequent failure of membrane insertion or lack of function and early degradation. A lack of functional CFTR leads to the production of viscous mucus that is hard to clear from the lungs and gastrointestinal tract, providing a medium for bacterial growth and infection. While treatments for Cystic Fibrosis have made advancements, the median survival age for patients with CF is still 40.In order to utilize a mouse model for human CF disease, we developed a protocol culturing mouse intestinal crypts into three dimensional organoids. Mature organoids are subsequently dosed with Forskolin, an adenylyl cyclase activator, to elicit ion and fluid transport into the lumen of the organoid, via stimulation of CFTR channels. The unidirectional flow of ions and fluid resulted in the swelling of the organoids as fluid is moved into the lumen. We were able to measure the change in volume and surface area as well as movement of ions across the epithelium using a modified two electrode voltage clamp technique. Forskolin induced swelling was inhibited by the presence of the CFTR inhibitor cftrinh172, consistent with the role of CFTR ion fluid transport. We will present data using intestinal organoids from mice lacking transport proteins, such as LMTK2.Support or Funding InformationNIH Heart Lung and Blood