High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids.

Sunny Xia,Zoltán Bozóky,Amy L Pitstick,Christine E Bear,Christopher N Mayhew,Onofrio Laselva,Michelle Di Paola,Chong Jiang,Nicola L Jones,Saumel Ahmadi,Daniela Rotin,Jia Xin Jiang

doi:10.3390/cells10123419

Abstract

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.

Highlights

There has been remarkable progress made in the use of patient tissue-derived primary organoids for the in vitro modeling of Cystic Fibrosis (CF) pathogenesis and testing of therapies targeting mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) [1,2,3,4,5]
Human Intestinal Organoids (HIO) were differentiated from Embryonic Stem Cells (H1 ESC line) and Induced Pluripotent Stem Cells (iPSCs) lines (non-CF1 and non-CF2 (n = 2) and CF1 (n = 1), see Table 1) using previously established differentiation protocols (Figure 1a) [13,14]
Immunostaining confirmed that the CF1 and non-CF1 HIOs expressed CDX2, and Zona Occludin-1 (ZO-1), which are characteristic of intestinal epithelial cells (Figure 1b)

Summary

Introduction

There has been remarkable progress made in the use of patient tissue-derived primary organoids for the in vitro modeling of Cystic Fibrosis (CF) pathogenesis and testing of therapies targeting mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) [1,2,3,4,5]. CFTR mutations lead to the loss of CFTR expression and/or function as a cyclic AMP-regulated anion channel at the cell surface. CFTR mutations that are associated with CF reduce forskolin-dependent swelling while enabling the ranking of therapeutic interventions targeting defective CFTR expression and function [2,6]. Organoid swelling has been shown to correlate with multiple clinical biomarkers of CF, such as high sweat chloride concentration and compromised lung function measured as FEV1 [6].

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Methods

Results