Three-Dimensional Airway Spheroids and Organoids for Cystic Fibrosis Research
Abstract
:1. Introduction
1.1. Novel Therapies for CF
1.2. In Vivo and In Vitro Models for CF
1.3. Spheroids and Organoids for CF
2. Airway Spheroids from Primary Airway Epithelial Cells
2.1. Spheroids from Differentiated Nasal Epithelial Cells
2.2. Spheroids from Airway Stem/Progenitor Cells
3. Airway Organoids from iPSCs
4. Concluding Remarks
Author Contributions
Funding
Conflicts of Interest
References
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Study | Spheroid Model | Morphology and Antigen Expression | Function | Pharmacological Treatment |
---|---|---|---|---|
Pedersen et al., 1999 [92] | CF (n = 9) and non-CF (n = 17) subjects. Free-floating spheroids from nasal polyp-epithelial sheets. | “Lumen-out” configuration. The apical ciliated membrane facing the bath, and the basolateral cell membrane pointing toward a fluid-filled lumen. | Transepithelial PD measurements compatible with the presence of an amiloride-sensitive Na+ absorption and ATP-sensitive Cl- channel in the apical membrane. CF spheroids PD was not changed by the increase in cAMP. | None. |
Pedersen et al., 1999 [93] | CF (n = 7) and non-CF (n = 15) subjects. Free-floating spheroids from nasal polyp-epithelial sheets. | “Lumen-out” configuration. The apical ciliated membrane facing the bath, and the basolateral cell membrane pointing toward a fluid-filled lumen. | Fluid absorption rates were equal in non-CF and CF spheroids. Amiloride inhibited fluid absorption to a lower residual level in non-CF than in CF spheroids. | None. |
Pedersen et al., 2007 [94] | CF (n = 4) and non-CF (n = 5) subjects. | “Lumen-out” configuration. The apical ciliated membrane facing the bath, and the basolateral cell membrane pointing toward a fluid-filled lumen. | Hyperosmotic treatment caused an increase in epithelial water permeability without changing fluid absorption rates. | None. |
Guimbellot et al., 2017 [68] | CF (n = 3) and non-CF (n = 9) subjects. CF genotypes: F508del/F508del; F508del/I618T; G551D/F508del | “Lumen-out” configuration. The apical ciliated membrane facing the bath, and the basolateral cell membrane pointing toward a fluid-filled lumen. CFTR expression at the level of apical region. | Shrinking of non-CF spheroids upon the increase in cAMP levels. CF spheroids showed diminished volume reduction following CFTR activation. | Lumacaftor–Ivacaftor treatment partially restored cross-sectional area reduction of CF nanospheroids. |
Brewington et al., 2018 [89] | CF (n = 19) and non-CF (n = 6) subjects. CF genotypes: F508del/F508del (n = 9); others with at least one non-F508del mutation | “Lumen-in” configuration. Cilia on the luminal surface. Positive for E-cadherin, luminal F actin and alpha tubulin, and the mucin MUC5AC. | Non-CF spheroids swelled upon CFTR stimulation. NEC spheroids from F508del homozygotes shrank following CFTR stimulation. | F508del homozygous spheroids swelled when pre-treated with Ivacaftor and Lumacaftor, or incubated at 27 °C. |
McCarthy et al., 2018 [95] | Nasal curettage. One CF patient heterozygous for Ser1159Pro and F508del. | “Lumen-in” configuration. | Nasospheroids did not swell in the FIS assay. | Nasospheroids swelled in response to Lumacaftor–Ivacaftor. Following ex vivo studies, the patients commenced in vivo therapy. |
Sachs et al., 2019 [70] | Lung biospies and BAL specimens (“bronchospheroids”). Non-CF and CF subjects. CF genotypes: F508el/F508del (n = 3), F508del/G542X (n = 1), R334W/R334W (n = 1). | “Lumen-in” configuration. Cilia on the luminal surface. Positive for basal marker keratin-5 (KRT5), club cell marker secretoglobin family 1A member 1 (SCGB1A1), cilia marker acetylated a-tubulin, or secretory cell marker mucin 5AC (MUC5AC). | In non-CF spheroids, spheroids swelled upon forskolin and Eact stimulation. | Forskolin-induced swelling was reduced in CF compared to wild-type spheroids, and correlated with the severity of the tested CFTR genotypes. It could be augmented with Lumacaftor and Ivacaftor. Eact induced a swelling similar to that induced by forskolin. |
Liu et al., 2020 [90] | Nasal brushing. CF (n = 36) and non-CF (n = 12) subjects. CF genotypes: F508del/F508del (n = 5); others with at least one minimal or residual function mutation | “Lumen-in” configuration. Cilia on the luminal surface. Positive for MUC5AC and MUC5B, ZO-1, CFTR, and FOXI1. | The baseline luminal ratio and the FIS assay distinguish between non-CF and CF spheroids and also between CF with different genotypes. | None. |
Amatngalim et al., 2021 [96] | Nasal brushing. CF (n = 22) and CF (n = 22) subjects (F508del/F508del). | 3D spheroids with a “lumen-in configuration” derived from 2D differentiated ALI-NEC cultures. β-tubulin IV+ cilia and MUC5AC+ secretory cells inside of the spheroid and p63+ and KRT5+ basal cells. | FIS measured in non-CF spheroids was significantly higher compared to CF spheroids, while Eact induced a more significant swelling in CF spheroids compared to non-CF ones. | No response of CF organoids to Ivacaftor–Lumacaftor while a detectable swelling was obtained when cells were grown at ALI in the presence of neuregulin and IL-1β. Under these conditions, a high increase in FIS was obtained with Ivacaftor–Tezacaftor–Elexacaftor treatment. |
Study | Organoid Model | Morphology and Antigen Expression | Function | Pharmacological Treatment |
---|---|---|---|---|
McCauley et al., [111] | Proximalized airway organoids. One non-CF subject and two CF patients homozygous for F508del. | “Lumen-in” configuration. | Little, if any, swelling was observed in either CF lines after exposure to forskolin as compared to the wild-type line. | The gene-corrected F508del/WT organoids significantly swelled in response to forskolin treatment. |
Berical et al., [115] | Proximalized airway organoids. Three non-CF subjects and five CF patients (1 homozygous for W1282X, three homozygous for F508del, one homozygous for G551D). | “Lumen-in” configuration. CFTR expression at similar levels to primary HBE cultures | A small but statistically significant basal swelling in G551D organoids, but no detectable basal FIS in F508del and W1282X organoids. | FIS increased in G551D organoids after treatment with Ivacaftor. Treatment of F508del organoids with the first-generation correctors (Lumacaftor, Tezacaftor) had a small effect on FIS, while a robust increase was obtained with the triple combination Ivacaftor–Tezacaftor–Elexacaftor. In W1282X organoids, combinatorial treatment with G418, SMG1i, and Ivacaftor–Tezacaftor–Elexacaftor led to a significant increase in FIS. |
Ngan et al., 2021 [117] | Fetal lung-derived organoids. Non-CF hiPSC lines. | “Lumen-in” configuration. By immunofluorescence: expression of basal cell marker KRT5, ciliated cell marker FOXJ1, luminal epithelial cell marker KRT8, secretory cell marker MUC16. By qPCR: expression of ΔNP63 and KRT14, acetylated a-tubulin, cytokeratin-8/18 (KRT8/18), and secretoglobulin 1A1 (SCGB1A1) and mucin 5ac (MUC5AC). CFTR co-expressed at the lumen side with ZO-1. | The FLiPR analysis on spheroids 2–3 days after seeding on collagen-coated plates found an increase in fluorescence activity indicative of CFTR function upon forskolin-induction that was inhibited with CFTR inhibitor-172. Significant swelling was found after 24 h of forskolin stimulation. | None. |
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Laselva, O.; Conese, M. Three-Dimensional Airway Spheroids and Organoids for Cystic Fibrosis Research. J. Respir. 2021, 1, 229-247. https://doi.org/10.3390/jor1040022
Laselva O, Conese M. Three-Dimensional Airway Spheroids and Organoids for Cystic Fibrosis Research. Journal of Respiration. 2021; 1(4):229-247. https://doi.org/10.3390/jor1040022
Chicago/Turabian StyleLaselva, Onofrio, and Massimo Conese. 2021. "Three-Dimensional Airway Spheroids and Organoids for Cystic Fibrosis Research" Journal of Respiration 1, no. 4: 229-247. https://doi.org/10.3390/jor1040022