Human pluripotent stem cell modeling of alveolar type 2 cell dysfunction caused by ABCA3 mutations
Yuliang L. Sun, … , Jennifer A. Wambach, Darrell N. Kotton
Yuliang L. Sun, … , Jennifer A. Wambach, Darrell N. Kotton
Published January 16, 2024
Citation Information: J Clin Invest. 2024;134(2):e164274. https://doi.org/10.1172/JCI164274.
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Research Article Cell biology Stem cells

Human pluripotent stem cell modeling of alveolar type 2 cell dysfunction caused by ABCA3 mutations

Abstract

Mutations in ATP-binding cassette A3 (ABCA3), a phospholipid transporter critical for surfactant homeostasis in pulmonary alveolar type II epithelial cells (AEC2s), are the most common genetic causes of childhood interstitial lung disease (chILD). Treatments for patients with pathological variants of ABCA3 mutations are limited, in part due to a lack of understanding of disease pathogenesis resulting from an inability to access primary AEC2s from affected children. Here, we report the generation of AEC2s from affected patient induced pluripotent stem cells (iPSCs) carrying homozygous versions of multiple ABCA3 mutations. We generated syngeneic CRISPR/Cas9 gene-corrected and uncorrected iPSCs and ABCA3-mutant knockin ABCA3:GFP fusion reporter lines for in vitro disease modeling. We observed an expected decreased capacity for surfactant secretion in ABCA3-mutant iPSC-derived AEC2s (iAEC2s), but we also found an unexpected epithelial-intrinsic aberrant phenotype in mutant iAEC2s, presenting as diminished progenitor potential, increased NFκB signaling, and the production of pro-inflammatory cytokines. The ABCA3:GFP fusion reporter permitted mutant-specific, quantifiable characterization of lamellar body size and ABCA3 protein trafficking, functional features that are perturbed depending on ABCA3 mutation type. Our disease model provides a platform for understanding ABCA3 mutation–mediated mechanisms of alveolar epithelial cell dysfunction that may trigger chILD pathogenesis.

Authors

Yuliang L. Sun, Erin E. Hennessey, Hillary Heins, Ping Yang, Carlos Villacorta-Martin, Julian Kwan, Krithi Gopalan, Marianne James, Andrew Emili, F. Sessions Cole, Jennifer A. Wambach, Darrell N. Kotton

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Figure 6

iAEC2s expressing E690K and W308R ABCA3 mutant proteins have increased canonical NFκB signaling activity and variable proliferative capacity.

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iAEC2s expressing E690K and W308R ABCA3 mutant proteins have increased c...
(A) Percentages of day 75 patient iAEC2s incorporating EdU after 24 hour incubation. Biological replicates n = 3. *P ≤ 0.05, 2-tailed Student’s t test. (B) CFE reported as percentages of total spheres divided by input cells, of indicated patient iAEC2s on day 57. Biological replicates (n = 3), separated at day 0. Bars represent mean ± SE. ***P ≤ 0.001, ns, not significant, 2-tailed Student’s t test. (C) Lentiviral vector containing 2 gene expression cassettes, one allowing for constitutive expression of GFP for purifying transduced cells, and the second expressing luciferase under the regulatory control of a minimal promoter (TAp) adjacent to p50/p65 heterodimer consensus binding sequence (NFκB). This vector (detailed in Alysandratos et al., 2021 [36]) was used to transduce each indicated iAEC2 sample. LTR, lentiviral long terminal repeat; RRE, Rev responsive element; CPPT, central polypurine tract; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; UBCp, ubiquitin protein-C promoter. (D) Relative NFκB pathway activity of lentiviral-transduced (GFP sorted) day 286 W308R and cW308 iAEC2s and day 158 E690K and cE690 iAEC2s measured by levels of D-luciferin luminescence. Replicates (n = 3), W308R and cW308 separated at day 148, E690K and cE690 separated at day 0. Graphs show mean ± SE. ***P ≤ 0.001, ****P ≤ 0.0001, 2-tailed Student’s t test. (E) Levels of indicated cytokines released in the culture supernatants of 2D monolayer cultured patient iAEC2s. Biological replicates (n = 3), separated at day 0. Graphs show mean ± SE. *P ≤ 0.05, **P ≤ 0.01, 2-tailed Student’s t test.