Human induced pluripotent stem cells for in vitro modeling of impaired mucociliary clearance in cystic fibrosis lung disease

Human induced pluripotent stem cells for in vitro modeling of impaired mucociliary clearance in cystic fibrosis lung disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Human induced pluripotent stem cells for in vitro modeling of impaired mucociliary clearance in cystic fibrosis lung disease Mark-Christian Jaboreck, Anita Balázs, Janina Zöllner, Nicole Cleve, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7184232/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 13 You are reading this latest preprint version Abstract Severely impaired mucociliary airway function is the primary pathomechanism in Cystic Fibrosis (CF) lung disease. Despite significant advances in CF therapy, there is still a critical need for alternative, individualized treatment options, especially for patients with untreatable CFTR mutations. Although intestinal organoids and primary airway cells are widely used as preclinical models of CF, both systems exhibit limitations with regard to the proper modelling of mucociliary clearance or the availability of sufficient cell quantities. Patient-specific human induced pluripotent stem cells (hiPSCs) are a promising alternative due to their unlimited expansion potential and capacity to differentiate into airway epithelia. However, cellular inhomogeneities in iPSC-derived airway cultures complicated conventional assays that determine CFTR function such as Ussing chamber measurements, and a comprehensive demonstration of CF pathophysiology in hiPSC-derived airway models has been largely lacking. This study provides comprehensive data demonstrating very similar gene expression, (ultra)structure and CFTR function in CF iPSC-derived airway (iALI) and primary airway (pALI) cultures. Addressing current limitations, we have implemented a sensitive, straightforward, and automatable ciliary beat frequency (CBF) assay, which is largely unaffected by inhomogeneities and directly reflects disturbed mucus viscosity and mucociliary transport in CF lung disease. Electron microscopy images confirmed the disease phenotype showing a highly dense and dehydrated mucus layer on top of CF iALI cultures. Furthermore, established CFTR modulator drugs partially rescued the disease phenotype in CF iALI cultures, which validated the utility of iALI cultures as a scalable, patient-specific platform for CF research and personalized drug development. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cystic fibrosis (CF) is a rare recessive genetic disorder that affects approximately 100,000 people worldwide. It is caused by mutations of the Cystic fibrosis transmembrane conductance regulator (CFTR) gene [ 1 – 4 ], which encodes a cAMPregulated chloride and bicarbonate channel protein. Mutations in the CFTR gene have been shown to affect the transepithelial ion transport in multiple organs, with CF lung disease being the primary cause of morbidity and mortality [ 5 – 14 ]. To date, over 700 CF-causing mutations of the CFTR gene have been identified [ 15 – 17 ]. In healthy individuals, the respiratory epithelium plays a critical role in the host defense against pulmonary infection. In the airways, the secreting cell types, particularly goblet cells, produce a protective layer of mucus that covers the epithelial surface and traps inhaled pathogens. Subsequent to this initial defensive barrier, mucociliary clearance (MCC) is initiated through ciliary beating, which effectively removes mucus and pathogens from the respiratory system [ 18 ]. In CF, CFTR mutations result in reduced chloride secretion and concurrent hyperabsorption of sodium by the CFTR-regulated epithelial sodium channel (ENaC). The reduction in apical ion secretion causes the dehydration of airway mucus and the subsequent increase in mucus viscosity [ 19 – 24 ]. This increased viscosity impairs ciliary movement and MCC, consequently leading to chronic airway infection, inflammation, and ultimately progressive loss of lung function and lung failure [ 11 , 25 – 28 ]. The development of highly effective small-molecule drugs, known as CFTR modulators, to restore CFTR function in CF patients has profoundly changed the clinical landscape [ 29 , 30 ]. The most prominent classes of CFTR modulators include corrector drugs, which correct the folding and trafficking of the mutant CFTR protein, and potentiator drugs, which enhance the activity of the CFTR protein [ 30 – 34 ]. Notably, the triple CFTR modulator combination (elexacaftor-tezacaftor-ivacaftor; ETI) has been shown to provide substantial clinical benefit in CF patients carrying a Phe508del CFTR allele. However, it should be noted that ETI treatment does not fully restore CFTR function and observational studies suggest that residual abnormalities persist in patients [ 31 , 32 , 35 – 42 ]. In addition, many of the patients with other mutations do not benefit from ETI. The development of novel drugs that restore CFTR function to patients carrying other so far untreatable mutations necessitates the use of improved in vitro systems that closely reflect CF lung disease including impaired MCC as the main pathomechanism. Furthermore, the identification of genetic modifiers of CF disease, which apparently lead to a wide range of mild to severe phenotypes in patients carrying the Phe508del mutation, is imperative [ 43 ]. Such genetic modifiers may represent novel therapeutic targets in CF. To date, intestinal organoids and primary airway cells are most commonly used for these purposes. Intestinal organoids, cultivated from rectal biopsies and analyzed by forskolin-induced swelling (FIS), function as a standard for drug testing [ 44 ]. However, their utility is limited in predicting the efficacy of CF lung disease drugs or studying airway-specific genetic modifiers [ 45 ]. This limitation also stems from their lack of cilia, which prevents them from modeling mucociliary clearance (MCC), a key feature of CF lung disease. Conversely, primary airway epithelia cultured in air-liquid interface (ALI) conditions exhibit a greater degree of similarity to the pathophysiology of CF in vivo and are instrumental in the development of personalized medicine and preclinical drug development [ 33 , 41 ]. However, the supply of sufficient quantities of these cells from patients remains challenging [ 46 ]. Moreover, gene editing, particularly at the clonal level, remains challenging in both systems, making the generation of isogeneic control lines impractical. In recent years, human induced pluripotent stem cells (hiPSCs) have emerged as a novel cell source for in vitro models of CF [ 47 – 51 ]. HiPSCs can easily be generated from patients that carry specific CFTR variants, for instance by reprogramming of small blood samples [ 50 , 52 ]. These cells are characterized by their virtually unlimited proliferation capacity and potential to differentiate into all cell types of the human body [ 53 , 54 ]. In contrast to primary intestinal and airway cells, hiPSCs facilitate gene editing on a clonal level to introduce seamless correction of mutations, gene knockouts and overexpression, and integration of reporter genes. This provides tools to study the physiologic and therapeutic relevance of candidate genes or to perform high throughput screens [ 50 , 55 – 59 ]. Although several studies have demonstrated the differentiation of hiPSCs into airway epithelial cells in organoid or ALI cultures, most differentiation protocols are relatively complex and still associated with considerably variable differentiation efficiencies [ 47 , 49 , 60 – 65 ]. While CFTR function has already been demonstrated in these cells [ 47 – 49 , 65 ], cellular impurities frequently complicated studies that aimed to demonstrate that hiPSCderived airway cells closely recapitulate CF lung disease comparable to primary airway epithelial cells, which are currently considered the gold standard in the field. In this study, we sought to expand upon the findings of recent investigations by undertaking a comprehensive characterization of CF patient-specific hiPSC-derived airway epithelial cells in ALI (iALI) cultures. Our objective was to demonstrate the potential of iALI cultures as a preclinical disease model and as a drug testing platform. Our results show that despite the use of a relatively simple and straightforward differentiation protocol, iALI cultures have a high degree of similarity to primary airway (pALI) cultures, including mRNA and protein expression, mucus (ultra)structure and ion channel function. Measurement of ciliary beat frequency (CBF), which directly reflects impaired mucus viscosity and ciliary transport as a major pathomechanism in CF lung disease, was applied to iALI cultures to overcome problems associated with cellular impurities due to variable differentiation efficiencies. The CF iALI cultures carrying the Phe508del CFTR mutation closely recapitulated CF lung disease in vitro, showing a severe impairment of mucociliary function and also a reduction of chloride conductance, which was partially rescued by the triple CFTR modulator combination ETI, comparable to clinical findings. By providing an unlimited supply of patient-specific cells, iALI cultures will serve as a valuable tool in CF research. Our iALI culture platform will facilitate individualized drug development and the identification of alternative therapeutic targets. It will also accelerate the development of personalized therapies and their clinical translation. Results iALI cultures derived from healthy individuals and CF patient-specific hiPSCs share characteristic features of airway epithelial cells Healthy (wild type, WT) hiPSCs [ 66 ] and CF patientspecific (CF) hiPSCs carrying a homozygous CFTR Phe508del mutation were differentiated into iALI cultures by applying a multistage differentiation protocol [ 64 , 67 – 69 ]. First, hiPSCs were differentiated into the definitive endoderm (DE) until day 3 of differentiation (Fig. 1 A). Flow cytometry analysis was performed, which confirmed high DE marker expression of > 97.0% in both WT and CF DE cells (Fig. 1 B). As an intermediate step in the differentiation process, cryopreservation of DE cells was implemented to create WT and CF DE batches for all subsequent differentiations into iALI cultures (Fig. 1 A). Following the thawing process, DE cells were differentiated into CPM + /NKX2.1 + lung progenitor (LP) cells yielding 41.6 ± 15.5% NKX2.1 + WT cells and 47.4 ± 20,5% NKX2.1 + CF cells. A magnetic-activated cell sorting (MACS) for CPM + cells was performed (Fig. 1 A) to enrich LP cells, yielding 79.9 ± 5.2% NKX2.1 + WT cells and 83.9 ± 6.8% NKX2.1 + CF cells (Fig. 1 C). Finally, LP cells were seeded on inserts for ALI cultivation and were matured by cultivation for a minimum of an additional 27 days (Fig. 1 A). To enable direct comparison with primary airway cultures as the current gold standard in CF research, we generated pALI cultures from a healthy (WT) donor and a CF patient, carrying a homozygous CFTR Phe508del mutation (CF). Our proof-of-concept study included each a single WT hiPSC line and CF hiPSC line as well as primary airway cells from a single WT and CF donor, which were all (hiPSC and primary cells) derived from independent donors. A combination of transcription analysis, immunofluorescence staining and electron microscopy was used to confirm the formation of airway epithelial cells in iALI cultures (Fig. 2 , Fig. 3 , Supplemental Fig. 1). qRT-PCR analysis demonstrated the expression of epithelial cell type markers in iALI and pALI cultures including markers for basal cells (p63, KRT5), club (CCSP) cells, goblet cells (MUC5AC) and ciliated cells (FOXJ1) (Fig. 2 ). In addition, the expression of NKX2.1, a lung development marker [ 70 ], CFTR and TMEM16A, another chloride channel and a potential alternative target for CF therapies [ 71 , 72 ], was detected (Fig. 2 ). The prominent features and morphology of airway epithelial cells were found in both iALI cultures and pALI cultures. iALI cultures exhibited a polarized morphology (Fig. 3 A) and contained p63 + /KRT5 + basal cells (Fig. 3 A) as well as more mature epithelial cell types, including MUC5AC + goblet cells, TUBB4 + ciliated cells and CCSP + club cells (Fig. 3 BD). Additionally, iALI cultures contained BSND + ionocytes, which were only found at a low frequency in pALI and iALI cultures (Supplemental Fig. 1). The immunofluorescence signal of the entire ALI insert membrane revealed comparable numbers of TUBB4 + ciliated cells in iALI and pALI cultures (Fig. 3 D). However, the number of MUC5AC + goblet cells was significantly lower in iALI cultures than in pALI cultures (Fig. 3 C). Additional imaging of the apical surface via scanning electron microscopy (SEM) validated these observations and confirmed the prevalent presence of ciliated cells in iALI cultures (Fig. 3 E). In accordance with the immunofluorescence staining in Fig. 3 B, ciliated cells in iALI cultures appeared to be more clustered and interspersed with groups of nonciliated cells. Morphologic differences between WT and CF epithelial cells were not observed in iALI or pALI cultures. Furthermore, we performed Western blot analyses to investigate the protein expression of CFTR, of TMEM16A, a calcium-activated chloride channel [ 72 , 73 ], and of the tight junction marker ZO-1, both important for proper function of the airway epithelium (Fig. 4 , Supplemental Fig. 2, Supplemental Fig. 3, Supplemental Fig. 4, Supplemental Fig. 5). In both iALI and pALI cultures we detected CFTR B band at 130 kDa and CFTR C band between 140 and 180 kDa (Fig. 4 A, Supplemental Fig. 2). As expected, the Phe508del mutation led to a decline in the expression of the CFTR protein, particularly the CFTR C band, in both the CF iALI and CF pALI cultures in comparison to the WT controls. Treatment with the CFTR modulator drugs ETI for 48 h (3µM elexacaftor, 18 µM tezacaftor and 1 µM ivacaftor) was applied to rescue the protein expression and function of the Phe508del mutated CFTR protein (CF DMSO – vehicle, CF ETI – ETItreated). Western blot analysis revealed that the expression of CFTR C band was increased to a varying degree in CF ETI iALI and CF ETI pALI cultures as shown in three out of four replicates (Fig. 4 A, Supplemental Fig. 2, Supplemental Fig. 5). In accordance with the qRT-PCR data, all iALI and pALI cultures showed protein expression of TMEM16A and ZO-1 (Fig. 4 B + B, Supplemental Fig. 3, Supplemental Fig. 4). Phe508del CF iALI cultures show an impaired CFTR-dependent transepithelial chloride conductance that can be partially rescued by treatment with ETI The bioelectric properties of WT and CF patientderived iALI and pALI cultures were studied in Ussing chamber experiments to evaluate the basic chloride transport defect in CF iALI and CF pALI cultures, as well as to assess the rescue of mutant CFTR chloride channel function by ETI treatment (48 h) through measurements of the short circuit current (I SC ) (Fig. 5 A + B). In both CF DMSO iALI and CF DMSO pALI cultures, the transepithelial electrical resistance (R TE ) was increased (Fig. 5 C), whereas the basal I SC and amilorideinsensitive I SC were decreased compared to respective WT ALI cultures (Fig. 5 D + E). While pALI cultures generally exhibited a more pronounced amiloride response compared to iALI cultures (Fig. 5 A + B + F), only minor variations in the ΔAmiloride were observed between WT iALI and CF DMSO iALI cultures (Fig. 5 F). This finding indicates that neither the Phe508del mutation nor ETI significantly impacted ENaC activity in the examined cultures (Fig. 5 F). Following activation of CFTR with forskolin/IBMX, both WT iALI and WT pALI cultures showed a sustained plateau phase of increased chloride transport (Fig. 5 A + B + G). In accordance with the impaired CFTRmediated chloride transport observed in individuals with CF, the recorded forskolin/IBMX-sensitive I SC (ΔFsk/IBMX) was reduced in CF DMSO iALI and CF DMSO pALI cultures compared to their respective WT controls (Fig. 5 A + B + G). iALI cultures exhibited a significant but substantially lower response after treatment with the CFTR inhibitor-172 (ΔCFTRinh-172) compared to pALI cultures. Nevertheless, the CFTR inhibitor-172 significantly reduced CFTR activity in all WT ALI cultures, and to a lesser extent in CF DMSO ALI cultures, reflecting residual activity of the Phe508del mutant CFTR (Fig. 5 A + B + H). Furthermore, UTPmediated activation of calcium activated chloride channels (CaCCs), such as TMEM16A, reached significantly greater amplitudes in pALI cultures than in iALI cultures (Fig. 5 A + B + I). CF DMSO iALI cultures exhibited an increased UTP-sensitive I SC (ΔUTP) in comparison to CF ETI iALI cultures, while only negligible to none differences were observed between WT and CF pALI cultures (Fig. 5 A + B + I). Finally, the unspecific inhibition of chloride conductance was performed by adding of GlyH-101. As expected because of the decreased chloride transport in CFTR mutated cells, the GlyH-101sensitive I SC (ΔGlyH-101) was found to be more prominent in WT ALI cultures than in CF DMSO ALI and could be observed in both cell systems (Fig. 5 J). Overall, the recorded I SC confirmed the activity of epithelial ion channels, such as ENaC, CFTR and CaCCs in iALI cultures, similarly to pALI cultures. iALI cultures generally produced lower currents than pALI cultures, and measurement of the I SC in iALI culture may be accompanied by a lower sensitivity for individual parameters (Fig. 5 ). Nonetheless, treatment with CFTR modulators ETI for 48 h resulted in a partial rescue of the impaired chloride conductance in both CF ETI iALI and CF ETI pALI cultures that was significant compared to untreated controls (Fig. 5 ). In comparison to CF DMSO ALI cultures, R TE was reduced in CF ETI iALI and CF ETI pALI cultures (Fig. 5 C). Concurrently, basal I SC and amilorideinsensitive I SC were increased (Fig. 5 D + E). Furthermore, the levels of ΔFsk/IBMX increased in both CF ETI iALI and CF ETI pALI cultures, accounting for approximately 62.0% and 44.9% of CFTR activity measured in WT iALI and WT pALI cultures, respectively (Fig. 5 G). In addition, both CF ETI iALI and CF ETI pALI cultures exhibited a significant increase of CFTR inhibition, as reflected by the ΔCFTRinh-172 (Fig. 5 H). Moreover, a reduction in ΔUTP was observed in CF ETI iALI cultures (Fig. 5 I). ΔGlyH-101 was increased in CF ETI pALI cultures but not in CF ETI iALI cultures (Fig. 5 J). CF iALI cultures show altered mucus structure and mucus layer height that can be partially rescued by treatment with CFTR modulators ETI Given that impaired mucociliary function is a hallmark of CF lung disease and appears to be at least partially rescued clinically through ETI treatment, we sought to determine whether the thickness of the mucus layer and mucus structure in CF iALI and CF pALI cultures reflect the CF disease phenotype. To this end, we characterized the ultrastructure of the mucus layer in iALI and pALI cultures from WT and CF donors by highpressure freezing transmission electron microscopy (TEM) (Fig. 6 A). The TEM imaging and mucus height measurements revealed that the mucus layer exhibited a comparable structure and properties in iALI and pALI cultures (Fig. 6 , Fig. 7 , Supplemental Fig. 6). In all ALI cultures, the mucus layer was composed of more or less densely packed molecular networks, which were visible throughout most of the prepared cross sections (Fig. 6 C, Fig. 7 , Supplemental Fig. 6), and exhibited a mucus layer height (MLH) of variable degree (Fig. 6 B). The CF DMSO ALI cultures exhibited a denser mucus layer that typically showed a distinctive surface lining, which closely reflected the in vivo phenotype of CF lung disease. In contrast, the mucus layer in WT ALI cultures was less dense and showed a loose surface lining (Fig. 6 B, Fig. 7 , Supplemental Fig. 6). The measured MLH was generally lower in CF DMSO ALI cultures than in WT cultures in both the iALI and pALI culture systems (Fig. 6 B + C). Treatment with ETI for 24 h not only increased the MLH in CF ETI iALI and CF ETI pALI cultures but resulted in an even thicker mucus layer in CF ETI iALI cultures than in WT iALI cultures (Fig. 6 B + C). In addition, the mucus structure was less dense and showed a loose surface lining in CF ETI iALI and CF ETI pALI cultures (Fig. 6 C, Fig. 7 , Supplemental Fig. 6). These findings suggest a rehydration of the mucus layer and complete rescue of the disease phenotype by ETI in the iALI culture system, which so far could not be observed or concluded in Ussing chamber measurements (Fig. 5 ). Furthermore, TEM imaging confirmed the presence of cilia and microvilli in both iALI and pALI cultures (Fig. 6 , Supplemental Fig. 6). CF iALI cultures show decreased ciliary beat frequency that can be partially rescued by treatment with CFTR modulators ETI CFTR dysfunction leads to airway surface dehydration, which results in the secretion of highly viscoelastic mucus that impairs proper ciliary function and thus clearance of mucus, pathogens and other inhaled particles. Due to the laborious and challenging nature of MLH measurements and mucus structure visualization via TEM (Fig. 6 ), we sought an alternative method to directly quantify impairment of MCC, which largely determines the phenotype of CF lung disease. Given that the decreased chloride transport in CF results in elevated mucus viscosity and subsequent impairment of ciliary movement and MCC, we opted to utilize high-speed video microscopy to assess the ciliary beat frequency ( CBF ). Notably our approach involved the utilization of a specialized software tool (SAVA) that facilitates automated analysis (Fig. 8 A). This software was configured to quantify the active areas of ciliary movement as well as the CBF. Motile cilia were detected in all iALI and pALI cultures and measurements confirmed a generally reduced active area of ciliary beating in iALI cultures compared to pALI cultures (Fig. 8 B + C). This finding corresponds with the more clustered appearance of ciliated cells (Fig. 3 B + E). However, the CBF levels in WT iALI cultures generally surpassed those observed in WT pALI cultures (Fig. 8 D). While CF iALI cultures measurements exhibited a less distinct phenotype in the Ussing chamber compared to CF pALI cultures (Fig. 5 ), CF iALI cultures demonstrated an even more pronounced disease phenotype in the CBF assay, characterized by a significant impairment of the ciliary beating (Fig. 8 C + D). Despite the presence of ciliated cells, the active area and CBF were significantly diminished in CF DMSO pALI cultures, and in CF DMSO iALI cultures the ciliary beating was either not visible in the majority of cultures, or below the detection limit (2 Hz) of the software (Fig. 8 BD). Following 24 h of ETI treatment, a significant augmentation in the active area and CBF was observed in both CF ETI iALI and CF ETI pALI cultures (Fig. 8 BD). Also, all CF ETI ALI cultures generally exhibited a greater CBF than the respective WT controls (Fig. 8 D). Similar to the MLH measurements (Fig. 6 ), these findings suggest a rehydration of the mucus layer that consequently reduced the mucus viscosity and accelerated the ciliary beating (Fig. 8 C + D). Again, ETI treatment led to a complete rescue of the disease phenotype and even overcompensation of the CBF in both CF ETI iALI and CF ETI pALI cultures (Fig. 8 D). Discussion In CF research, reliable patient-specific in vitro models are a crucial to developing novel CFTR modulator drug against currently untreatable CFTR mutations and identifying genetic modifiers as novel therapeutic targets. Although, intestinal organoids and primary airway cells are currently considered the gold standards in the field, both systems face particular limitations concerning either their ability to reflect important aspects of CF lung disease or the availability of sufficient quantities of patient-specific cells, respectively. In principle, all these issues and limitations can be adequately addressed in organotypic models consisting of airway epithelial cells derived from patientspecific hiPSCs. Several studies demonstrated the differentiation of hiPSCs into airway epithelial cells. However, currently available differentiation protocols face two major difficulties. On the one hand, many of the available protocols results in highly variable outcomes in terms of efficiency and cellular purity. As a result, the sensitivity of FIS assays and electrophysiological measurements are compromised [ 47 , 49 , 64 , 65 ]. On the other hand, more advanced protocols, namely those of Hawkins and colleagues, are difficult to establish due to their relatively high complexity and are more cost- and time-intensive than others [ 47 , 49 , 60 – 65 ], which is critical in particular on a higher throughput scale as often required in drug development. Going beyond published work, we focused on a comprehensive comparison of organotypic hiPSCderived airway cultures (iALI cultures) and primary airway cultures (pALI cultures), the current gold standard in CF research, in terms of proper gene and protein expression, structural characteristics and functionality. We demonstrate the usefulness of these iALI cultures, generated via a relatively simple and fast protocol that is easily scalable in 2D and does not require intermediate organoid cultivation. Application of an automatable CBF assay allows determination of decreased CBF which directly reflects reduced mucociliary clearance due to increased mucus viscosity as key pathomechanism of CF lung disease. Our results show that this assay is not affected by a variable cellular composition of iALI cultures and suggest an even higher sensitivity than measurement of pALI cultures. We verified that iALI cultures contain a structured and polarized airway epithelium, which is composed of basal cells, ciliated cells, club cells, goblet cells and ionocytes, and which exhibit expression of CFTR and TMEM16A. In both iALI and pALI cultures, minor variations in the expression levels were observed between WT and CF cells, which however are likely not dependent on the mutated CFTR gene. It can be assumed that these variations reflect donor differences. In case of iALI cultures they may also reflect the outcome of differentiation, which depends on the donor or cell clone, and may lead to different cell composition and maturity. The low magnification images of the entire wells stained for MUC5AC and TUBB4 illustrate that iALI cultures contain a high abundance of ciliated and goblet cells often arranged as clusters. Electron microscopy revealed that in iALI cultures, in contrast to pALI cultures, regions covered with ciliated cells were interspersed with flat cells that may comprise non-ciliated epithelia as well as other cell types. Of particular note are vimentin + mesenchymal cell lineages (data not shown), which represent cellular contaminants that are to a limited degree carried over during MACS of CPM + /NKX2.1 + LP cells. While the significance of ionocytes in CF lung disease remains to be elucidated, a low number of ionocytes were also detected in iALI cultures. It is noteworthy that these ionocytes have been described as a rare epithelial cell type that exhibits exceptionally high levels of CFTR expression and might be of particular interest for CF research in the future [ 74 , 75 ]. Typical for WT-CFTR, we detected a much lower expression of the immature coreglycosylated glycoisoform B (CFTR B) in both WT pALI and iALI cultures, than of the mature complex glycosylated glycoisoform C (CFTR C). According to the findings of previous studies [ 76 , 77 ], our CF cultures both exhibit a substantially lower level of the CFTR C compared to the CFTR B. Notably, our data indicate an even more pronounced effect of the Phe508del mutation compared to the recent published findings [ 76 ]. Finally, although a substantial variation was observed between biological replicates for iALI and pALI cultures, ETI treatment increased the proportion of the CFTR C, which is consistent with previously published data [ 77 , 78 ]. These findings provide further evidence that iALI cultures behave comparable with regard to the effect of CFTR correctors (elexcaftor and tezacaftor) on CFTR glycosylation and maturation, both crucial for trafficking and stability. Aside from this, we identified protein expression for TMEM16A, a potential alternative target for CF therapies [ 6 , 79 ], and ZO-1. While the expression levels of ZO-1 remained consistent across all samples, TMEM16A expression exhibited significant variability. This variability appeared to be independent of the presence of the Phe508del mutation and may rather be donor- or, in case of iALI cultures, clone-dependent, and may additionally be influenced by the differentiation outcome. Overall, iALI cultures demonstrated a significant degree of molecular similarity to the epithelial cells present in pALI cultures. The Phe508del mutation resulted in reduced expression of CFTR C protein, while other variations between WT and CF cultures were likely due to cell line- and clone-specific factors. Additionally, cellular contaminants carried over during MACS of CPM + /NKX2.1 + LP cells were the most probable source of molecular differences between iALI and pALI cultures. However, these contaminants did not compromise the utility of iALI cultures for modeling CF lung disease, as discussed below. At the functional level of the airway epithelial cells, Ussing chamber experiments have demonstrated the activity of epithelial ion channels, including ENaC, CFTR, and CaCCs, in iALI cultures. While the activity of CFTR could be confirmed by means of stimulation with forskolin/IBMX, we also assume the activity of other GlyH 101-sensitive chloride channels, such as SLC26A9 [ 80 ], in iALI and pALI cultures, as CFTR inhibitor-172 did not fully inhibit CFTR. In addition, our findings are consistent with previous reports, demonstrating that ETI only partially restores chloride transport in CF ALI cultures despite its considerable therapeutic benefit for patients with at least one Phe508del mutation [ 32 , 36 ]. Nonetheless, we also detected that iALI cultures generally produced lower currents than pALI cultures. Therefore, electrophysiological measurements in iALI cultures may be accompanied by a lower sensitivity for individual parameters. It is possible that the lower degree of maturity of the epithelial cells in iALI cultures contributes to this observation. However, it is more likely that contaminating non-epithelial cells, especially vimentin + mesenchymal cell lineages typically detectable at varying numbers, account for the lower currents and correspondingly lower assay sensitivity. Depending on the differentiation outcome, cells that lack expression of relevant ion channels may actually cover a considerable part of the ALI membranes between clusters of ciliated epithelia. These cells may substantially influence the measurement of I SC . According to these results, we anticipated that the lower purity of our iALI cultures, compared to pALI cultures, would negatively affect the manifestation of CF-typical impairments in mucociliary function, such as reduced MLH, increased mucus density, and reduced CBF (due to dehydration and leading to increased viscosity). However, contaminating non-epithelial cells did not negatively affect MLH or mucus density, enabling accurate modeling of mucus impairment in CF lung disease. The mucus layer in iALI cultures was thicker than in pALI cultures, and the effect of the Phe508del-mutated CFTR was evident in both systems. In contrast to the measured chloride transport and CFTR activity, ETI treatment fully restored the MLH of CF ALI cultures to WT levels, even exceeding them in iALI cultures. Although not quantitatively assessed, ETI also restored the looser mucus density in CF iALI cultures, as observed in WT controls. Together, these findings indicate rehydration of the mucus layer following ETI treatment and highlight the significant clinical benefits of ETI treatment observed in CF patients [ 30 – 32 , 36 , 40 ]. Compared to electrophysiological measurements or the FIS assay in organoids, the measurement of MLH and mucus density may provide a more accurate reflection of the therapeutic effect of CF modulators on CF lung disease. Still, utilizing electron microscopy to evaluate the MLH and mucus density remains a very laborious and time-consuming process. We further sought an alternative approach to evaluate the impairment of MCC in CF. We considered tracking of mucociliary transport by the use of fluorescent particles as not suitable due to the reduced areas covered by ciliated cells in iALI cultures [ 69 ]. Therefore, we employed the CBF assay as a significantly more useful, straightforward and time-efficient tool. In particular, the CBF assay could potentially be automated and adapted for medium- and high-throughput applications, making it a promising tool for large-scale studies of MCC impairment in CF models. While pALI cultures exhibited substantially more active areas of ciliary beating, CBF levels remained comparable in iALI and pALI cultures. In comparison to pALI cultures, the effect of the Phe508del mutation was even more pronounced in iALI cultures, suggesting a more sensitive readout in iALI cultures. Certainly, further analyses of primary cells and iPSCderived airway cells from different patients have to confirm whether iALI cultures are generally more sensitive than primary airway cells. Nevertheless, we can confidently conclude that analysis of CBF on iPSCderived airway cells largely eliminates the current problem of variable differentiation efficiencies and closely reflects changes in mucociliary transport in the airways caused by CFTR mutations and CFTR modulator drugs. Notably, the application of ETI resulted in a CBF that surpassed the CBF in WT iALI cultures, suggesting that this approach may more accurately reflect the substantial therapeutic efficacy of ETI than the observed partial rescue of chloride transport function as measured by electrophysiological techniques or FIS assays. We believe that our iALI culture system, in combination with the CBF assay, offers a user-friendly platform that can be easily adopted by other laboratories and the broader CF research community. First, the relative simplicity of our differentiation protocol provides significant advantages, making our platform more time- and resource-efficient compared to existing protocols. For example, the current state-of-the-art protocol developed by Hawkins and colleagues includes a targeted basal cell generation step prior to ALI culture seeding, which significantly increases culture purity [ 49 ]. However, this protocol necessitates additional differentiation steps in Matrigel-based organoid cultures, which are both highly time- and cost-intensive even before ALI seeding. In contrast, our iALI cultures are derived from 2D cultures that require less expensive materials and can be easily scaled to produce larger quantities of cells. Second, despite the presence of considerable cellular contaminants in iALI cultures, our CBF assay remains unaffected by cellular contaminations, providing a robust readout of the primary defect in CF lung disease and demonstrating sensitivity to clinically relevant CFTR modulator drugs. Moreover, the CBF assay utilizes a straightforward setup consisting of an incubator chamber microscope equipped with a high-speed camera and the SAVA software for video recording and analysis. As mentioned before, the CBF assay could even be automated to enable screenings for novel CFTR modulator drugs. In comparison, other standard methods such as electrophysiological measurements in Ussing chambers require greater technical expertise, enable only a low sample throughput and provide no conclusive evidence for effects on the main impairment of MCC in CF lung disease. Conclusions In summary, our study demonstrates that patient-specific iALI cultures are indeed an excellent in vitro model of CF lung disease. We have leveraged the vast majority of the theoretical advantages of hiPSCs over immortalized cell lines, intestinal organoids, and primary airway cells. Taking advantage of the unlimited expansion potential of hiPSCs, which allows even largescale production of individual patientderived cells [ 81 , 82 ], we demonstrated that complex organotypic cultures can be generated with characteristic (ultra)structural features of highly polarized airway epithelial cells, including ciliated cells, goblet cells, club cells and underlying basal cells, covered with mucus whose height and density are contingent on CFTR function. We have demonstrated the functionality of CFTR and other characteristic ion channels relevant for CF lung disease at the electrophysiological level, including the effect of CF modulators that closely reflect clinical data. These functional studies have been complemented by an innovative in vitro assay that is suitable for high throughput applications to determine CBF. The CBF assay in iALI cultures has been shown to more closely reflect impaired MCC as the predominant pathomechanism of CF lung disease than existing in vitro systems. Furthermore, it has the capacity to directly visualize the effects of genomic modifiers of CF, as well as of novel drugs and drug combinations, on rare CFTR mutations that have remained untreatable to date. Methods Cell Culture of hiPSCs . A CFTR wild type hiPSC line (MHHi001A) [ 66 ] (wild type, WT) and a hiPSC line (MHHi002A) carrying a homozygous CFTR Phe508del mutation as a diseased reference (CF) were used for all experiments. HiPSC lines were cultured in 5–8 mL of E8 medium (self-made) on Geltrex® (Thermo Fisher Science, A1413202)-coated culture vessels (25 cm² surface area). Media changes were conducted daily. Every three to four days the cells were passaged by dissociation with 1 mL Accutase™ (Sigma-Aldrich, A6964) and seeded at a density of 3.2 x 10 4 cells cm − 2 while supplementing the medium with 10 µM ROCK inhibitor Y27632 (RI) (Tocris; 1254). All cells were maintained at 37°C and 5% CO 2 . Differentiation of hiPSCs into airway epithelial cells (iALI cultures) . HiPSCs were differentiated towards definitive endoderm (DE) utilizing the STEMdiff™ Definitive Endoderm Kit (STEMCELL Technologies, 05115). Seven days (day − 7) before induction of the DE differentiation, hiPSCs were passaged and seeded at a density of 1.2 x 10 4 cells cm − 2 on a 25 cm² culture vessel. From day − 3 onward the 8 mL E8 medium was supplemented with STEMdiff™ Definitive Endoderm TeSR™-E8™ Supplement (1:20). On day − 1 the cells were passaged and seeded at a density of 3.33 x 10 4 cells cm − 2 on a 25 cm² culture vessel. In intervals of 24 h the media was changes to 5 mL of STEMdiff™ Endoderm Basal Medium supplemented with MR (1:100) and CJ (1:100) for the first 24 h and then supplement CJ for further 48 h. On day 3, the DE cells were dissociated with 1 mL Accutase™ and seeded at a density of 1.35 x 10 5 cells cm − 2 on Geltrex®-coated 6well plates in 2 mL of basis medium (BM) supplemented with 3 µM Dorsomorphin (Merck, P5499), 10 µM SB431542 (kindly provided by A. Kirschning, Leibniz University Hannover) and 10 µM RI. All further media changes were conducted in intervals of 24 h. On day 4, media was changed to BM supplemented with 2 µM IWP2 (Torcis, 3533) and 10 µM SB431542. From day 5 until day 12, media changes were conducted with BM supplemented with 10 µm BMP4 (R&D Systems, 314-BP), 3 µM CHIR99021 (kindly provided by A. Kirschning, Leibniz University Hannover) and 10 nM FGF10 (R&D Systems; 345-FG). On day 13, cells were dissociated with 2 mL of Accutase™ per well and magneticactivated cell sorting (MACS) was applied to enrich CPM + lung progenitor cells. The following quantities are appropriate for MACS of 10 7 cells. MACS was performed by resuspension of cells in 320 µL MACS buffer (selfmade), initial blocking of the cells with 80 µL FcR blocking reagents (Miltenyi Biotec, 130-059-901) for 20 min and staining with 2µL anti-CPM antibody (FUJIFILM Wako, 014-27501) for 20 min. After centrifugation, cells were resuspended in 150 µL MACS buffer and labelling with 50 µL anti-mouse IgG MicroBeads (Miltenyi Biotec, 130-047-201) was performed. Cells were centrifuged and resuspended in 3 mL of Knockout™ DMEM (Thermo Fisher Science, 10829018) before performing cell separation in LS columns (Miltenyi Biotec, 130-042-401). For further maturation in ALI culture, CPM + cells were either seeded at a density of 2.65 x 10 5 cells cm − 2 on ThinCert® inserts (Greiner Bio-One, 665641) or Costar® Snapwell™ inserts (Corning, 3801) coated with 804G medium (self-made) [ 83 ]. Cells were cultured in Small Airway Epithelial Cell Growth medium (PromoCell, C-21070) supplemented with 1.0 µM A 83 − 01 (Tocris, 2939), 0.2 µM DMH-1 (Tocris, 4126) and 5 µM RI (ADRI) for 96 h (adapted from Mou et al. 2016 and McCauley et al. 2017) [ 64 , 83 ] and 48 h in in PneumaCult™-ALI medium (STEMCELL Technologies, 05001) before apical medium was removed to induce airliquidinterface (ALI) cultures. ALI cultures were provided with 0.5 mL of medium from the apical side and 1–3 mL from the basolateral side. Media changes on the basolateral side were conducted every two to three days. Molecular and functional analyses were performed earliest on day 40/41 of differentiation. All iALI cultures were stimulated IL-13 (10 ng mL − 1 ; PeproTech, 200 − 13). In addition, CF ALI cultures were treated with elexacaftor (3 µM, Selleckchem, S8851), tezacaftor (18 µM, Selleckchem, S7059) and ivacaftor (1 µM, Selleckchem, S1144) (ETI) for 24 h or 48 h before analysis. All cells were maintained at 37°C and 5% CO 2 . Isolation, Expansion and Differentiation of Primary Human Airway Epithelial Cells (pALI cultures). Human explanted lung tissue was obtained from the Hannover Lung Transplant Program after patients informed written consent. The tissue samples were thoroughly cleared of excess tissue to isolated the bronchial airways, cut into small pieces of a few millimetres in diameter and transferred into 30 mL of 0.18% Protease Type XIV (SigmaAdlrich, P5147) in Hank’s buffer (Thermo Fisher Science, 14175095). After dissociation for 2 h at 37°C, cells were transferred to PureCol®(Advanced BioMatrix, 5005)-coated flasks (75 cm²) and cultured in Small Airway Epithelial Cell Growth medium supplemented with 1.0 µM A 83 − 01, 0.48 µM CHIR99021, 0.2 µM DMH-1 and 5 µM RI (ACDRI). After expansion, cells were dissociated with TrypLE™ (Thermo Fisher Science, 12604013) and seeded at a density of 1.77 x 10 5 cells cm − 2 on ThinCert® inserts or Costar® Snapwell™ inserts coated with PureCol®. Cells were first cultured in Small Airway Epithelial Cell Growth medium supplemented with ACDRI for 48 h. Then, the media was changed to PneumaCult™-ALI medium. ALI cultures were provided with 0.5 mL of medium from the apical side and 1–3 mL from the basolateral side. ALI cultures were provided with 0.5 mL of medium from the apical side and 1–3 mL from the basolateral side. After 48 h the apical medium was removed to induce proper ALI cultivation. Following cells were matured and media changes were conducted every two to three days. Molecular and functional analyses were performed on day 37/38 of differentiation. Before the analyses, all pALI cultures were treated with IL-13 (10 ng mL − 1 ) and partially ETI as previously described for iALI cultures. All cells were maintained at 37°C and 5% CO 2 . Immunofluorescence Staining. iALI and pALI cultures were fixed with 0.5 mL 4% paraformaldehyde (Morphisto, 11762) at room temperature (RT). The insert membrane was utilized to perform staining of either the top viewed whole membrane or side viewed membrane sections. For section staining, the insert membrane was first paraffinembedded, cut and then further processed for staining as described in von Schledorn et al. 2023 [ 69 ]. For whole membrane staining, permeabilization was performed with 0.5 mL TBS buffer with donkey serum (GeneTex, GTX73245) for 20min at RT. The following primary antibodies were utilized and diluted in PBS plus 1% bovine serum albumin (BSA) (Sigma-Aldrich, A9418): MUC5AC (Thermo Fisher Science, MA5-12178, 1:200), TUBB4 (Cell Signaling Technology, 5335S, 1:800). The following secondary antibodies were utilized and diluted in PBS plus 1% BSA: Cy™3 anti-rabbit IgG (Jackson ImmunoResearch, 711-165-152, 1:200), Alexa Fluor® 488 anti-mouse IgG (Jackson ImmunoResearch, 715-545-151, 1:200). 0.5mL of primary antibodies were incubated over night at 4°C. 0.5mL of secondary antibodies were incubated for 30 min at RT. Nuclei were stained with DAPI for 15 min at RT. For imaging the membrane was mounted on a glass slide in Fluorescence mounting medium (Agilent Dako, S3023). For membrane section staining, the membrane was first dehydrated and then embedded in paraffin. The membrane was cut into sections of 3 µm in thickness and transferred onto glass slides for staining. Primary and secondary antibodies as well as DAPI were applied as described above. Fluorescence imaging and image processing were performed with an AxioObserver A1 fluorescence microscope and AxioObserver Z1 fluorescence microscope and the ZENPro Sofware 3.0. For BSND staining samples were blocked and permeabilized using 0.25% (v/v) Triton-X 100 in PBS with 3% BSA for 60 min at RT, then incubated overnight at 4°C with the anti-BSND antibody (Abcam, ab196017, 1:500) diluted in the Triton/BSA buffer. The samples were rinsed three times for 5 min with PBS before incubation with secondary antibody (Invitrogen, A11008, 1:500) diluted in Triton/BSA buffer for 1 h at 37°C, followed by a triple 5 min wash with PBS. Then, samples were incubated for 1 hour at 37°C in Triton/BSA buffer containing directly conjugated anti- TUBB4 (Santa Cruz, sc-23950, 1:200) followed by a triple 5 min wash with PBS. The samples were stored at 4°C until mounting. The samples were mounted by removing the cell culture membrane from the insert using a scalpel and placing the membrane onto a glass slide with the cells facing upwards. The cell was coated with a drop of ProLong™ Diamond Antifade Mountant (Invitrogen, P36965) and covered with a round number 1.5 glass coverslip. Composition Analysis of ALI Cultures via Quantification of Immunofluorescence Signal Area. Analysis was performed on ALI cultures after immunofluorescence staining of TUBB4 and MUC5AC, as earlier described. ImageJ/Fiji software was utilized to determine the area of TUBB4 + and MUC5AC + signal after fluorescence microscopy. Input images were used in 16 bit file format. First, threshold was manually set using the ‘moments’ function. Afterwards signalpositive area was measured. Analysis was performed on multiple regions of interest (each 879µm x 879µm) per sample group. Flow Cytometry Analysis. Flow cytometry analysis was performed to quantify the expression of definitive endoderm and lung progenitor markers on day 3 and day 13 of hiPSCs differentiation into respiratory epithelial cells, respectively. On day 3, live cell staining was performed. On day 13, cells were fixed and permeabilized using the FoxP3 staining buffer set (Miltenyi Biotec, 130-093-142). Each 10 5 cells were taken and suspended in 100 µL PBS with 1% BSA. The following directly-labelled primary antibodies were utilized: APC anti-CXCR4 (Thermo Fisher Science; 17-9999-42, 1:25), APC anti-NXK2.1 (Miltenyi Biotec, 130-118-309, 1:2,000), PE anti-c-Kit (Thermo Fisher Science, 12-1178-42, 1:33), PE anti-EpCAM (BD Biosciences, 347198, 1:33). Primary antibodies were diluted in 1% BSA (Sigma-Aldrich, A9418) in PBS w/o (Thermo Fisher Science, 70011044) and incubated for 30 min on ice. Flow cytometry analysis was performed with a MACSQuant Analyzer 10 and FlowJo analysis software. Real-Time Quantitative PCR (RT-qPCR). RNA samples from cells were collected in Trizol® reagent (Invitrogen, 15596018) RNA isolation was performed using the NucleoSpin® RNA II kit (Macherey-Nagel, 740955.50) and cDNA synthesis was performed using the RevertAid™ H Minus First Strand cDNA Synthesis kit (Thermo Fisher Science, K1631). Real-time qPCR analysis was carried out using the SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, 1725270) and CTX Connect Real-Time PCR Detection system (Bio-Rad Laboratories). Target gene expression was normalized to the expression of housekeeping genes bACT and GAPDH. Applied primer pairs and sequences are listed in supplemental data set. Western Blot Analysis. Western blot analyses were performed as previously described [ 56 ]. The following antibodies were utilized: Beta-actin (Abcam, ab8226), CFTR (Cystic Fibrosis Foundation CFTR Antibody Distribution Program, Chapel Hill, North Carolina; antibody mix: 596 + 570 + 217 + 660), TMEM16A (Abcam, ab64085); ZO-1 (Invitrogen, 33-9100). 16HBE14o- cells (human bronchial epithelial cells; HBE) were utilized as a control for protein expression in airway epithelial cells. Measurement of the Transepithelial Ion Conductance. Recordings of the transepithelial ion conductance in iALI and pALI cultures was performed in EasyMount Ussing chambers (Physiologic Instruments) using voltage clamp configuration to measure the transepithelial short-circuit current ( I SC ). The I SC was continuously recorded using Lab-Chart8 (AD Instruments), and transepithelial resistance ( R TE ) was monitored by application of short voltage pulses (1 mV) every 60 s. Experiments were performed in Ringer buffer as previously described [ 84 , 85 ]. After 10–15 min equilibration, basal I SC was measured and amiloride (100 µM; Sigma-Aldrich, A7410) was added to inhibit sodium absorption via ENaC. Next, forskolin (10 µM; Sigma-Aldrich, F6886) and 3isobutyl-1methylxanthin (IBMX; 100 µM; Sigma-Aldrich, I5879) were added together, followed by CFTRinhibitor 172 (20 µM; CFTRinh-172) (TargetMol, T2355) to assess CFTR-mediated chloride conductance. Uridine-triphosphate (UTP; 10 µM; Thermo Fisher Science, R1471) was added to evaluate the calcium-activated chloride conductance. Both, the activation of CFTR by forskolin and IBMX as well as the activation of CaCCs were measured as the peak response after compound addition. Lastly, GlyH-101 (50 µM; AbMole, M6754) and subsequently niflumic acid (NFA; 500 µM; Cayman Chemical Company, 70650) were applied to inhibited residual anion conductance. Measurement of the Ciliary Beat Frequency (CBF). Measurement of the CBF on iALI and pALI cultures was performed via high-frequency video microscopy imaging using a Zeiss Axiovert A.1 microscope equipped with a Basler SCA640 120FM camera in a humidified environmental chamber at 37°C and in 5% CO2. Videos were recorded with a 40x magnification objective at 100 frames per second using a phase contrast filter. Videos were recorded in at least 15 positions per insert, selected in a meandering pattern throughout the whole insert, avoiding the edges with the meniscus. CBF and cilia coverage were analyzed using Sisson-Ammons video analysis (SAVA) software [ 86 ], where average CBF and area of moving pixels were measured by whole field analysis. Inserts with less than 10.000 moving pixels (< 0.01%) of total imaged area) were determined as non-motile. Data are show as average per insert. Apical washes were performed with PBS 24 h before the measurement. Transmission Electron Microscopy. ALI cultures were fixed in 150 mM HEPES buffer (pH 7.35) containing 1.5% glutaraldehyde and formaldehyde at RT for 20 min and at 4°C over night. 1.5 mm sized pieces of the fixed cultures were high pressure frozen in a HPM 100 (Leica Microsystems, Wetzlar). Freeze substitution was carried out in a Leica AFS (Leica Microsystems, Wetzlar) in acetone containing 0,1% tannic acid at -90°C over night and after washing in acetone continued in acetone containing 2% osmiumtetroxide. Temperature was raised to -20°C and after 2 h to 4°C. After washing in acetone, samples were transferred to RT and embedded in EPON. 50 nm thick cross-sections of the ALI-cultures were poststained with uranyl acetate and lead citrate (Reynolds et al. 1963) and observed in a Zeiss EM 900 (Zeiss, Oberkochen), operated in the bright field mode at 80 kV. Images were recorded with a side-mounted 4k CCD-camera (TRS, Dünzelbach). For estimation of the mucus layer height, complete 1.5 mm profiles were recorded iteratively and the mucus layer was estimated every 6 µm at 90 degree to the median cell surface on the respective images. Scanning Electron Microscopy. After fixation as for TEM, ALI cultures were washed in water, critical point dried and sputtered with gold. Examination was done in a Crossbeam 540 (Zeiss, Oberkochen) at 10 kV. Statistical Analyses. GraphPad Prism6 was utilized to perform statistical analyses. Results are presented as means ± SD unless otherwise noted. Significance of two sample groups was analyzed as noted in the figure descriptions by using the unpaired or paired t test. Use of Artificial Intelligence. For the purpose of supporting the writing process of this manuscript, Meta LLaMA 3.1 8B Instruct and OpenAI ChatGPT-4 and DeepL were sporadically used to rephrase individual sentences. Abbreviations ALI: Air-liquid-interface BSND: Barttin CLCNK Type Accessory Subunit Beta CaCCs: Calcium-activated chloride channel cAMP: Cyclic adenosine monophosphate CBF: Ciliary beat frequency CCSP: Clara-cell secretory protein CF: Cystic fibrosis CFTR: Cystic fibrosis transmembrane conductance regulator CXCR4: C-X-C chemokine receptor type 4 DE: Definitive endoderm ENaC: Epithelial sodium channel EpCAM: Epithelial cell adhesion molecule ETI: Elexacaftor-tezacaftor-ivacaftor FIS: Forskolin-induced swelling hiPSC: Human induced pluripotent stem cell iALI culture: hiPSC-derived ALI culture I SC : Short-circuit current LP: Lung progenitor MACS: Magnetic-activated cell sorting MCC: Mucociliary clearance MLH: Mucus layer height MUC5AC: Mucin 5AC n: Replicate NKX2.1: NK2 homeobox 1 ns: None significant pALI culture: primary ALI culture SD: Standard deviation SEM: Scanning electron microscopy SLC26A9: Solute carrier family 26 member 9 TEM: Transmission electron microscopy TMEM16A: Transmembrane member 16A TUBB4: Acetylated tubulin beta-4 chain WT: Wild type (healthy) Declarations Ethics approval and consent to participate Human anonymized blood samples for generation of hiPSCs were collected based on the approvals by the Hannover Medical School (MHH) Ethics Committee (No. 409; approval date: 21.09.2010) as part of the project ‘Nutzung von anonymisierten Patientenzellen für die biomedizinische Forschung / Generierung von induzierten pluripotenten Stammzellen’ and following the donor’s written informed consent, or in the case of newborns, following parental consent. Human anonymized explanted lung tissue samples for isolation of primary bronchial epithelial cells were collected based on the approvals by the Hannover Medical School (MHH) Ethics Committee and following the donor’s written informed consent. Data availability and supplemental information The underlying data to all graphs is available in the supplementary data sheets Supplementary Table 1. Supplementary material information are available in Supplementary Table 2. Author contribution MCJ designed, performed and analyzed a large part of experiments and wrote the manuscript. AB designed the measurements of the transepithelial chloride conductance, performed parts of the measurements of the transepithelial chloride conductance and ciliary beat frequency, provided scientific input and wrote the manuscript. JZ and NC performed RT‑qPCR, immunofluorescence staining and generated iALI and pALI cultures. LC performed parts of the measurements of the transepithelial chloride conductance and ciliary beating frequency. LvS performed immunofluorescence staining. JH performed transmitted electron microscopy, analyzed the data and wrote the manuscript. JN and DR performed immunofluorescence staining and microscopy for ionocyte detection. MM and SH performed the western blot experiments. FS analyzed the western blot data and revised manuscript critically for scientific content. TR performed parts of the measurements of the transepithelial chloride conductance and ciliary beating frequency. FI, DJ and AR prepared and provided lung material for primary cell isolation. JH performed the isolation of CF patient‑specific primary human respiratory epithelial cells. MAM supervised the study, provided scientific input, provided his laboratory equipment to performed measurements of the transepithelial chloride conductance and ciliary beating frequency, and wrote the manuscript. 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Sisson JH, et al. All-digital image capture and whole-field analysis of ciliary beat frequency. J Microsc. 2003;211(Pt 2):103–11. Additional Declarations No competing interests reported. Supplementary Files SupplementalFiguresJaboreckCFdiseasemodel.docx SupplementaryTables1Dataavailablility.xlsx SupplementaryTable2Supplemtarymaterials.xlsx Cite Share Download PDF Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted Editorial decision: Revision requested 10 Sep, 2025 Reviews received at journal 09 Sep, 2025 Reviews received at journal 03 Sep, 2025 Reviewers agreed at journal 02 Sep, 2025 Reviewers agreed at journal 28 Aug, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers invited by journal 27 Aug, 2025 Editor assigned by journal 18 Aug, 2025 Submission checks completed at journal 10 Aug, 2025 First submitted to journal 08 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Intermediate cryopreservation was performed at day 3 of differentiation. (B-C) Flow cytometry was performed to check the efficiency of definitive endoderm (DE) and lung progenitor (LP) cell formation on day 3 and 13 of differentiation, respectively. Results are shown for differentiations from WT and CF hiPSCs. (\u003cstrong\u003eB\u003c/strong\u003e) Flow cytometry analysis of DE generation on day 3 of differentiation (before cryopreservation). Dot blots showing expression of DE markers c-Kit, CXCR4 and EpCAM (number of differentiations: n=1 per group); grey – isotype control; red – target antibody. (\u003cstrong\u003eC\u003c/strong\u003e) Flow cytometry analysis of NKX2.1\u003csup\u003e+\u003c/sup\u003e LP cells before and after MACS on day 13 of differentiation (Pre – before sorting, Neg – negative fraction after sorting, Pos – positive fraction after sorting). Results are shown as mean ± SD, replicates represent number of differentiations: n=6 per group; Paired two-tailed t test: ns – none significant, * p\u0026lt;0.05, ** p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/34d47faddd442485495f06d9.png"},{"id":90543745,"identity":"51321e75-8b4d-4195-b85d-1f441ad825c9","added_by":"auto","created_at":"2025-09-04 00:14:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":196923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eiALI cultures show transcription of mature epithelial cell type markers and epithelial ion channels.\u003c/strong\u003e mRNA expression of airway epithelial markers for basal cells (p63, KRT5), goblet cells (MUC5AC), ciliated cells (FOXJ1), club cells (CCSP) and respiratory ion channels (CFTR, TMEM16A). Results are shown as mean ± SD, replicates represent number of ALI cultures derived from independent differentiations: n=3-4 per group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/5bdbace102f52d9b52a9bd3d.png"},{"id":90543755,"identity":"fa79e7e7-f5b1-485c-b109-cd7195ec9ae5","added_by":"auto","created_at":"2025-09-04 00:14:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":693765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eiALI cultures contain typical cellular components of mature human airway epithelium incl. a large number of ciliated cells, goblet cells and basal cells.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Immunofluorescence staining of paraffin sections (scale bar: 50\u0026nbsp;µm) of p63\u003csup\u003e+\u003c/sup\u003e/KRT5\u003csup\u003e+\u003c/sup\u003e basal cells, MUC5AC\u003csup\u003e+\u003c/sup\u003e goblet cells, TUBB4\u003csup\u003e+\u003c/sup\u003e ciliated cells and CCSP\u003csup\u003e+\u003c/sup\u003e club cells. (\u003cstrong\u003eB\u003c/strong\u003e) Whole mount immunofluorescence staining of complete ALI insert membranes (scale bar: 2000\u0026nbsp;µm) showing the expression of MUC5AC\u003csup\u003e+\u003c/sup\u003e goblet cells and TUBB4\u003csup\u003e+\u003c/sup\u003e ciliated cells. Quantification of area of (\u003cstrong\u003eC\u003c/strong\u003e) MUC5AC signal and (\u003cstrong\u003eD\u003c/strong\u003e) TUBB4 signal on whole mount immunofluorescence staining of ALI insert membranes. Results are shown as mean ± SD, replicates represent number of different field‑of‑views of multiple ALI cultures: n=9-13 per group; Unpaired two-tailed t test: ns – none significant, *p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001. (\u003cstrong\u003eE\u003c/strong\u003e) Scanning electron microscopy (SEM) of the apical surface of ALI cultures showing multi ciliated cells and other microvilli carrying cells reflecting the more clustered distribution of ciliated cells in iALI cultures obvious also after TUBB4 staining in (B) (scale bar: 10\u0026nbsp;µm).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/d37a6e869bc592f2f032899b.png"},{"id":90543757,"identity":"2dc0871a-042e-4261-a1d3-7672baddbc07","added_by":"auto","created_at":"2025-09-04 00:14:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eiALI cultures show protein expression of epithelial ion channels CFTR and TMEM16A as well as tight junction marker ZO‑1.\u003c/strong\u003e Western blot analyses were performed on iALI and pALI cultures as well as human bronchial epithelial cells (HBE; 16HBE14o-) as a control. Treatment with CFTR modulators ETI was applied for 48 h (WT – Healthy, CF – homozygous CFTR Phe508del, - vehicle, + ETI‑treated). (\u003cstrong\u003eA\u003c/strong\u003e) Expression of CFTR B band at 130 kDa and CFTR C band at approx. 140-180 kDa. WT and CF\u003csup\u003eETI\u003c/sup\u003e iALI cultures expressed CFTR C band at approx. 140 kDa. (\u003cstrong\u003eB\u003c/strong\u003e) Staining for TMEM16A shows the expected band at approx. 120-140 kDa. (\u003cstrong\u003eC\u003c/strong\u003e) ZO‑1 detection at approx. 195-250 kDa. (\u003cstrong\u003eD\u003c/strong\u003e) β-actin reference staining. All lanes were loaded with 40 µg of protein. Presented exposure times were selected for optimal display of banding pattern and are highlighted below the blot lanes. Western blot analyses have been performed as replicates of n=3-4. Replicates and images of the complete blot membrane are provided in the supplementary data of Supplemental Figure 2, Supplemental Figure 3, Supplemental Figure 4 and Supplemental Figure 5.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/f91bc23186142b81e5cc26a0.png"},{"id":90543749,"identity":"b2a341a3-fb8a-4a00-b82c-458cb46fc2c8","added_by":"auto","created_at":"2025-09-04 00:14:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":252810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCF iALI and CF pALI cultures show an impaired transepithelial chloride transport that is partially recovered by treatment with ETI.\u003c/strong\u003e Measurements of the transepithelial ion transport in iALI and pALI cultures by recording of the short circuit current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e) in Ussing chamber experiments. Treatment with CFTR modulators ETI was applied for 48 h (WT – Healthy, CF – homozygous CFTR Phe508del, CF\u003csup\u003eDMSO\u003c/sup\u003e\u0026nbsp;– vehicle, CF\u003csup\u003eETI\u003c/sup\u003e\u0026nbsp;–\u0026nbsp;ETI-treated). Exemplary original recordings of \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e measurements in (A) iALI cultures and (B) pALI cultures. (\u003cstrong\u003eC–J\u003c/strong\u003e) data summary of \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e measurements in iALI and pALI cultures. (C) transepithelial electrical resistance (\u003cem\u003eR\u003c/em\u003e\u003csub\u003eTE\u003c/sub\u003e), (D) basal \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e, (\u003cstrong\u003eE\u003c/strong\u003e) amiloride‑insensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e, (\u003cstrong\u003eF\u003c/strong\u003e) amiloride‑sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e (\u003cem\u003eΔAmiloride\u003c/em\u003e), (\u003cstrong\u003eG\u003c/strong\u003e) forskolin/IBMX‑sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e (\u003cem\u003eΔFsk/IBMX\u003c/em\u003e), (\u003cstrong\u003eH\u003c/strong\u003e) CFTR\u0026nbsp;inhibitor‑172‑sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e (\u003cem\u003eΔCFTRinh-172\u003c/em\u003e), (\u003cstrong\u003eI\u003c/strong\u003e) UTP‑sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e (\u003cem\u003eΔUTP\u003c/em\u003e) and (\u003cstrong\u003eJ\u003c/strong\u003e) GlyH‑101‑sensitive \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e (\u003cem\u003eΔGlyH‑101\u003c/em\u003e). Right y‑axis only applies to pALI cultures if shown. Results are shown as mean ± SD, replicates represent individual ALI cultures that were derived from multiple independent differentiations: n=30-57 for iALI cultures, n=10-27 for pALI cultures; Unpaired two-tailed t test: ns – none significant, *p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/86633add4abae133a757b778.png"},{"id":90543747,"identity":"d10e6d90-dfb6-422e-ac1f-ce49ec9fa972","added_by":"auto","created_at":"2025-09-04 00:14:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":581067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCF iALI and CF pALI cultures show a reduction of the mucus layer height compared to WT ALI cultures.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Sample preparation before imaging of the mucus layer by TEM (generated with Biorender). (\u003cstrong\u003eB\u003c/strong\u003e) Mucus layer height (\u003cem\u003eMLH\u003c/em\u003e) measurements were manually performed on TEM images as presented in (\u003cstrong\u003eC\u003c/strong\u003e). Results are shown as mean ± SD, replicates represent technical replicates measured on single biological replicates (ALI cultures): n=61-238 per group. Unpaired two-tailed t test: ns – none significant, *p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001. (\u003cstrong\u003eC\u003c/strong\u003e) Transmission electron microscopy images of the mucus layer in iALI and pALI cultures. Results are shown at low magnification for complete overview of the cellular surface (at the bottom) and mucus layer. Structural labelling: A, air; C, cilia; M, mucus; dotted line, surface lining of the mucus layer. CF\u003csup\u003eDMSO\u003c/sup\u003e iALI and CF\u003csup\u003eDMSO\u003c/sup\u003e pALI cultures showed notable differences in \u003cem\u003eMLH\u003c/em\u003e and quality. The mucus layer generally appeared denser and lower, with a distinctively sharp air facing surface lining (white arrowheads). On WT ALI cultures and CF\u003csup\u003eETI\u003c/sup\u003e ALI cultures, the mucus layer was higher and more dispersed, with a fissured air facing surface lining.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/6f556c2967dba73f69cbd65d.png"},{"id":90544866,"identity":"35500138-8547-4974-b53a-4b2cb0e3e38b","added_by":"auto","created_at":"2025-09-04 00:22:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":703456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHighly dense mucus layer in CF iALI and CF pALI cultures is loosened up after treatment with CFTR modulators ETI.\u003c/strong\u003e Transmission electron microscopy was performed on (\u003cstrong\u003eA+C+E\u003c/strong\u003e) iALI and (\u003cstrong\u003eB+D+F\u003c/strong\u003e) pALI cultures. (\u003cstrong\u003eA+B\u003c/strong\u003e): WT; (\u003cstrong\u003eC+D\u003c/strong\u003e): CF\u003csup\u003eDMSO\u003c/sup\u003e; (\u003cstrong\u003eE+F\u003c/strong\u003e): CF\u003csup\u003eETI\u003c/sup\u003e. Treatment with CFTR modulators ETI was applied for 24 h (WT – Healthy, CF – homozygous CFTR ΔF508, CF\u003csup\u003eDMSO\u003c/sup\u003e – vehicle, CF\u003csup\u003eETI\u003c/sup\u003e – ETI-treated). Results are shown at high magnification of the mucus layer. Boxed areas are depicted at doubled magnification. Scale bar shown in (\u003cstrong\u003eA\u003c/strong\u003e). (\u003cstrong\u003eB+E\u003c/strong\u003e) the mucus on CF\u003csup\u003eDMSO\u003c/sup\u003e iALI and CF\u003csup\u003eDMSO\u003c/sup\u003e pALI cultures showed a distinctively higher density and contain inclusions of vesicles and cellular debris. Analysis was performed on one biological replicate (ALI culture) per group (n=1).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/1f31f9e8f47cb55d4398c90e.png"},{"id":90543751,"identity":"04fdcc92-c6fe-494e-8eef-fe1613bc855c","added_by":"auto","created_at":"2025-09-04 00:14:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":216534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCF iALI and CF pALI cultures show an reduction of ciliary beating and corresponding impairment of mucociliary function that could be recovered by treatment with ETI.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Essential components for the measurement of the ciliary beat frequency (\u003cem\u003eCBF\u003c/em\u003e) and corresponding active area of ciliary motion using high‑speed video recordings and SAVA software‑based analysis (generated with Biorender). Treatment with CFTR modulators ETI was applied for 24 h (WT – Healthy, CF – homozygous CFTR ΔF508, CF\u003csup\u003eDMSO\u003c/sup\u003e – vehicle, CF\u003csup\u003eETI\u003c/sup\u003e – ETI-treated). (\u003cstrong\u003eB\u003c/strong\u003e) Heat maps showing exemplary fields of view of the \u003cem\u003eCBF\u003c/em\u003e measurement. Data summary of (\u003cstrong\u003eC\u003c/strong\u003e) the active area of ciliary motion and (\u003cstrong\u003eD\u003c/strong\u003e) the \u003cem\u003eCBF\u003c/em\u003e recorded in iALI and pALI cultures. Results are shown as mean ± SD, replicates represent individual ALI cultures that were derived from multiple independent differentiations: n=8-14 per group. Unpaired two-tailed t test: ns – none significant, \u003cem\u003e*p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/d0730e2b2fa79c31021b19a7.png"},{"id":94490424,"identity":"5fdce00f-0a68-467f-9f2f-20f6d3c1ec7a","added_by":"auto","created_at":"2025-10-27 17:09:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3934228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/902cf410-204b-4cc5-a216-d39a100c64ee.pdf"},{"id":90543758,"identity":"df1a68da-4db7-4f7a-8fe2-1888be10c24f","added_by":"auto","created_at":"2025-09-04 00:14:15","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":8463951,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFiguresJaboreckCFdiseasemodel.docx","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/cf0c479f286c286c1593a335.docx"},{"id":90543743,"identity":"79660ecc-5198-44d9-b605-6b13abdddee1","added_by":"auto","created_at":"2025-09-04 00:14:15","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":89565,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables1Dataavailablility.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/398ae7635ea48364035eb938.xlsx"},{"id":90544867,"identity":"c0eb2a51-09cb-47af-912f-f407f8edc77e","added_by":"auto","created_at":"2025-09-04 00:22:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15364,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2Supplemtarymaterials.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7184232/v1/f569e88961a3571502c7cce1.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Human induced pluripotent stem cells for in vitro modeling of impaired mucociliary clearance in cystic fibrosis lung disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCystic fibrosis (CF) is a rare recessive genetic disorder that affects approximately 100,000 people worldwide. It is caused by mutations of the Cystic fibrosis transmembrane conductance regulator (CFTR) gene [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which encodes a cAMPregulated chloride and bicarbonate channel protein. Mutations in the CFTR gene have been shown to affect the transepithelial ion transport in multiple organs, with CF lung disease being the primary cause of morbidity and mortality [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To date, over 700 CF-causing mutations of the CFTR gene have been identified [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn healthy individuals, the respiratory epithelium plays a critical role in the host defense against pulmonary infection. In the airways, the secreting cell types, particularly goblet cells, produce a protective layer of mucus that covers the epithelial surface and traps inhaled pathogens. Subsequent to this initial defensive barrier, mucociliary clearance (MCC) is initiated through ciliary beating, which effectively removes mucus and pathogens from the respiratory system [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn CF, CFTR mutations result in reduced chloride secretion and concurrent hyperabsorption of sodium by the CFTR-regulated epithelial sodium channel (ENaC). The reduction in apical ion secretion causes the dehydration of airway mucus and the subsequent increase in mucus viscosity [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This increased viscosity impairs ciliary movement and MCC, consequently leading to chronic airway infection, inflammation, and ultimately progressive loss of lung function and lung failure [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe development of highly effective small-molecule drugs, known as CFTR modulators, to restore CFTR function in CF patients has profoundly changed the clinical landscape [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The most prominent classes of CFTR modulators include corrector drugs, which correct the folding and trafficking of the mutant CFTR protein, and potentiator drugs, which enhance the activity of the CFTR protein [\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Notably, the triple CFTR modulator combination (elexacaftor-tezacaftor-ivacaftor; ETI) has been shown to provide substantial clinical benefit in CF patients carrying a Phe508del CFTR allele. However, it should be noted that ETI treatment does not fully restore CFTR function and observational studies suggest that residual abnormalities persist in patients [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In addition, many of the patients with other mutations do not benefit from ETI. The development of novel drugs that restore CFTR function to patients carrying other so far untreatable mutations necessitates the use of improved in vitro systems that closely reflect CF lung disease including impaired MCC as the main pathomechanism. Furthermore, the identification of genetic modifiers of CF disease, which apparently lead to a wide range of mild to severe phenotypes in patients carrying the Phe508del mutation, is imperative [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Such genetic modifiers may represent novel therapeutic targets in CF.\u003c/p\u003e\u003cp\u003eTo date, intestinal organoids and primary airway cells are most commonly used for these purposes. Intestinal organoids, cultivated from rectal biopsies and analyzed by forskolin-induced swelling (FIS), function as a standard for drug testing [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, their utility is limited in predicting the efficacy of CF lung disease drugs or studying airway-specific genetic modifiers [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This limitation also stems from their lack of cilia, which prevents them from modeling mucociliary clearance (MCC), a key feature of CF lung disease. Conversely, primary airway epithelia cultured in air-liquid interface (ALI) conditions exhibit a greater degree of similarity to the pathophysiology of CF in vivo and are instrumental in the development of personalized medicine and preclinical drug development [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, the supply of sufficient quantities of these cells from patients remains challenging [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, gene editing, particularly at the clonal level, remains challenging in both systems, making the generation of isogeneic control lines impractical.\u003c/p\u003e\u003cp\u003eIn recent years, human induced pluripotent stem cells (hiPSCs) have emerged as a novel cell source for in vitro models of CF [\u003cspan additionalcitationids=\"CR48 CR49 CR50\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. HiPSCs can easily be generated from patients that carry specific CFTR variants, for instance by reprogramming of small blood samples [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These cells are characterized by their virtually unlimited proliferation capacity and potential to differentiate into all cell types of the human body [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In contrast to primary intestinal and airway cells, hiPSCs facilitate gene editing on a clonal level to introduce seamless correction of mutations, gene knockouts and overexpression, and integration of reporter genes. This provides tools to study the physiologic and therapeutic relevance of candidate genes or to perform high throughput screens [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56 CR57 CR58\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Although several studies have demonstrated the differentiation of hiPSCs into airway epithelial cells in organoid or ALI cultures, most differentiation protocols are relatively complex and still associated with considerably variable differentiation efficiencies [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan additionalcitationids=\"CR61 CR62 CR63 CR64\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. While CFTR function has already been demonstrated in these cells [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], cellular impurities frequently complicated studies that aimed to demonstrate that hiPSCderived airway cells closely recapitulate CF lung disease comparable to primary airway epithelial cells, which are currently considered the gold standard in the field.\u003c/p\u003e\u003cp\u003eIn this study, we sought to expand upon the findings of recent investigations by undertaking a comprehensive characterization of CF patient-specific hiPSC-derived airway epithelial cells in ALI (iALI) cultures. Our objective was to demonstrate the potential of iALI cultures as a preclinical disease model and as a drug testing platform. Our results show that despite the use of a relatively simple and straightforward differentiation protocol, iALI cultures have a high degree of similarity to primary airway (pALI) cultures, including mRNA and protein expression, mucus (ultra)structure and ion channel function. Measurement of ciliary beat frequency (CBF), which directly reflects impaired mucus viscosity and ciliary transport as a major pathomechanism in CF lung disease, was applied to iALI cultures to overcome problems associated with cellular impurities due to variable differentiation efficiencies. The CF iALI cultures carrying the Phe508del CFTR mutation closely recapitulated CF lung disease in vitro, showing a severe impairment of mucociliary function and also a reduction of chloride conductance, which was partially rescued by the triple CFTR modulator combination ETI, comparable to clinical findings. By providing an unlimited supply of patient-specific cells, iALI cultures will serve as a valuable tool in CF research. Our iALI culture platform will facilitate individualized drug development and the identification of alternative therapeutic targets. It will also accelerate the development of personalized therapies and their clinical translation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eiALI cultures derived from healthy individuals and CF patient-specific hiPSCs share characteristic features of airway epithelial cells\u003c/p\u003e\u003cp\u003eHealthy (wild type, WT) hiPSCs [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] and CF patientspecific (CF) hiPSCs carrying a homozygous CFTR Phe508del mutation were differentiated into iALI cultures by applying a multistage differentiation protocol [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. First, hiPSCs were differentiated into the definitive endoderm (DE) until day 3 of differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Flow cytometry analysis was performed, which confirmed high DE marker expression of \u0026gt;\u0026thinsp;97.0% in both WT and CF DE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). As an intermediate step in the differentiation process, cryopreservation of DE cells was implemented to create WT and CF DE batches for all subsequent differentiations into iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following the thawing process, DE cells were differentiated into CPM\u003csup\u003e+\u003c/sup\u003e/NKX2.1\u003csup\u003e+\u003c/sup\u003e lung progenitor (LP) cells yielding 41.6\u0026thinsp;\u0026plusmn;\u0026thinsp;15.5% NKX2.1\u003csup\u003e+\u003c/sup\u003e WT cells and 47.4\u0026thinsp;\u0026plusmn;\u0026thinsp;20,5% NKX2.1\u003csup\u003e+\u003c/sup\u003e CF cells. A magnetic-activated cell sorting (MACS) for CPM\u003csup\u003e+\u003c/sup\u003e cells was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) to enrich LP cells, yielding 79.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2% NKX2.1\u003csup\u003e+\u003c/sup\u003e WT cells and 83.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8% NKX2.1\u003csup\u003e+\u003c/sup\u003e CF cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Finally, LP cells were seeded on inserts for ALI cultivation and were matured by cultivation for a minimum of an additional 27 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To enable direct comparison with primary airway cultures as the current gold standard in CF research, we generated pALI cultures from a healthy (WT) donor and a CF patient, carrying a homozygous CFTR Phe508del mutation (CF). Our proof-of-concept study included each a single WT hiPSC line and CF hiPSC line as well as primary airway cells from a single WT and CF donor, which were all (hiPSC and primary cells) derived from independent donors.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA combination of transcription analysis, immunofluorescence staining and electron microscopy was used to confirm the formation of airway epithelial cells in iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplemental Fig.\u0026nbsp;1). qRT-PCR analysis demonstrated the expression of epithelial cell type markers in iALI and pALI cultures including markers for basal cells (p63, KRT5), club (CCSP) cells, goblet cells (MUC5AC) and ciliated cells (FOXJ1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, the expression of NKX2.1, a lung development marker [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], CFTR and TMEM16A, another chloride channel and a potential alternative target for CF therapies [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe prominent features and morphology of airway epithelial cells were found in both iALI cultures and pALI cultures. iALI cultures exhibited a polarized morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and contained p63\u003csup\u003e+\u003c/sup\u003e/KRT5\u003csup\u003e+\u003c/sup\u003e basal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) as well as more mature epithelial cell types, including MUC5AC\u003csup\u003e+\u003c/sup\u003e goblet cells, TUBB4\u003csup\u003e+\u003c/sup\u003e ciliated cells and CCSP\u003csup\u003e+\u003c/sup\u003e club cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e BD). Additionally, iALI cultures contained BSND\u003csup\u003e+\u003c/sup\u003e ionocytes, which were only found at a low frequency in pALI and iALI cultures (Supplemental Fig.\u0026nbsp;1). The immunofluorescence signal of the entire ALI insert membrane revealed comparable numbers of TUBB4\u003csup\u003e+\u003c/sup\u003e ciliated cells in iALI and pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, the number of MUC5AC\u003csup\u003e+\u003c/sup\u003e goblet cells was significantly lower in iALI cultures than in pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additional imaging of the apical surface via scanning electron microscopy (SEM) validated these observations and confirmed the prevalent presence of ciliated cells in iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In accordance with the immunofluorescence staining in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, ciliated cells in iALI cultures appeared to be more clustered and interspersed with groups of nonciliated cells. Morphologic differences between WT and CF epithelial cells were not observed in iALI or pALI cultures.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, we performed Western blot analyses to investigate the protein expression of CFTR, of TMEM16A, a calcium-activated chloride channel [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], and of the tight junction marker ZO-1, both important for proper function of the airway epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplemental Fig.\u0026nbsp;2, Supplemental Fig.\u0026nbsp;3, Supplemental Fig.\u0026nbsp;4, Supplemental Fig.\u0026nbsp;5). In both iALI and pALI cultures we detected CFTR B band at 130 kDa and CFTR C band between 140 and 180 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Supplemental Fig.\u0026nbsp;2). As expected, the Phe508del mutation led to a decline in the expression of the CFTR protein, particularly the CFTR C band, in both the CF iALI and CF pALI cultures in comparison to the WT controls. Treatment with the CFTR modulator drugs ETI for 48 h (3\u0026micro;M elexacaftor, 18 \u0026micro;M tezacaftor and 1 \u0026micro;M ivacaftor) was applied to rescue the protein expression and function of the Phe508del mutated CFTR protein (CF\u003csup\u003eDMSO\u003c/sup\u003e \u0026ndash; vehicle, CF\u003csup\u003eETI\u003c/sup\u003e \u0026ndash; ETItreated). Western blot analysis revealed that the expression of CFTR C band was increased to a varying degree in CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures as shown in three out of four replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Supplemental Fig.\u0026nbsp;2, Supplemental Fig.\u0026nbsp;5). In accordance with the qRT-PCR data, all iALI and pALI cultures showed protein expression of TMEM16A and ZO-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026thinsp;+\u0026thinsp;B, Supplemental Fig.\u0026nbsp;3, Supplemental Fig.\u0026nbsp;4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePhe508del CF iALI cultures show an impaired CFTR-dependent transepithelial chloride conductance that can be partially rescued by treatment with ETI\u003c/p\u003e\u003cp\u003eThe bioelectric properties of WT and CF patientderived iALI and pALI cultures were studied in Ussing chamber experiments to evaluate the basic chloride transport defect in CF iALI and CF pALI cultures, as well as to assess the rescue of mutant CFTR chloride channel function by ETI treatment (48 h) through measurements of the short circuit current (I\u003csub\u003eSC\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026thinsp;+\u0026thinsp;B).\u003c/p\u003e\u003cp\u003eIn both CF\u003csup\u003eDMSO\u003c/sup\u003e iALI and CF\u003csup\u003eDMSO\u003c/sup\u003e pALI cultures, the transepithelial electrical resistance (R\u003csub\u003eTE\u003c/sub\u003e) was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), whereas the basal I\u003csub\u003eSC\u003c/sub\u003e and amilorideinsensitive I\u003csub\u003eSC\u003c/sub\u003e were decreased compared to respective WT ALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026thinsp;+\u0026thinsp;E). While pALI cultures generally exhibited a more pronounced amiloride response compared to iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;F), only minor variations in the ΔAmiloride were observed between WT iALI and CF\u003csup\u003eDMSO\u003c/sup\u003e iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). This finding indicates that neither the Phe508del mutation nor ETI significantly impacted ENaC activity in the examined cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eFollowing activation of CFTR with forskolin/IBMX, both WT iALI and WT pALI cultures showed a sustained plateau phase of increased chloride transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;G). In accordance with the impaired CFTRmediated chloride transport observed in individuals with CF, the recorded forskolin/IBMX-sensitive I\u003csub\u003eSC\u003c/sub\u003e (ΔFsk/IBMX) was reduced in CF\u003csup\u003eDMSO\u003c/sup\u003e iALI and CF\u003csup\u003eDMSO\u003c/sup\u003e pALI cultures compared to their respective WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;G). iALI cultures exhibited a significant but substantially lower response after treatment with the CFTR inhibitor-172 (ΔCFTRinh-172) compared to pALI cultures. Nevertheless, the CFTR inhibitor-172 significantly reduced CFTR activity in all WT ALI cultures, and to a lesser extent in CF\u003csup\u003eDMSO\u003c/sup\u003e ALI cultures, reflecting residual activity of the Phe508del mutant CFTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;H).\u003c/p\u003e\u003cp\u003eFurthermore, UTPmediated activation of calcium activated chloride channels (CaCCs), such as TMEM16A, reached significantly greater amplitudes in pALI cultures than in iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;I). CF\u003csup\u003eDMSO\u003c/sup\u003e iALI cultures exhibited an increased UTP-sensitive I\u003csub\u003eSC\u003c/sub\u003e (ΔUTP) in comparison to CF\u003csup\u003eETI\u003c/sup\u003e iALI cultures, while only negligible to none differences were observed between WT and CF pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;I).\u003c/p\u003e\u003cp\u003eFinally, the unspecific inhibition of chloride conductance was performed by adding of GlyH-101. As expected because of the decreased chloride transport in CFTR mutated cells, the GlyH-101sensitive I\u003csub\u003eSC\u003c/sub\u003e (ΔGlyH-101) was found to be more prominent in WT ALI cultures than in CF\u003csup\u003eDMSO\u003c/sup\u003e ALI and could be observed in both cell systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ).\u003c/p\u003e\u003cp\u003eOverall, the recorded I\u003csub\u003eSC\u003c/sub\u003e confirmed the activity of epithelial ion channels, such as ENaC, CFTR and CaCCs in iALI cultures, similarly to pALI cultures. iALI cultures generally produced lower currents than pALI cultures, and measurement of the I\u003csub\u003eSC\u003c/sub\u003e in iALI culture may be accompanied by a lower sensitivity for individual parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Nonetheless, treatment with CFTR modulators ETI for 48 h resulted in a partial rescue of the impaired chloride conductance in both CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures that was significant compared to untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In comparison to CF\u003csup\u003eDMSO\u003c/sup\u003e ALI cultures, R\u003csub\u003eTE\u003c/sub\u003e was reduced in CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Concurrently, basal I\u003csub\u003eSC\u003c/sub\u003e and amilorideinsensitive I\u003csub\u003eSC\u003c/sub\u003e were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026thinsp;+\u0026thinsp;E). Furthermore, the levels of ΔFsk/IBMX increased in both CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures, accounting for approximately 62.0% and 44.9% of CFTR activity measured in WT iALI and WT pALI cultures, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). In addition, both CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures exhibited a significant increase of CFTR inhibition, as reflected by the ΔCFTRinh-172 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Moreover, a reduction in ΔUTP was observed in CF\u003csup\u003eETI\u003c/sup\u003e iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). ΔGlyH-101 was increased in CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures but not in CF\u003csup\u003eETI\u003c/sup\u003e iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCF iALI cultures show altered mucus structure and mucus layer height that can be partially rescued by treatment with CFTR modulators ETI\u003c/p\u003e\u003cp\u003eGiven that impaired mucociliary function is a hallmark of CF lung disease and appears to be at least partially rescued clinically through ETI treatment, we sought to determine whether the thickness of the mucus layer and mucus structure in CF iALI and CF pALI cultures reflect the CF disease phenotype. To this end, we characterized the ultrastructure of the mucus layer in iALI and pALI cultures from WT and CF donors by highpressure freezing transmission electron microscopy (TEM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe TEM imaging and mucus height measurements revealed that the mucus layer exhibited a comparable structure and properties in iALI and pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Supplemental Fig.\u0026nbsp;6). In all ALI cultures, the mucus layer was composed of more or less densely packed molecular networks, which were visible throughout most of the prepared cross sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Supplemental Fig.\u0026nbsp;6), and exhibited a mucus layer height (MLH) of variable degree (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The CF\u003csup\u003eDMSO\u003c/sup\u003e ALI cultures exhibited a denser mucus layer that typically showed a distinctive surface lining, which closely reflected the in vivo phenotype of CF lung disease. In contrast, the mucus layer in WT ALI cultures was less dense and showed a loose surface lining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Supplemental Fig.\u0026nbsp;6). The measured MLH was generally lower in CF\u003csup\u003eDMSO\u003c/sup\u003e ALI cultures than in WT cultures in both the iALI and pALI culture systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026thinsp;+\u0026thinsp;C). Treatment with ETI for 24 h not only increased the MLH in CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures but resulted in an even thicker mucus layer in CF\u003csup\u003eETI\u003c/sup\u003e iALI cultures than in WT iALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026thinsp;+\u0026thinsp;C). In addition, the mucus structure was less dense and showed a loose surface lining in CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Supplemental Fig.\u0026nbsp;6). These findings suggest a rehydration of the mucus layer and complete rescue of the disease phenotype by ETI in the iALI culture system, which so far could not be observed or concluded in Ussing chamber measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, TEM imaging confirmed the presence of cilia and microvilli in both iALI and pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Supplemental Fig.\u0026nbsp;6).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCF iALI cultures show decreased ciliary beat frequency that can be partially rescued by treatment with CFTR modulators ETI\u003c/p\u003e\u003cp\u003eCFTR dysfunction leads to airway surface dehydration, which results in the secretion of highly viscoelastic mucus that impairs proper ciliary function and thus clearance of mucus, pathogens and other inhaled particles. Due to the laborious and challenging nature of \u003cem\u003eMLH\u003c/em\u003e measurements and mucus structure visualization via TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), we sought an alternative method to directly quantify impairment of MCC, which largely determines the phenotype of CF lung disease. Given that the decreased chloride transport in CF results in elevated mucus viscosity and subsequent impairment of ciliary movement and MCC, we opted to utilize high-speed video microscopy to assess the ciliary beat frequency (\u003cem\u003eCBF\u003c/em\u003e). Notably our approach involved the utilization of a specialized software tool (SAVA) that facilitates automated analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). This software was configured to quantify the active areas of ciliary movement as well as the CBF.\u003c/p\u003e\u003cp\u003eMotile cilia were detected in all iALI and pALI cultures and measurements confirmed a generally reduced active area of ciliary beating in iALI cultures compared to pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u0026thinsp;+\u0026thinsp;C). This finding corresponds with the more clustered appearance of ciliated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026thinsp;+\u0026thinsp;E). However, the CBF levels in WT iALI cultures generally surpassed those observed in WT pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). While CF iALI cultures measurements exhibited a less distinct phenotype in the Ussing chamber compared to CF pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), CF iALI cultures demonstrated an even more pronounced disease phenotype in the CBF assay, characterized by a significant impairment of the ciliary beating (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u0026thinsp;+\u0026thinsp;D). Despite the presence of ciliated cells, the active area and CBF were significantly diminished in CF\u003csup\u003eDMSO\u003c/sup\u003e pALI cultures, and in CF\u003csup\u003eDMSO\u003c/sup\u003e iALI cultures the ciliary beating was either not visible in the majority of cultures, or below the detection limit (2 Hz) of the software (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e BD). Following 24 h of ETI treatment, a significant augmentation in the active area and CBF was observed in both CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e BD). Also, all CF\u003csup\u003eETI\u003c/sup\u003e ALI cultures generally exhibited a greater CBF than the respective WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Similar to the MLH measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), these findings suggest a rehydration of the mucus layer that consequently reduced the mucus viscosity and accelerated the ciliary beating (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u0026thinsp;+\u0026thinsp;D). Again, ETI treatment led to a complete rescue of the disease phenotype and even overcompensation of the CBF in both CF\u003csup\u003eETI\u003c/sup\u003e iALI and CF\u003csup\u003eETI\u003c/sup\u003e pALI cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn CF research, reliable patient-specific in vitro models are a crucial to developing novel CFTR modulator drug against currently untreatable CFTR mutations and identifying genetic modifiers as novel therapeutic targets.\u003c/p\u003e\u003cp\u003eAlthough, intestinal organoids and primary airway cells are currently considered the gold standards in the field, both systems face particular limitations concerning either their ability to reflect important aspects of CF lung disease or the availability of sufficient quantities of patient-specific cells, respectively.\u003c/p\u003e\u003cp\u003eIn principle, all these issues and limitations can be adequately addressed in organotypic models consisting of airway epithelial cells derived from patientspecific hiPSCs. Several studies demonstrated the differentiation of hiPSCs into airway epithelial cells. However, currently available differentiation protocols face two major difficulties. On the one hand, many of the available protocols results in highly variable outcomes in terms of efficiency and cellular purity. As a result, the sensitivity of FIS assays and electrophysiological measurements are compromised [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. On the other hand, more advanced protocols, namely those of Hawkins and colleagues, are difficult to establish due to their relatively high complexity and are more cost- and time-intensive than others [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan additionalcitationids=\"CR61 CR62 CR63 CR64\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], which is critical in particular on a higher throughput scale as often required in drug development.\u003c/p\u003e\u003cp\u003eGoing beyond published work, we focused on a comprehensive comparison of organotypic hiPSCderived airway cultures (iALI cultures) and primary airway cultures (pALI cultures), the current gold standard in CF research, in terms of proper gene and protein expression, structural characteristics and functionality. We demonstrate the usefulness of these iALI cultures, generated via a relatively simple and fast protocol that is easily scalable in 2D and does not require intermediate organoid cultivation. Application of an automatable CBF assay allows determination of decreased CBF which directly reflects reduced mucociliary clearance due to increased mucus viscosity as key pathomechanism of CF lung disease. Our results show that this assay is not affected by a variable cellular composition of iALI cultures and suggest an even higher sensitivity than measurement of pALI cultures.\u003c/p\u003e\u003cp\u003eWe verified that iALI cultures contain a structured and polarized airway epithelium, which is composed of basal cells, ciliated cells, club cells, goblet cells and ionocytes, and which exhibit expression of CFTR and TMEM16A. In both iALI and pALI cultures, minor variations in the expression levels were observed between WT and CF cells, which however are likely not dependent on the mutated CFTR gene. It can be assumed that these variations reflect donor differences. In case of iALI cultures they may also reflect the outcome of differentiation, which depends on the donor or cell clone, and may lead to different cell composition and maturity. The low magnification images of the entire wells stained for MUC5AC and TUBB4 illustrate that iALI cultures contain a high abundance of ciliated and goblet cells often arranged as clusters. Electron microscopy revealed that in iALI cultures, in contrast to pALI cultures, regions covered with ciliated cells were interspersed with flat cells that may comprise non-ciliated epithelia as well as other cell types. Of particular note are vimentin\u003csup\u003e+\u003c/sup\u003e mesenchymal cell lineages (data not shown), which represent cellular contaminants that are to a limited degree carried over during MACS of CPM\u003csup\u003e+\u003c/sup\u003e/NKX2.1\u003csup\u003e+\u003c/sup\u003e LP cells.\u003c/p\u003e\u003cp\u003eWhile the significance of ionocytes in CF lung disease remains to be elucidated, a low number of ionocytes were also detected in iALI cultures. It is noteworthy that these ionocytes have been described as a rare epithelial cell type that exhibits exceptionally high levels of CFTR expression and might be of particular interest for CF research in the future [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTypical for WT-CFTR, we detected a much lower expression of the immature coreglycosylated glycoisoform B (CFTR B) in both WT pALI and iALI cultures, than of the mature complex glycosylated glycoisoform C (CFTR C). According to the findings of previous studies [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], our CF cultures both exhibit a substantially lower level of the CFTR C compared to the CFTR B. Notably, our data indicate an even more pronounced effect of the Phe508del mutation compared to the recent published findings [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Finally, although a substantial variation was observed between biological replicates for iALI and pALI cultures, ETI treatment increased the proportion of the CFTR C, which is consistent with previously published data [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. These findings provide further evidence that iALI cultures behave comparable with regard to the effect of CFTR correctors (elexcaftor and tezacaftor) on CFTR glycosylation and maturation, both crucial for trafficking and stability.\u003c/p\u003e\u003cp\u003eAside from this, we identified protein expression for TMEM16A, a potential alternative target for CF therapies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e], and ZO-1. While the expression levels of ZO-1 remained consistent across all samples, TMEM16A expression exhibited significant variability. This variability appeared to be independent of the presence of the Phe508del mutation and may rather be donor- or, in case of iALI cultures, clone-dependent, and may additionally be influenced by the differentiation outcome.\u003c/p\u003e\u003cp\u003eOverall, iALI cultures demonstrated a significant degree of molecular similarity to the epithelial cells present in pALI cultures. The Phe508del mutation resulted in reduced expression of CFTR C protein, while other variations between WT and CF cultures were likely due to cell line- and clone-specific factors. Additionally, cellular contaminants carried over during MACS of CPM\u003csup\u003e+\u003c/sup\u003e/NKX2.1\u003csup\u003e+\u003c/sup\u003e LP cells were the most probable source of molecular differences between iALI and pALI cultures. However, these contaminants did not compromise the utility of iALI cultures for modeling CF lung disease, as discussed below.\u003c/p\u003e\u003cp\u003eAt the functional level of the airway epithelial cells, Ussing chamber experiments have demonstrated the activity of epithelial ion channels, including ENaC, CFTR, and CaCCs, in iALI cultures. While the activity of CFTR could be confirmed by means of stimulation with forskolin/IBMX, we also assume the activity of other GlyH 101-sensitive chloride channels, such as SLC26A9 [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e], in iALI and pALI cultures, as CFTR inhibitor-172 did not fully inhibit CFTR. In addition, our findings are consistent with previous reports, demonstrating that ETI only partially restores chloride transport in CF ALI cultures despite its considerable therapeutic benefit for patients with at least one Phe508del mutation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNonetheless, we also detected that iALI cultures generally produced lower currents than pALI cultures. Therefore, electrophysiological measurements in iALI cultures may be accompanied by a lower sensitivity for individual parameters. It is possible that the lower degree of maturity of the epithelial cells in iALI cultures contributes to this observation. However, it is more likely that contaminating non-epithelial cells, especially vimentin\u003csup\u003e+\u003c/sup\u003e mesenchymal cell lineages typically detectable at varying numbers, account for the lower currents and correspondingly lower assay sensitivity. Depending on the differentiation outcome, cells that lack expression of relevant ion channels may actually cover a considerable part of the ALI membranes between clusters of ciliated epithelia. These cells may substantially influence the measurement of I\u003csub\u003eSC\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eAccording to these results, we anticipated that the lower purity of our iALI cultures, compared to pALI cultures, would negatively affect the manifestation of CF-typical impairments in mucociliary function, such as reduced MLH, increased mucus density, and reduced CBF (due to dehydration and leading to increased viscosity). However, contaminating non-epithelial cells did not negatively affect MLH or mucus density, enabling accurate modeling of mucus impairment in CF lung disease. The mucus layer in iALI cultures was thicker than in pALI cultures, and the effect of the Phe508del-mutated CFTR was evident in both systems. In contrast to the measured chloride transport and CFTR activity, ETI treatment fully restored the MLH of CF ALI cultures to WT levels, even exceeding them in iALI cultures. Although not quantitatively assessed, ETI also restored the looser mucus density in CF iALI cultures, as observed in WT controls. Together, these findings indicate rehydration of the mucus layer following ETI treatment and highlight the significant clinical benefits of ETI treatment observed in CF patients [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Compared to electrophysiological measurements or the FIS assay in organoids, the measurement of MLH and mucus density may provide a more accurate reflection of the therapeutic effect of CF modulators on CF lung disease. Still, utilizing electron microscopy to evaluate the MLH and mucus density remains a very laborious and time-consuming process.\u003c/p\u003e\u003cp\u003eWe further sought an alternative approach to evaluate the impairment of MCC in CF. We considered tracking of mucociliary transport by the use of fluorescent particles as not suitable due to the reduced areas covered by ciliated cells in iALI cultures [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Therefore, we employed the CBF assay as a significantly more useful, straightforward and time-efficient tool. In particular, the CBF assay could potentially be automated and adapted for medium- and high-throughput applications, making it a promising tool for large-scale studies of MCC impairment in CF models. While pALI cultures exhibited substantially more active areas of ciliary beating, CBF levels remained comparable in iALI and pALI cultures. In comparison to pALI cultures, the effect of the Phe508del mutation was even more pronounced in iALI cultures, suggesting a more sensitive readout in iALI cultures. Certainly, further analyses of primary cells and iPSCderived airway cells from different patients have to confirm whether iALI cultures are generally more sensitive than primary airway cells. Nevertheless, we can confidently conclude that analysis of CBF on iPSCderived airway cells largely eliminates the current problem of variable differentiation efficiencies and closely reflects changes in mucociliary transport in the airways caused by CFTR mutations and CFTR modulator drugs. Notably, the application of ETI resulted in a CBF that surpassed the CBF in WT iALI cultures, suggesting that this approach may more accurately reflect the substantial therapeutic efficacy of ETI than the observed partial rescue of chloride transport function as measured by electrophysiological techniques or FIS assays.\u003c/p\u003e\u003cp\u003eWe believe that our iALI culture system, in combination with the CBF assay, offers a user-friendly platform that can be easily adopted by other laboratories and the broader CF research community. First, the relative simplicity of our differentiation protocol provides significant advantages, making our platform more time- and resource-efficient compared to existing protocols. For example, the current state-of-the-art protocol developed by Hawkins and colleagues includes a targeted basal cell generation step prior to ALI culture seeding, which significantly increases culture purity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, this protocol necessitates additional differentiation steps in Matrigel-based organoid cultures, which are both highly time- and cost-intensive even before ALI seeding. In contrast, our iALI cultures are derived from 2D cultures that require less expensive materials and can be easily scaled to produce larger quantities of cells. Second, despite the presence of considerable cellular contaminants in iALI cultures, our CBF assay remains unaffected by cellular contaminations, providing a robust readout of the primary defect in CF lung disease and demonstrating sensitivity to clinically relevant CFTR modulator drugs. Moreover, the CBF assay utilizes a straightforward setup consisting of an incubator chamber microscope equipped with a high-speed camera and the SAVA software for video recording and analysis. As mentioned before, the CBF assay could even be automated to enable screenings for novel CFTR modulator drugs. In comparison, other standard methods such as electrophysiological measurements in Ussing chambers require greater technical expertise, enable only a low sample throughput and provide no conclusive evidence for effects on the main impairment of MCC in CF lung disease.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our study demonstrates that patient-specific iALI cultures are indeed an excellent in vitro model of CF lung disease. We have leveraged the vast majority of the theoretical advantages of hiPSCs over immortalized cell lines, intestinal organoids, and primary airway cells. Taking advantage of the unlimited expansion potential of hiPSCs, which allows even largescale production of individual patientderived cells [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], we demonstrated that complex organotypic cultures can be generated with characteristic (ultra)structural features of highly polarized airway epithelial cells, including ciliated cells, goblet cells, club cells and underlying basal cells, covered with mucus whose height and density are contingent on CFTR function. We have demonstrated the functionality of CFTR and other characteristic ion channels relevant for CF lung disease at the electrophysiological level, including the effect of CF modulators that closely reflect clinical data. These functional studies have been complemented by an innovative in vitro assay that is suitable for high throughput applications to determine CBF. The CBF assay in iALI cultures has been shown to more closely reflect impaired MCC as the predominant pathomechanism of CF lung disease than existing in vitro systems. Furthermore, it has the capacity to directly visualize the effects of genomic modifiers of CF, as well as of novel drugs and drug combinations, on rare CFTR mutations that have remained untreatable to date.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eCell Culture of hiPSCs\u003c/em\u003e. A CFTR wild type hiPSC line (MHHi001A) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] (wild type, WT) and a hiPSC line (MHHi002A) carrying a homozygous CFTR Phe508del mutation as a diseased reference (CF) were used for all experiments. HiPSC lines were cultured in 5–8 mL of E8 medium (self-made) on Geltrex® (Thermo Fisher Science, A1413202)-coated culture vessels (25 cm² surface area). Media changes were conducted daily. Every three to four days the cells were passaged by dissociation with 1 mL Accutase™ (Sigma-Aldrich, A6964) and seeded at a density of 3.2 x 10\u003csup\u003e4\u003c/sup\u003e cells cm\u003csup\u003e− 2\u003c/sup\u003e while supplementing the medium with 10 µM ROCK inhibitor Y27632 (RI) (Tocris; 1254). All cells were maintained at 37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eDifferentiation of hiPSCs into airway epithelial cells (iALI cultures)\u003c/em\u003e. HiPSCs were differentiated towards definitive endoderm (DE) utilizing the STEMdiff™ Definitive Endoderm Kit (STEMCELL Technologies, 05115). Seven days (day − 7) before induction of the DE differentiation, hiPSCs were passaged and seeded at a density of 1.2 x 10\u003csup\u003e4\u003c/sup\u003e cells cm\u003csup\u003e− 2\u003c/sup\u003e on a 25 cm² culture vessel. From day − 3 onward the 8 mL E8 medium was supplemented with STEMdiff™ Definitive Endoderm TeSR™-E8™ Supplement (1:20). On day − 1 the cells were passaged and seeded at a density of 3.33 x 10\u003csup\u003e4\u003c/sup\u003e cells cm\u003csup\u003e− 2\u003c/sup\u003e on a 25 cm² culture vessel. In intervals of 24 h the media was changes to 5 mL of STEMdiff™ Endoderm Basal Medium supplemented with MR (1:100) and CJ (1:100) for the first 24 h and then supplement CJ for further 48 h. On day 3, the DE cells were dissociated with 1 mL Accutase™ and seeded at a density of 1.35 x 10\u003csup\u003e5\u003c/sup\u003e cells cm\u003csup\u003e− 2\u003c/sup\u003e on Geltrex®-coated 6well plates in 2 mL of basis medium (BM) supplemented with 3 µM Dorsomorphin (Merck, P5499), 10 µM SB431542 (kindly provided by A. Kirschning, Leibniz University Hannover) and 10 µM RI. All further media changes were conducted in intervals of 24 h. On day 4, media was changed to BM supplemented with 2 µM IWP2 (Torcis, 3533) and 10 µM SB431542. From day 5 until day 12, media changes were conducted with BM supplemented with 10 µm BMP4 (R\u0026amp;D Systems, 314-BP), 3 µM CHIR99021 (kindly provided by A. Kirschning, Leibniz University Hannover) and 10 nM FGF10 (R\u0026amp;D Systems; 345-FG). On day 13, cells were dissociated with 2 mL of Accutase™ per well and magneticactivated cell sorting (MACS) was applied to enrich CPM\u003csup\u003e+\u003c/sup\u003e lung progenitor cells. The following quantities are appropriate for MACS of 10\u003csup\u003e7\u003c/sup\u003e cells. MACS was performed by resuspension of cells in 320 µL MACS buffer (selfmade), initial blocking of the cells with 80 µL FcR blocking reagents (Miltenyi Biotec, 130-059-901) for 20 min and staining with 2µL anti-CPM antibody (FUJIFILM Wako, 014-27501) for 20 min. After centrifugation, cells were resuspended in 150 µL MACS buffer and labelling with 50 µL anti-mouse IgG MicroBeads (Miltenyi Biotec, 130-047-201) was performed. Cells were centrifuged and resuspended in 3 mL of Knockout™ DMEM (Thermo Fisher Science, 10829018) before performing cell separation in LS columns (Miltenyi Biotec, 130-042-401). For further maturation in ALI culture, CPM\u003csup\u003e+\u003c/sup\u003e cells were either seeded at a density of 2.65 x 10\u003csup\u003e5\u003c/sup\u003e cells cm\u003csup\u003e− 2\u003c/sup\u003e on ThinCert® inserts (Greiner Bio-One, 665641) or Costar® Snapwell™ inserts (Corning, 3801) coated with 804G medium (self-made) [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Cells were cultured in Small Airway Epithelial Cell Growth medium (PromoCell, C-21070) supplemented with 1.0 µM A 83 − 01 (Tocris, 2939), 0.2 µM DMH-1 (Tocris, 4126) and 5 µM RI (ADRI) for 96 h (adapted from Mou \u003cem\u003eet al.\u003c/em\u003e 2016 and McCauley \u003cem\u003eet al.\u003c/em\u003e 2017) [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] and 48 h in in PneumaCult™-ALI medium (STEMCELL Technologies, 05001) before apical medium was removed to induce airliquidinterface (ALI) cultures. ALI cultures were provided with 0.5 mL of medium from the apical side and 1–3 mL from the basolateral side. Media changes on the basolateral side were conducted every two to three days. Molecular and functional analyses were performed earliest on day 40/41 of differentiation. All iALI cultures were stimulated IL-13 (10 ng mL\u003csup\u003e− 1\u003c/sup\u003e; PeproTech, 200 − 13). In addition, CF ALI cultures were treated with elexacaftor (3 µM, Selleckchem, S8851), tezacaftor (18 µM, Selleckchem, S7059) and ivacaftor (1 µM, Selleckchem, S1144) (ETI) for 24 h or 48 h before analysis. All cells were maintained at 37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIsolation, Expansion and Differentiation of Primary Human Airway Epithelial Cells (pALI cultures).\u003c/em\u003e Human explanted lung tissue was obtained from the Hannover Lung Transplant Program after patients informed written consent. The tissue samples were thoroughly cleared of excess tissue to isolated the bronchial airways, cut into small pieces of a few millimetres in diameter and transferred into 30 mL of 0.18% Protease Type XIV (SigmaAdlrich, P5147) in Hank’s buffer (Thermo Fisher Science, 14175095). After dissociation for 2 h at 37°C, cells were transferred to PureCol®(Advanced BioMatrix, 5005)-coated flasks (75 cm²) and cultured in Small Airway Epithelial Cell Growth medium supplemented with 1.0 µM A 83 − 01, 0.48 µM CHIR99021, 0.2 µM DMH-1 and 5 µM RI (ACDRI). After expansion, cells were dissociated with TrypLE™ (Thermo Fisher Science, 12604013) and seeded at a density of 1.77 x 10\u003csup\u003e5\u003c/sup\u003e cells cm\u003csup\u003e− 2\u003c/sup\u003e on ThinCert® inserts or Costar® Snapwell™ inserts coated with PureCol®. Cells were first cultured in Small Airway Epithelial Cell Growth medium supplemented with ACDRI for 48 h. Then, the media was changed to PneumaCult™-ALI medium. ALI cultures were provided with 0.5 mL of medium from the apical side and 1–3 mL from the basolateral side. ALI cultures were provided with 0.5 mL of medium from the apical side and 1–3 mL from the basolateral side. After 48 h the apical medium was removed to induce proper ALI cultivation. Following cells were matured and media changes were conducted every two to three days. Molecular and functional analyses were performed on day 37/38 of differentiation. Before the analyses, all pALI cultures were treated with IL-13 (10 ng mL\u003csup\u003e− 1\u003c/sup\u003e) and partially ETI as previously described for iALI cultures. All cells were maintained at 37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eImmunofluorescence Staining.\u003c/em\u003e iALI and pALI cultures were fixed with 0.5 mL 4% paraformaldehyde (Morphisto, 11762) at room temperature (RT). The insert membrane was utilized to perform staining of either the top viewed whole membrane or side viewed membrane sections. For section staining, the insert membrane was first paraffinembedded, cut and then further processed for staining as described in \u003cem\u003evon Schledorn et al.\u003c/em\u003e 2023 [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. For whole membrane staining, permeabilization was performed with 0.5 mL TBS buffer with donkey serum (GeneTex, GTX73245) for 20min at RT. The following primary antibodies were utilized and diluted in PBS plus 1% bovine serum albumin (BSA) (Sigma-Aldrich, A9418): MUC5AC (Thermo Fisher Science, MA5-12178, 1:200), TUBB4 (Cell Signaling Technology, 5335S, 1:800). The following secondary antibodies were utilized and diluted in PBS plus 1% BSA: Cy™3 anti-rabbit IgG (Jackson ImmunoResearch, 711-165-152, 1:200), Alexa Fluor® 488 anti-mouse IgG (Jackson ImmunoResearch, 715-545-151, 1:200). 0.5mL of primary antibodies were incubated over night at 4°C. 0.5mL of secondary antibodies were incubated for 30 min at RT. Nuclei were stained with DAPI for 15 min at RT. For imaging the membrane was mounted on a glass slide in Fluorescence mounting medium (Agilent Dako, S3023). For membrane section staining, the membrane was first dehydrated and then embedded in paraffin. The membrane was cut into sections of 3 µm in thickness and transferred onto glass slides for staining. Primary and secondary antibodies as well as DAPI were applied as described above. Fluorescence imaging and image processing were performed with an AxioObserver A1 fluorescence microscope and AxioObserver Z1 fluorescence microscope and the ZENPro Sofware 3.0. For BSND staining samples were blocked and permeabilized using 0.25% (v/v) Triton-X 100 in PBS with 3% BSA for 60 min at RT, then incubated overnight at 4°C with the anti-BSND antibody (Abcam, ab196017, 1:500) diluted in the Triton/BSA buffer. The samples were rinsed three times for 5 min with PBS before incubation with secondary antibody (Invitrogen, A11008, 1:500) diluted in Triton/BSA buffer for 1 h at 37°C, followed by a triple 5 min wash with PBS. Then, samples were incubated for 1 hour at 37°C in Triton/BSA buffer containing directly conjugated anti- TUBB4 (Santa Cruz, sc-23950, 1:200) followed by a triple 5 min wash with PBS. The samples were stored at 4°C until mounting. The samples were mounted by removing the cell culture membrane from the insert using a scalpel and placing the membrane onto a glass slide with the cells facing upwards. The cell was coated with a drop of ProLong™ Diamond Antifade Mountant (Invitrogen, P36965) and covered with a round number 1.5 glass coverslip.\u003c/p\u003e\u003cp\u003e\u003cem\u003eComposition Analysis of ALI Cultures via Quantification of Immunofluorescence Signal Area.\u003c/em\u003e Analysis was performed on ALI cultures after immunofluorescence staining of TUBB4 and MUC5AC, as earlier described. ImageJ/Fiji software was utilized to determine the area of TUBB4\u003csup\u003e+\u003c/sup\u003e and MUC5AC\u003csup\u003e+\u003c/sup\u003e signal after fluorescence microscopy. Input images were used in 16 bit file format. First, threshold was manually set using the ‘moments’ function. Afterwards signalpositive area was measured. Analysis was performed on multiple regions of interest (each 879µm x 879µm) per sample group.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFlow Cytometry Analysis.\u003c/em\u003e Flow cytometry analysis was performed to quantify the expression of definitive endoderm and lung progenitor markers on day 3 and day 13 of hiPSCs differentiation into respiratory epithelial cells, respectively. On day 3, live cell staining was performed. On day 13, cells were fixed and permeabilized using the FoxP3 staining buffer set (Miltenyi Biotec, 130-093-142). Each 10\u003csup\u003e5\u003c/sup\u003e cells were taken and suspended in 100 µL PBS with 1% BSA. The following directly-labelled primary antibodies were utilized: APC anti-CXCR4 (Thermo Fisher Science; 17-9999-42, 1:25), APC anti-NXK2.1 (Miltenyi Biotec, 130-118-309, 1:2,000), PE anti-c-Kit (Thermo Fisher Science, 12-1178-42, 1:33), PE anti-EpCAM (BD Biosciences, 347198, 1:33). Primary antibodies were diluted in 1% BSA (Sigma-Aldrich, A9418) in PBS w/o (Thermo Fisher Science, 70011044) and incubated for 30 min on ice. Flow cytometry analysis was performed with a MACSQuant Analyzer 10 and FlowJo analysis software.\u003c/p\u003e\u003cp\u003e\u003cem\u003eReal-Time Quantitative PCR (RT-qPCR).\u003c/em\u003e RNA samples from cells were collected in Trizol® reagent (Invitrogen, 15596018) RNA isolation was performed using the NucleoSpin® RNA II kit (Macherey-Nagel, 740955.50) and cDNA synthesis was performed using the RevertAid™ H Minus First Strand cDNA Synthesis kit (Thermo Fisher Science, K1631). Real-time qPCR analysis was carried out using the SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, 1725270) and CTX Connect Real-Time PCR Detection system (Bio-Rad Laboratories). Target gene expression was normalized to the expression of housekeeping genes bACT and GAPDH. Applied primer pairs and sequences are listed in supplemental data set.\u003c/p\u003e\u003cp\u003e\u003cem\u003eWestern Blot Analysis.\u003c/em\u003e Western blot analyses were performed as previously described [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The following antibodies were utilized: Beta-actin (Abcam, ab8226), CFTR (Cystic Fibrosis Foundation CFTR Antibody Distribution Program, Chapel Hill, North Carolina; antibody mix: 596 + 570 + 217 + 660), TMEM16A (Abcam, ab64085); ZO-1 (Invitrogen, 33-9100). 16HBE14o- cells (human bronchial epithelial cells; HBE) were utilized as a control for protein expression in airway epithelial cells.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMeasurement of the Transepithelial Ion Conductance.\u003c/em\u003e Recordings of the transepithelial ion conductance in iALI and pALI cultures was performed in EasyMount Ussing chambers (Physiologic Instruments) using voltage clamp configuration to measure the transepithelial short-circuit current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e). The \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e was continuously recorded using Lab-Chart8 (AD Instruments), and transepithelial resistance (\u003cem\u003eR\u003c/em\u003e\u003csub\u003eTE\u003c/sub\u003e) was monitored by application of short voltage pulses (1 mV) every 60 s. Experiments were performed in Ringer buffer as previously described [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. After 10–15 min equilibration, basal \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSC\u003c/sub\u003e was measured and amiloride (100 µM; Sigma-Aldrich, A7410) was added to inhibit sodium absorption via ENaC. Next, forskolin (10 µM; Sigma-Aldrich, F6886) and 3isobutyl-1methylxanthin (IBMX; 100 µM; Sigma-Aldrich, I5879) were added together, followed by CFTRinhibitor 172 (20 µM; CFTRinh-172) (TargetMol, T2355) to assess CFTR-mediated chloride conductance. Uridine-triphosphate (UTP; 10 µM; Thermo Fisher Science, R1471) was added to evaluate the calcium-activated chloride conductance. Both, the activation of CFTR by forskolin and IBMX as well as the activation of CaCCs were measured as the peak response after compound addition. Lastly, GlyH-101 (50 µM; AbMole, M6754) and subsequently niflumic acid (NFA; 500 µM; Cayman Chemical Company, 70650) were applied to inhibited residual anion conductance.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMeasurement of the Ciliary Beat Frequency (CBF).\u003c/em\u003e Measurement of the \u003cem\u003eCBF\u003c/em\u003e on iALI and pALI cultures was performed via high-frequency video microscopy imaging using a Zeiss Axiovert A.1 microscope equipped with a Basler SCA640 120FM camera in a humidified environmental chamber at 37°C and in 5% CO2. Videos were recorded with a 40x magnification objective at 100 frames per second using a phase contrast filter. Videos were recorded in at least 15 positions per insert, selected in a meandering pattern throughout the whole insert, avoiding the edges with the meniscus. \u003cem\u003eCBF\u003c/em\u003e and cilia coverage were analyzed using Sisson-Ammons video analysis (SAVA) software [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e], where average \u003cem\u003eCBF\u003c/em\u003e and area of moving pixels were measured by whole field analysis. Inserts with less than 10.000 moving pixels (\u0026lt; 0.01%) of total imaged area) were determined as non-motile. Data are show as average per insert. Apical washes were performed with PBS 24 h before the measurement.\u003c/p\u003e\u003cp\u003e\u003cem\u003eTransmission Electron Microscopy.\u003c/em\u003e ALI cultures were fixed in 150 mM HEPES buffer (pH 7.35) containing 1.5% glutaraldehyde and formaldehyde at RT for 20 min and at 4°C over night. 1.5 mm sized pieces of the fixed cultures were high pressure frozen in a HPM 100 (Leica Microsystems, Wetzlar). Freeze substitution was carried out in a Leica AFS (Leica Microsystems, Wetzlar) in acetone containing 0,1% tannic acid at -90°C over night and after washing in acetone continued in acetone containing 2% osmiumtetroxide. Temperature was raised to -20°C and after 2 h to 4°C. After washing in acetone, samples were transferred to RT and embedded in EPON. 50 nm thick cross-sections of the ALI-cultures were poststained with uranyl acetate and lead citrate (Reynolds et al. 1963) and observed in a Zeiss EM 900 (Zeiss, Oberkochen), operated in the bright field mode at 80 kV. Images were recorded with a side-mounted 4k CCD-camera (TRS, Dünzelbach). For estimation of the mucus layer height, complete 1.5 mm profiles were recorded iteratively and the mucus layer was estimated every 6 µm at 90 degree to the median cell surface on the respective images.\u003c/p\u003e\u003cp\u003e\u003cem\u003eScanning Electron Microscopy.\u003c/em\u003e After fixation as for TEM, ALI cultures were washed in water, critical point dried and sputtered with gold. Examination was done in a Crossbeam 540 (Zeiss, Oberkochen) at 10 kV.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStatistical Analyses.\u003c/em\u003e GraphPad Prism6 was utilized to perform statistical analyses. Results are presented as means ± SD unless otherwise noted. Significance of two sample groups was analyzed as noted in the figure descriptions by using the unpaired or paired t test.\u003c/p\u003e\u003cp\u003e\u003cem\u003eUse of Artificial Intelligence.\u003c/em\u003e For the purpose of supporting the writing process of this manuscript, Meta LLaMA 3.1 8B Instruct and OpenAI ChatGPT-4 and DeepL were sporadically used to rephrase individual sentences.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALI:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Air-liquid-interface\u003c/p\u003e\n\u003cp\u003eBSND:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Barttin CLCNK Type Accessory Subunit Beta\u003c/p\u003e\n\u003cp\u003eCaCCs:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Calcium-activated chloride channel\u003c/p\u003e\n\u003cp\u003ecAMP:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cyclic adenosine monophosphate\u003c/p\u003e\n\u003cp\u003eCBF:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ciliary beat frequency\u003c/p\u003e\n\u003cp\u003eCCSP:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Clara-cell secretory protein\u003c/p\u003e\n\u003cp\u003eCF:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cystic fibrosis\u003c/p\u003e\n\u003cp\u003eCFTR:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cystic fibrosis transmembrane conductance regulator\u003c/p\u003e\n\u003cp\u003eCXCR4:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;C-X-C chemokine receptor type 4\u003c/p\u003e\n\u003cp\u003eDE:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Definitive endoderm\u003c/p\u003e\n\u003cp\u003eENaC:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Epithelial sodium channel\u003c/p\u003e\n\u003cp\u003eEpCAM:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Epithelial cell adhesion molecule\u003c/p\u003e\n\u003cp\u003eETI:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Elexacaftor-tezacaftor-ivacaftor\u003c/p\u003e\n\u003cp\u003eFIS:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Forskolin-induced swelling\u003c/p\u003e\n\u003cp\u003ehiPSC:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Human induced pluripotent stem cell\u003c/p\u003e\n\u003cp\u003eiALI culture:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;hiPSC-derived ALI culture\u003c/p\u003e\n\u003cp\u003eI\u003csub\u003eSC\u003c/sub\u003e:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Short-circuit current\u003c/p\u003e\n\u003cp\u003eLP:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Lung progenitor\u003c/p\u003e\n\u003cp\u003eMACS:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Magnetic-activated cell sorting\u003c/p\u003e\n\u003cp\u003eMCC:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mucociliary clearance\u003c/p\u003e\n\u003cp\u003eMLH:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mucus layer height\u003c/p\u003e\n\u003cp\u003eMUC5AC:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mucin 5AC\u003c/p\u003e\n\u003cp\u003en:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Replicate\u003c/p\u003e\n\u003cp\u003eNKX2.1:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;NK2 homeobox 1\u003c/p\u003e\n\u003cp\u003ens:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;None significant\u003c/p\u003e\n\u003cp\u003epALI culture:\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;primary ALI culture\u003c/p\u003e\n\u003cp\u003eSD:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Standard deviation\u003c/p\u003e\n\u003cp\u003eSEM:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Scanning electron microscopy\u003c/p\u003e\n\u003cp\u003eSLC26A9:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Solute carrier family 26 member 9\u003c/p\u003e\n\u003cp\u003eTEM:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Transmission electron microscopy\u003c/p\u003e\n\u003cp\u003eTMEM16A:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Transmembrane member 16A\u003c/p\u003e\n\u003cp\u003eTUBB4:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Acetylated tubulin beta-4 chain\u003c/p\u003e\n\u003cp\u003eWT: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Wild type (healthy)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman anonymized blood samples for generation of hiPSCs were collected based on the approvals by the Hannover Medical School (MHH) Ethics Committee (No. 409; approval date: 21.09.2010) as part of the project \u0026lsquo;Nutzung von anonymisierten Patientenzellen f\u0026uuml;r die biomedizinische Forschung / Generierung von induzierten pluripotenten Stammzellen\u0026rsquo; and following the donor\u0026rsquo;s written informed consent, or in the case of newborns, following parental consent.\u003c/p\u003e\n\u003cp\u003eHuman anonymized explanted lung tissue samples for isolation of primary bronchial epithelial cells were collected based on the approvals by the Hannover Medical School (MHH) Ethics Committee and following the donor\u0026rsquo;s written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability and supplemental information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe underlying data to all graphs is available in the supplementary data sheets Supplementary Table 1. Supplementary material information are available in Supplementary Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMCJ designed, performed and analyzed a large part of experiments and wrote the manuscript. AB designed the measurements of the transepithelial chloride conductance, performed parts of the measurements of the transepithelial chloride conductance and ciliary beat frequency, provided scientific input and wrote the manuscript. JZ and NC performed RT‑qPCR, immunofluorescence staining and generated iALI and pALI cultures. LC performed parts of the measurements of the transepithelial chloride conductance and ciliary beating frequency. LvS performed immunofluorescence staining. JH performed transmitted electron microscopy, analyzed the data and wrote the manuscript. JN and DR performed immunofluorescence staining and microscopy for ionocyte detection. MM and SH performed the western blot experiments. FS analyzed the western blot data and revised manuscript critically for scientific content. TR performed parts of the measurements of the transepithelial chloride conductance and ciliary beating frequency. FI, DJ and AR prepared and provided lung material for primary cell isolation. JH performed the isolation of CF patient‑specific primary human respiratory epithelial cells. MAM supervised the study, provided scientific input, provided his laboratory equipment to performed measurements of the transepithelial chloride conductance and ciliary beating frequency, and wrote the manuscript. RO, SM and UM conceptualized and supervised the study, provided scientific input and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur sincere appreciation goes to Bob J Scholte\u0026dagger;, whose insightful scientific guidance and advice greatly benefited to our study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuo J, Garratt A, Hill A. Worldwide rates of diagnosis and effective treatment for cystic fibrosis. J Cyst Fibros. 2022;21(3):456\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKerem B, et al. Identification of the cystic fibrosis gene: genetic analysis. Science. 1989;245(4922):1073\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRiordan JR, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. 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J Microsc. 2003;211(Pt 2):103\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7184232/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7184232/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeverely impaired mucociliary airway function is the primary pathomechanism in Cystic Fibrosis (CF) lung disease. Despite significant advances in CF therapy, there is still a critical need for alternative, individualized treatment options, especially for patients with untreatable CFTR mutations.\u003c/p\u003e\u003cp\u003eAlthough intestinal organoids and primary airway cells are widely used as preclinical models of CF, both systems exhibit limitations with regard to the proper modelling of mucociliary clearance or the availability of sufficient cell quantities. Patient-specific human induced pluripotent stem cells (hiPSCs) are a promising alternative due to their unlimited expansion potential and capacity to differentiate into airway epithelia. However, cellular inhomogeneities in iPSC-derived airway cultures complicated conventional assays that determine CFTR function such as Ussing chamber measurements, and a comprehensive demonstration of CF pathophysiology in hiPSC-derived airway models has been largely lacking.\u003c/p\u003e\u003cp\u003eThis study provides comprehensive data demonstrating very similar gene expression, (ultra)structure and CFTR function in CF iPSC-derived airway (iALI) and primary airway (pALI) cultures. Addressing current limitations, we have implemented a sensitive, straightforward, and automatable ciliary beat frequency (CBF) assay, which is largely unaffected by inhomogeneities and directly reflects disturbed mucus viscosity and mucociliary transport in CF lung disease. Electron microscopy images confirmed the disease phenotype showing a highly dense and dehydrated mucus layer on top of CF iALI cultures. Furthermore, established CFTR modulator drugs partially rescued the disease phenotype in CF iALI cultures, which validated the utility of iALI cultures as a scalable, patient-specific platform for CF research and personalized drug development.\u003c/p\u003e","manuscriptTitle":"Human induced pluripotent stem cells for in vitro modeling of impaired mucociliary clearance in cystic fibrosis lung disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-04 00:14:10","doi":"10.21203/rs.3.rs-7184232/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-10T09:43:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-09T18:02:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-03T10:49:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117698479507725851780004410925966606251","date":"2025-09-02T13:36:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303278397719487333340360986765921059238","date":"2025-08-28T10:15:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18758785754346185281271094357167014781","date":"2025-08-27T22:19:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192233058643889847952742792983955812380","date":"2025-08-27T14:50:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160760187827115211727310280172695249639","date":"2025-08-27T13:47:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193669211089596068073857841163771474284","date":"2025-08-27T12:46:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-27T12:39:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-18T13:51:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-11T02:45:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-08-08T09:54:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"620fca0a-ae08-459e-b5af-bd878249d674","owner":[],"postedDate":"September 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:23:36+00:00","versionOfRecord":{"articleIdentity":"rs-7184232","link":"https://doi.org/10.1186/s13287-025-04737-0","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-10-21 16:16:22","publishedOnDateReadable":"October 21st, 2025"},"versionCreatedAt":"2025-09-04 00:14:10","video":"","vorDoi":"10.1186/s13287-025-04737-0","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04737-0","workflowStages":[]},"version":"v1","identity":"rs-7184232","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7184232","identity":"rs-7184232","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}