Airway and Lung Organoids from Human-Induced Pluripotent Stem Cells Can Be Used to Assess CFTR Conductance.

Airway and lung organoids derived from human-induced pluripotent stem cells (hiPSCs) are current models for personalized drug screening, cell-cell interaction studies, and lung disease research. We analyzed the existing differentiation protocols and identified the optimal conditions for obtaining organoids. In this article, we describe a step-by-step protocol for differentiating hiPSCs into airway and lung organoids. We obtained airway and lung organoids from a healthy donor and from five donors with cystic fibrosis. Analysis of the cellular composition of airway and lung organoids showed that airway organoids contain proximal lung epithelial cells, while lung organoids contain both proximal and distal lung epithelial cells. Forskolin-induced swelling of organoids derived from a healthy donor showed that lung organoids, as well as airway organoids, contain functional epithelial cells and swell after 24 h exposure to forskolin, which makes it a suitable model for analyzing the cystic fibrosis transmembrane conductance regulator (CFTR) channel conductance in vitro. Thus, our results demonstrate the feasibility of generating and characterizing airway and lung organoids from hiPSCs, which can be used for a variety of future applications.

Health Characteristics of Patients with Cystic Fibrosis whose Genotype Includes a Variant of the Nucleotide Sequence c.3140-16T>A and Functional Analysis of this Variant.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease, associated with pathogenic variants in the CFTR gene. The splicing variant c.3140-16T>A (3272-16T>A) has been described previously and, according to the Russian CF Patients Registry, occurs with a frequency of 0.34%. The phenotypic features of CF patients with the c.3140-16T>A variant were compared with those of patients with the genotype F508del/F508del. Patients with the allele c.3140-16T>A had higher average age and age at diagnosis, and the allele was present in a greater proportion of adults. Patients carrying the c.3140-16T>A allele were characterised by better physical development indicators, both in adults and in children, had preserved pancreatic function, as well as the absence of a number of complications, and required pancreatic enzyme replacement therapy less often than patients with the F508del/F508del genotype. Sweat test values also were lower in patients with the c.3140-16T>A genotype. According to the results of clinical and laboratory studies, the phenotype of patients with the genetic variant c.3140-16T>A can be considered "mild". Functional CFTR protein activity in the presence of c.3140-16T>A was evaluated using intestinal current measurements (ICM) and the forskolin-induced swelling assay on organoids obtained from patients' rectal biopsies. c.3140-16T>A had high residual CFTR channel activity and was amenable to effective pharmacological correction with thea VX-770 potentiator. To evaluate the effect of the variant on CFTR pre-mRNA splicing we performed a minigene assay, as well as RT-PCR analysis of RNA isolated from the nasal epithelium and rectal biopsy of patients. We showed that the c.3140-16T>A variant creates a novel acceptor AG dinucleotide within CFTR intron 19, resulting in a 14-nucleotide extension of exon 20. This frameshift produces a premature termination codon and triggers mRNA degradation by the nonsense-mediated decay (NMD) mechanism. Moreover, we observed that the c.3140-16T>A allele could produce a residual amount of normally spliced transcript, thus explaining the patient's mild phenotype.