Human pluripotent stem cell modeling of alveolar type 2 cell dysfunction caused by ABCA3 mutations
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
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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Research Article Cell biology

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 1

Generation and directed differentiation of patient-specific ABCA3 mutant and syngeneic gene-corrected iPSC lines produces SFTPCtdTomato-expressing iAEC2s.

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Generation and directed differentiation of patient-specific ABCA3 mutant...
(A) H&E staining of lung tissue from each patient of indicated ABCA3 genotype showing extensive alveolar remodeling, type II cell hyperplasia, interstitial thickening, lymphoid aggregates, and neutrophilic infiltrates (Left), and intraalveolar macrophages (Right). Scale bars: 100 μm. AW, bronchiolar airway. See also Supplemental Figure 1. (B) TEM images showing irregular, small lamellar bodies (LBs, black arrow heads) with some abnormal LBs containing dense bodies (white arrow heads) in homozygous E690K ABCA3 patient lung explant. Scale bar: 1 μm. (C) TEM image showing AEC2 containing normal lamellar bodies with loose concentric whorled membranes (black arrow heads) in a patient with pediatric pulmonary hypertension. Scale bar: 600 nm. (D) ABCA3 mutant patient-specific iPSCs reprogrammed from dermal fibroblasts. Using TALENS gene-editing tools, patient-iPSCs are then targeted with SFTPCtdTomato knockin reporter, followed by biallelic gene-correction using CRISPR/Cas9 induced DNA strand break and homologous recombination (HR) to generate syngeneic control iPSC lines. (E) Directed differentiation timeline of all patient iPSC lines, biological replicates separated at day 0 (n = 3). iPSCs were differentiated as indicated into distal lung epithelium using the protocol described in Jacob et al. (32, 33) with additional CPM sorting followed by CHIR modulation. DE, definitive endoderm; AFE, anterior foregut endoderm. (F) Fluorescent images of day 22 to 23 live alveolospheres derived from patient iPSC lines following distal lung differentiation, showing detection of SFTPCtdTomato reporter (red). G) Representative flow cytometry analyses of day 43 to 44 cells showing similar frequency of SFTPCtdTomato-expressing cells across all lines following CHIR modulation. (H) Frequency of SFTPCtdTomato-expressing cells from day 29–30 to day 43–44 after CHIR modulation. Biological replicates (n = 3). Graphs show mean ± SE. See also Supplemental Figure 2.