Electrophysiological approaches to study cystic fibrosis transmembrane conductance regulator function from human induced pluripotent stem cell-derived airway basal cells

ABSTRACT

Background: Induced pluripotent stem cells (iPSCs), self-renewable and reprogrammed from somatic cells using different transcription factors, are considered an ideal resource for regenerative medicine to replace diseased or damaged tissues. Airway basal cells not only serve as precursors for secretory and multiciliated cells, but also contribute to maintenance and regeneration of the airway epithelial barrier. Recently, it was reported that induced basal cells (iBCs) from human iPSCs recapitulate molecular and functional features from human iPSCs of airway basal cells, including selfrenewal and multilineage differentiation [1]. iBCs as in vitro model can be used in research on diseases affecting the airway, including COVID-19, influenza, asthma, and cystic fibrosis, although despite these advantages in generating iBCs, this is insufficient to support electrophysiological evidence. Our goal in this study is to define CFTR function in iPSCderived iBCs using electrophysiological methods. Method(s) An iPSC line containing dual reporter NKX2.1GFP and TP63tdTOMATO were used to generate iBCs according to a previously published protocol [1]). iBCs were differentiated into ciliated cells using air-liquid interface (ALI) culture. Short-circuit current measurements were taken on the cells cultured in ALI culture using an Ussing chamber using a previously described protocol [2]). To measure CFTR current using electrophysiological studies, fully differentiated monolayers on filters were dissociated into single cells, which were fixed onto a collagencoated cover glass using cytospin. Whole-cell patch-clamp recordingswere performed according to a previous published protocol [3]. Result(s) We generated proximal airway iBCs from iPSCs with the dualfluorescent reporter system of green fluorescent protein (marks lung progenitors) and tdTomato (marks subsequent airway progenitor) (Figure 1a). These cells on ALI culture demonstrated CFTR function using short-circuit current measurements (Figure 1b). We also measured CFTRdependent currents in iPSC-airway basal cells using whole-cell patchclamp recording (Figure 2). Conclusion(s) We identified CFTR function in 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 allowfor development of individual, diseasespecific airway basal stem cells, leading to drug development and a platform on which targeted drug approaches can be tested. Copyright © 2022, European Cystic Fibrosis Society. All rights reserved