(652) Electrophysiological approaches to study CFTR function from human induced pluripotent stem cells (iPSC)-derived airway Basal Cells.

Background: Induced pluripotent stem cells (iPSCs), self-renewable and reprogrammed from somatic cells using different transcription factors, are considered as an ideal resource for regenerative medicine to replace diseased or damaged tissues. Airway Basal Cells (iBCs) not only serves as precursors for secretory and multi-ciliated cells but also contribute to maintenance and regeneration of the airway epithelial barrier. Recently, it was reported that induced BCs (iBCs) from human iPSCs recapitulate molecular and functional phenotype of airway basal cells, including self-renewal and multilineage differentiation [1]. iBCs as in vitro model can be especially used in research on diseases impacting the airway, including COVID-19, influenza, asthma, and cystic fibrosis. However, despite these advantage on generation of iBCs, it’s still insufficient to support electrophysiological evidence. Our goal in this study is to define CFTR function in iPSCs-derived iBCs using electrophysiological methods.

Methods: Induced pluripotent stem cell (iPSC) line containing dual reporter, NKX2.1 ^GFP and TP63 ^tdTOMATO were used to generate IBCs (previously published protocol from F. J. Hawkins et al. [1]). Further, iBCs were differentiated into ciliated cells by using ALI culture. Short-circuit current measurements were conducted on the cells cultured in ALI culture using Ussing chamber (by previously described protocol from Li et al. [2]). In order to measure CFTR current using electrophysiological studies, fully differentiated monolayers on filters were dissociated into single cells. Further these cells were fixed onto a collagen coated cover glass using cytospin. Whole-cell patch-clamp recordings were performed according to a previous published protocol from Yunji et al. [3].

Results: We generated proximal airway iBCs from iPSCs with the dual fluorescent reporter system of GFP (marks lung progenitors) and tdTomato (marks subsequent airway progenitor) [Fig 1, left]. These cells on ALI culture demonstrated CFTR function using short-circuit current measurements (Fig 1, right]. In addition, we also measured CFTR dependent currents in iPSC-airway BCs using whole cell patch clamp recording [Fig 2].

Conclusions: We identified CFTR function using electrophysiological experiments using airway iBCs in vitro from iPSCs. Therefore, our study helps advance the field of regenerative medicine, benefiting airway and lung diseases. This may ultimately allow for the development of individual, disease-specific airway basal stem cells, leading to drug development and a platform in which targeted drug approaches can be tested.

Acknowledgements:

References: [1] Hawkins FJ, et al. Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells. Cell Stem Cell. 2021 Jan 7;28(1):79-95.e8. doi: 10.1016/j.stem.2020.09.017. Epub 2020 Oct 23. PMID: 33098807; PMCID: PMC7796997.Whitsett, J.A., Airway epithelial differentiation and mucociliary clearance. Annals of the American Thoracic Society, 2018. 15(Supplement 3): p. S143-S148.

[2] Li C, et al. Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions. J Exp Med. 2005 Oct 3;202(7):975-86. doi: 10.1084/jem.20050421. PMID: 16203867; PMCID: PMC2213164.

[3] Huang Y, et al. Elexacaftor/Tezacaftor/Ivacaftor Improved Clinical Outcomes in a Patient with N1303K-CFTR Based on In Vitro Experimental Evidence. Am J Respir Crit Care Med. 2021 Nov 15;204(10):1231-1235. doi: 10.1164/rccm.202101-0090LE. PMID: 34379998; PMCID: PMC8759307.