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In vitro platform to model the function of ionocytes in the human airway epithelium
Respiratory Research volume 25, Article number: 180 (2024)
Abstract
Background
Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium.
Methods
hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties.
Results
Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells.
Conclusion
Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.
Background
Pulmonary ionocytes were described in 2018 as a small population of airway epithelial cells (AECs) that express high levels of ion channels and transporters, including CFTR (cystic fibrosis transmembrane conductance regulator), the protein mutated in cystic fibrosis (CF) [1,2,3]. Thus, it has been hypothesised that ionocytes might have a role in the pathogenesis of CF and understanding their function could be key in identifying new therapies for CF and other respiratory diseases. So far, available information on ionocytes and their function in the human airway epithelium is limited. Specific markers for this cell type include FOXI1 (forkhead box I1), high CFTR expression, ASCL3 (achaete-scute family BHLH transcription factor 3) and STAP1 (signal transducing adaptor family member 1) [2]. Ionocytes also express high levels of the vacuolar H+ ATP-ase (VATPase), barttin (BSND)/ClC-K channels and the large conductance Ca2+-activated K+ channel (KCa1.1) [2]. They seem to be more abundant in the nasal epithelium and proximal airways [4, 5] where they are more commonly found in the ducts of submucosal glands [3].
Lineage tracing analysis suggests that ionocytes differentiate from basal cells [2]. By showing that knock out (KO) of POU2F3 leads to air liquid interface (ALI) cultures with decreased numbers of ionocytes and pulmonary neuroendocrine cells (PNECs), Goldfarbmuren et al. [6] suggested that tuft cells give rise to both ionocytes and PNECs. By contrast, Plasschaert et al. [1] showed that the transcription factor FOXI1 is sufficient to drive ionocyte differentiation, while the inhibition of Notch signalling in ALI cultures leads to a reduction in their number. This pathway for ionocyte differentiation seems to be conserved between species [7]. More recently, Wang et al. [8] reported no changes in ionocyte marker expression after they overexpressed NOTCH in AECs derived from human induced pluripotent stem cell (hiPSCs). This could indicate that lower levels of Notch signalling are needed for ionocyte specification than those required by secretory cells and that signalling is finely tuned to achieve the complex composition of the airway epithelium [9,10,11]. Finally, Cai et al. [12] demonstrated that the Sonic hedgehog pathway is involved in ionocyte specification by showing that the inhibition of this pathway reduces the amount of ionocytes in culture, while its activation using the chemical agonist SAG (Sonic hedgehog agonist) increases their numbers. Thus, crosstalk between these two signalling pathways seems to be involved in ionocyte specification.
Early studies of the function of ionocytes by Plasschaert et al. showed that reduction of the number of ionocytes could affect CFTR-mediated Cl− currents in Ussing chamber assays [1], which was recently verified by the study of Cai et al. [12]. Additionally, a more recent study demonstrated ionocyte-specific regulation of CFTR by the phosphodiesterase PDE1C [13]. In a Foxi1 KO mouse model, absence of Foxi1 led to higher mucus viscosity and ciliary beat frequency (CBF), indicating that ionocytes could have a role in regulating airway physiology [2]. This has been further studied in a recent report by Lei et al. [14]. , where they describe a role of ionocytes in fluid and electrolyte absorption, and in a study by Yuan et al. [15] that demonstrates a pivotal role for ionocytes in homeostatic mechanisms regulating airway surface liquid (ASL) volume, pH and viscosity and mucociliary clearance. Furthermore, the observation that ionocytes have cellular extensions [2, 16], suggests the hypothesis that they could interact directly with other AEC types. However, the precise mechanisms by which ionocytes control these multiple functions are still not fully understood.
The challenge to further understand ionocyte function in human lungs is aggravated by the lack of appropriate models and the limited availability of primary tissue. AECs derived from hiPSCs (hiPSC-AECs) could provide unique opportunities for respiratory research since hiPSCs can grow indefinitely while maintaining their capacity to differentiate into any cell type. However, the differentiation of hiPSCs into AECs lacks standardised protocols and different methods often lead to divergent results [17,18,19]. Until recently, protocols failed to consistently produce rare AECs such as ionocytes [18]. Hor et al. published a protocol to generate PNECs from hiPSCs in vitro, without identifying ionocytes in their cultures [20]. In a recent report, Wang et al. [8] identified ionocytes in their hiPSC-AEC cultures using a protocol with 3 sorting steps which extended the length of the protocol to almost 80 days. Here, we present a platform to study the role of ionocytes in the airway epithelium in vitro using hiPSCs. We describe a protocol which produces AECs including ionocytes within 60 days and then perform loss of function experiments. Our results show that the KO of FOXI1 in hiPSCs using CRISPR/Cas9 reduced the number of ciliated cells after hiPSC-AEC maturation, indicating that ionocytes could be important in lung lineage specification and homeostasis.
Methods
Full descriptions of the methods used to differentiate hiPSCs into AECs and evaluate them biochemically and functionally are provided in the Supplementary Materials and Methods.
hiPSC differentiation to AECs
To derive AECs from hiPSCs, we used FS13B hiPSC lines generated as described previously [21] and the CF17/NKX2.1-GFP hiPSC line (kindly gifted by UTHEALTH and Dr. Jed Mahoney, Cystic Fibrosis Foundation lab, Lexington, MA, USA). hiPSCs were differentiated by driving cells through definitive endoderm and anterior foregut endoderm to reach a lung progenitor state. At day 16 of differentiation, cells were sorted to enrich for NKX2.1 expressing progenitors using anti-carboxypeptidase-M (CPM) antibody [17], anti-CD26/anti-CD47 sorting strategy [22] or sorting for GFP: NKX2.1 reporter cells [18]. Sorted cells were seeded in 3D Matrigel domes for expansion and cryopreservation. After at least 8 days of growth under expansion conditions, cells were seeded on Transwell® inserts to form mature polarised airway epithelia. Once cells were confluent in the Transwell®, medium bathing the apical membrane was removed to form an ALI. After 28 days of ALI culture, hiPSC-AEC epithelia were characterised biochemically and functionally.
Analysis of mRNA and protein expression
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence staining and Western blotting were performed to characterise mRNA and protein expression at different stages of the protocol and to investigate the effects of FOXI1 KO.
Lung progenitor transplantation into a mouse model of airway injury
Experiments using a mouse model of airway injury were approved by local ethical review committees and conducted according to Home Office project license PPL PEEE9B8E4 (Emma L. Rawlins, University of Cambridge). For these experiments, 9 male 9-week-old immune-compromised NOD-scid-IL2rg−/− (NSG; RRID: IMSR_JAX:005557) mice were used [23, 24]. Mice were treated with 2% polidocanol oropharyngeally and transplanted with a suspension of 1 million GFP + hiPSC-derived lung progenitor cells on the next day. At different time points (1, 7 or 10 days) after cell transplantation, mice were sacrificed and tracheas harvested for wholemount immunofluorescence staining to visualise cells.
CRISPR/Cas9-based FOXI1 KO and phenotypical assays
Single guide RNA (sgRNA) and CRISPR/Cas9 were used to KO FOXI1 in hiPSCs. The functional consequences of FOXI1 KO were evaluated using hiPSC-AEC epithelia and pH and transepithelial resistance (Rt) measurements, high-speed microscopy analysis of ciliary dynamics and Ussing chamber studies of epithelial ion transport.
Statistical analysis
Results are expressed as means ± SD of n observations. Statistical analyses were performed either using Prism 9 (GraphPad Software Inc., San Diego, CA, USA) or SigmaPlot 14 (Systat Software Inc., San Jose, CA, USA). The type of statistical analysis performed in each experiment and the number of replicates used are described in the figure legends. Differences were considered statistically significant when P < 0.05. Significance in each analysis is represented by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.
Results
Method to differentiate hiPSCs into AECs including ionocytes
For this study, we used two different hiPSC lines or genetic backgrounds: the previously described FS13B hiPSCs [21] and the CF17/NKX2.1-GFP, which has a GFP reporter for NKX2.1. We first differentiated these two hiPSCs lines into AECs following a natural path of development including definitive endoderm, anterior foregut endoderm and lung progenitors (Fig. 1A). RT-qPCR analyses confirmed the mRNA expression of specific markers for each stage (Fig. 1B and S1A) and cells formed a characteristic network pattern after 16 days of differentiation (Fig. 1C and D). The resulting lung progenitors were then sorted using anti-CPM staining to enrich for NKX2.1 expressing cells in FS13B cells (Fig. 1E and F) while CF17/NKX2.1-GFP cells were sorted for NKX2.1-GFP expression. To expand lung progenitors, sorted cells were grown as 3D organoids (Fig. 2A) in medium supplemented with the GSK3b inhibitor CHIR-99021, the Rho-associated protein kinase inhibitor Y-27632 and fibroblast growth factor 10 (FGF10). These organoids could be cryopreserved and thawed for further experiments while maintaining the expression of lung progenitor markers (Figure S1B). In some instances, organoids were maintained for up to 8 passages or + 6 passages after thawing without losing NKX2.1 expression (Figure S1C). Overall, our approach allowed the production and the expansion of lung progenitors in vitro thereby bypassing the need to systematically differentiate hiPSCs.
AEC maturation was performed by dissociating the organoids and seeding lung progenitors in Transwell® inserts. Once confluent, ALI was established and cells were differentiated for an additional 28 days (Fig. 2B and C). To promote the differentiation of ciliated cells, the Notch pathway inhibitor (2 S)-N-[2(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl-glycine 1,1-Dimethylethyl ester (DAPT) was added to the Maturation Medium for the first 14 days after initiating ALI culture. From day 14, PneumaCult™-ALI (PALI) Medium was used to further promote ciliation. The resulting epithelia showed an increase in the expression of TP63, CFTR, FOXJ1, MUC5AC and SCGB3A2 (Fig. 2D) and maintained expression of epithelial markers (Figure S1D). The presence of basal cells (CK5, p63), secretory cells (MUC5AC, SCGB3A2), ciliated cells (FOXJ1, acetylated tubulin (AcTub)), PNECs (ASCL1, CRP) and ionocytes (FOXI1, CFTR high expression, BSND) was confirmed by immunostaining (Fig. 2E and S1E). Importantly, the epithelium was polarised, with cilia (AcTub) located on the apical side and basal cells (CK5) at the basal side of the epithelium (Fig. 2F). Finally, functional analyses confirmed that the hiPSC-AEC epithelium had Rt values comparable to that of primary AECs [25] (Fig. 2G). Analysis of CBF by a robust Fourier Transform method [26], described in the Supplementary Materials and Methods, showed that the cilia in hiPSC-AEC cultures beat at a frequency comparable to that of primary human bronchial epithelial cells (HBECs) (Fig. 2H). The same analysis indicated that hiPSC-AECs were covered by fewer cilia than HBEC cultures (Fig. 2I), consistent with RT-qPCR results for FOXJ1 expression (Fig. 2D). Taken together, these results show that our protocol allows the production of a polarised airway epithelium containing a diversity of cell types, including ionocytes.
The engraftment capacity of hiPSC-derived lung progenitors
hiPSC-derived lung progenitors have been previously successfully transplanted into the respiratory airways of murine models [27,28,29], highlighting their potential in regenerative medicine. To further demonstrate the functionality of our cells, we explored the engraftment potential of our hiPSC-derived airway progenitors in vivo with a short-term transplantation experiment. We generated lung progenitors from GFP-expressing hiPSC lines (FS13B GFP) and GFP + CPM + sorted cells were cultured as 3D organoids for 8 days before cryopreservation. After thawing and expansion for at least 8 additional days, GFP + organoids expressed similar levels of the lung progenitor and basal cell markers NKX2.1 and TP63 when compared to passage 0 organoids (Figure S2A). These organoids were then dissociated into a single cell suspension and 1 million cells were transplanted oropharyngeally into the tracheas of mice that had been topically treated with polidocanol 18 h before transplantation (Fig. 3A). Tracheas were harvested at 1, 7 or 10 days after transplantation and we observed GFP + cells in the tracheas of the 9 mice that had received hiPSC-derived cells (Fig. 3B and Figure S2B). The appearance of clusters of cells indicates that the cells replicated after engraftment (Fig. 3B left). Interestingly, GFP + cells co-expressed CK5 at 7 and 10 days after transplantation (Fig. 3B right), indicating that lung progenitors generated with our approach can not only survive in a mouse model of acute airway injury, but also differentiate towards basal cells. Although longer time points would be needed to assess the full regeneration and differentiation potential of these cells, these results confirm their engraftment capacity.
KO of FOXI1 in hiPSCs leads to hiPSC-AECs lacking ionocytes
Based on the results generated above, we decided to use our platform to study the importance of ionocytes during the formation of the human airway epithelium. Of note, genetic studies in the mouse have shown that FOXI1 is necessary for the generation of ionocytes in vivo [2]. Thus, we hypothesised that the absence of FOXI1 will stop the production of ionocytes in vitro (Fig. 4A). Using CRISPR/Cas9 genome editing, we generated two hiPSC KOs for the FOXI1 gene by designing sgRNAs that target the DNA binding domain of FOXI1, which is found in exon 1 and is shared by both transcript variants of the gene (Fig. 4B). This approach generated hiPSC lines carrying a loss of function mutation caused by an early stop codon (Figure S3A) as confirmed by genotyping using PCR and Sanger sequencing. Importantly, each of the hiPSC lines was targeted with a different sgRNA to rule out off-target effects, while unedited clones that had gone through the same targeting and clone isolation process were used as isogenic controls (Fig. 4B). The resulting FOXI1−/− hiPSCs were then differentiated in parallel with their isogenic wild-type (WT) counterparts. There were no statistically significant differences in the expression of specific markers at key timepoints between the WT and KO up to day 16 of differentiation (Fig. 4C). Thus, the absence of FOXI1 does not affect lung progenitor production. Lung progenitor cells were then enriched by sorting (Fig. 4C) and the resulting organoids were further differentiated using ALI cultures after expansion. After 28 days of culture, WT and KO epithelia showed similar levels of airway markers including NKX2.1, TP63, CFTR, SCGB3A2, FOXJ1 and MUC5AC (Fig. 4D). However, the absence of FOXI1 seemed to induce a limited decrease in the expression of FOXJ1 in ALI cultures (Fig. 4D and S3B). Of note, we could not detect changes in FOXI1 expression by RT-qPCR as ionocytes represent only 0.5–1.5% of the epithelium. Immunofluorescence analyses showed the absence of ionocytes in the FOXI1 KO ALI cultures in contrast to the presence of FOXI1 + CFTR high-expressing ionocytes in WT ALI cultures (Fig. 4E and F). Furthermore, Western blotting indicated absence of FOXI1 protein in FOXI1 KO ALI cultures compared to its presence in WT epithelia (Fig. 4G and S3C). Together, these data show that FOXI1 KO leads to hiPSC-AEC epithelia without ionocytes and that their absence does not affect the early differentiation of the lung epithelium.
KO of FOXI1 reduces ciliation of hiPSC-AECs
We next tested whether the KO of FOXI1 could affect the function of hiPSC-AECs beyond that of CFTR function, which has already been extensively studied [1, 2, 12,13,14]. In this study, we first assessed the effect of FOXI1 KO on the pH of the ASL in hiPSC-AEC ALI cultures and we confirmed that there were no statistical differences between FOXI1 WT and KO cultures (Fig. 5A). We next evaluated whether the KO of FOXI1 impaired the barrier properties in hiPSC-AEC ALI cultures by measuring Rt. Although FOXI1 KO epithelia had significantly reduced Rt values compared to those of FOXI1 WT epithelia (Fig. 5B), Ussing chamber studies revealed that they functionally expressed the epithelial Na+ channel (ENaC), the Ca2+-activated Cl− channel TMEM16A and CFTR (Figure S6). Next, we assessed cilia coverage and motility as described in Supplementary Materials and Methods. FOXI1 KO ALI cultures showed a similar CBF to their WT counterparts (Fig. 5C and S4). Intriguingly, the coverage of cilia in FOXI1 KO epithelia was reduced (Fig. 5D and S4). Although this difference was not statistically significant, it suggested that either the number or function of ciliated cells was decreased in FOXI1 KO ALI cultures. To distinguish between these possibilities, we performed flow cytometry analyses and observed that the absence of ionocytes resulted in a significant reduction in the number of FOXJ1 expressing cells (Fig. 5E and S5A). The reduced number of ciliated cells in FOXI1 KO cells was validated with hiPSC-AECs from two other FOXI1 KO hiPSC clones of the same genetic background (Figure S5B and S5C) and with the clones from the second genetic background (Figure S5D and S5E). Because the FOXJ1 + cell number decrease was not as striking in the second genetic background (Figure S5D and S5E), we further investigated this phenotype in CF17/NKX2.1-GFP cells by assessing the expression of mature ciliated cell markers both at protein and mRNA levels. Western blot analysis indicated a decrease of DNAI1 in FOXI1 KO cells compared to their WT controls (Fig. 5F). RT-qPCR analysis showed decreased expression of the ciliated cell markers NEK10, DNAH5 and CP110, but only the differences in DNAH5 and CP110 expression were statistically significant (Figure S5F). Finally, immunofluorescence staining confirmed the presence of FOXJ1 + AcTub + ciliated cells in FOXI1 KO ALI cultures, but these had a more scattered distribution compared to their WT controls (Fig. 5G). Taken together, these results suggest that ionocytes and/or the expression of FOXI1 could be involved in the production of functional ciliated cells and could be necessary to establish the normal cellular composition of the lung epithelium.
Discussion
In this study, we described a protocol to differentiate hiPSCs not only into the most abundant cell types of the airway epithelium (basal, ciliated and secretory cells) but also into PNECs and ionocytes. To our knowledge, this is the first report of hiPSC-AECs including these rare cell types in the same culture system.
To date, only one other protocol for hiPSC-AEC differentiation producing ionocytes has been published [8]. In that study, Wang et al. reported the presence of FOXI1 + ionocytes using a protocol that requires 3 subsequent sorting steps. Thus, the generation of hiPSC-AEC cultures with ionocytes has proven challenging. One of the reasons for this might be the use of specialised media developed to produce highly ciliated cultures, which probably contain inhibitors of Notch that could reduce the presence of ionocytes [1]. By contrast, our protocol is based on a chemically defined medium combined with short-term culture with PneumaCult™-ALI Medium. This combination leads to hiPSC-AEC cultures that might be less ciliated, but that contain ionocytes expressing FOXI1 and high levels of CFTR or co-expressing FOXI1 and BSND.
We used our hiPSC-AEC cultures to study the impact of FOXI1 KO on the development and functionality of the airway epithelium. Although ionocytes constitute a rare population in the epithelium, several studies have shown that their impairment can lead to significant phenotypes [1, 2, 12,13,14]. Consistent with previous results [2], we found that the KO of FOXI1 does not impact the pH of ASL in ALI cultures. By contrast, we found that FOXI1 KO impacts Rt values of hiPSC-AEC epithelia. The lower Rt values of FOXI1 KO epithelia might be due to impaired epithelial barrier function and/or increased numbers/activity of ion channels. According to Pou Casellas et al. [30], transcriptional analysis of ionocytes revealed their involvement in various signalling pathways, including those involving occludin and junctional adhesion molecules, which could potentially explain why their absence affects the formation of a tight epithelial barrier. Additionally, Yuan et al. [15] reported the compensatory overexpression of ion and water channel encoding genes in airway cultures from a FOXI1 KO ferret model. Although our results differ from those published by Goldfarbmuren et al. [6] and Lei et al. [14], who reported increased Rt in FOXI1 KO cultures, their data are based on mosaic KOs, while Yuan et al. [15] did not report Rt measurements. The different Rt values of FOXI1 KO airway epithelia are reminiscent of earlier reports about the effects of the predominant CF-causing CFTR variant F508del on Rt. LeSimple et al. [31] found that epithelia heterologously expressing F508del-CFTR had reduced Rt values compared to those expressing wild-type CFTR, whereas Li et al. [32] found the converse. As with these previous studies, differences in the cells studied and the experimental conditions used likely explain the distinct results obtained with FOXI1 KO airway epithelia.
We found that ALI cultured hiPSC-AECs without ionocytes show reduced cilia motility properties compared to cultures with ionocytes. More importantly, we showed that cultures without ionocytes displayed a smaller number of ciliated cells. This could be the reason for the slower movement of cilia in FOXI1 KO cultures and it suggests that ionocytes could play a role in mucociliary clearance by influencing the production of ciliated cells. These results do not contradict the previous report by Montoro et al. showing that the absence of Foxi1 in mice led to more viscous mucous secretions in the airway epithelium and, in turn, higher CBF [2]. Our ALI cultured hiPSC-AECs do not produce abundant mucus, and CBF measurements did not change after washing the epithelia with PBS. Therefore, we cannot exclude the possibility that the absence of FOXI1 expression could also increase mucus viscosity. However, it could be interesting to confirm if the number of ciliated cells is also decreased in a mouse KO for Foxi1. In the study by Goldfarbmuren et al. [6], FOXI1 KO did not significantly affect FOXJ1 mRNA expression, consistent with our RT-qPCR results. However, changes in ciliated cell numbers or expression of key markers at a protein level were not tested in their study. Importantly, our results are reinforced by studies in Xenopus laevis epidermis [33] which reported that the almost complete absence of Foxi1 led to a reduced number and aberrant morphology of cilia. Interestingly, engraftment of Foxi1 WT epidermis patches rescued the ciliation of the nearby KO epidermis. Thus, the importance of ionocytes in the production of ciliated cells could be conserved between species and tissues.
Various mechanisms could be driving the decrease of ciliated cells in the absence of FOXI1. First, the KO of FOXI1 could be directly interfering with differentiation of ciliated cells. However, FOXI1 is not expressed during the production of these cells [1, 2] and there is no evidence that the lineage of these two cell types is interdependent even if they both originate from basal cells [2, 6]. Second, cell-to-cell contact could be necessary between ionocytes and ciliated cells for the proper differentiation of the latter. This hypothesis would fit with the results obtained with Xenopus laevis epidermis [33]. Furthermore, ionocyte and ciliated cell differentiation is tightly controlled by Notch signalling. Thus, Notch-related crosstalk between the two cell types could play a role in the maturation of ciliated cells. Finally, it has been shown that ion channels and transporters highly expressed in ionocytes, such as the VATPase, are important in the regulation of Wnt signalling [34,35,36]. Interestingly, canonical Wnt/beta-catenin signalling has a role in the activation of the cilia development machinery via the regulation of FOXJ1 expression [37,38,39], while the Wnt planar cell polarity signalling pathway is responsible for actin organisation and cilia beat alignment and coordination [40, 41]. The lack of ionocytes could affect the acidification of the microenvironment thereby blocking Wnt signalling and ciliated cell differentiation. Further investigation of AECs will help elucidate how such pathways can be controlled by pulmonary ionocytes.
Conclusion
Overall, our study confirms that hiPSCs can be differentiated into an airway epithelium containing ionocytes and that FOXI1 KO leads to a depletion of these cells. We show that the absence of ionocytes leads to impairment of epithelial barrier properties and ciliated cell homeostasis, revealing their potential role in the formation of the airway epithelium. This information represents an important step toward understanding the function of these cells in normal homeostasis and in lung disease, paving the way for new therapeutic applications focusing on ionocytes control.
Data availability
The single guide RNA and RT-qPCR primer sequences used for this study are provided in the Supplementary Materials and Methods. The code used to analyse cilia motility and coverage is publicly available [26] and the obtained data can be found in the Zenodo repository (DOI: https://0-doi-org.brum.beds.ac.uk/10.5281/zenodo.8309970). Other datasets and materials used and/or analysed in this study are available from the corresponding authors on reasonable request.
Abbreviations
- AcTub:
-
acetylated tubulin
- AEC:
-
airway epithelial cell
- ALI:
-
air liquid interface
- ASCL1:
-
achaete-scute family bHLH transcription factor 1
- ASCL3:
-
achaete-scute family bHLH transcription factor 3
- ASL:
-
airway surface liquid
- BSND:
-
barttin CLCNK type accessory subunit beta
- CBF:
-
ciliary beat frequency
- CF:
-
cystic fibrosis
- CFTR:
-
cystic fibrosis transmembrane conductance regulator
- CK5:
-
cytokeratin 5
- CPM:
-
carboxypeptidase-M
- CP110:
-
centrosomal protein of 110 kDa
- CRISPR:
-
clustered regularly interspaced short palindromic repeats
- DAPT:
-
(2 S)-N-[2(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl-glycine 1,1-Dimethylethyl ester
- DNAI1:
-
dynein axonemal intermediate chain 1
- DNAH5:
-
dynein axonemal heavy chain 5
- FGF10:
-
fibroblast growth factor 10
- FOV:
-
field of view
- FOXI1:
-
forkhead box I1
- FOXJ1:
-
forkhead box J1
- GCRP:
-
calcitonin gene-related peptide
- GFP:
-
green fluorescent protein
- hiPSC:
-
human induced pluripotent stem cell
- HBEC:
-
human bronchial epithelial cell
- hiPSC-AECs:
-
human induced pluripotent stem cell-derived airway epithelial cells
- HKG:
-
housekeeping gene
- ISC :
-
short circuit current
- KO:
-
knock-out
- mRNA:
-
messenger RNA
- MUC5AC:
-
mucin 5AC
- NEK10:
-
NIMA related kinase 10
- NKX2.1:
-
NK2 homeobox 1
- PBS:
-
phosphate-buffered saline
- PNEC:
-
pulmonary neuroendocrine cell
- Rt :
-
transepithelial resistance
- RT-qPCR:
-
reverse transcription-quantitative polymerase chain reaction
- SAG:
-
Smoothened agonist
- SCGB3A2:
-
secretoglobin family 3 A member 2
- sgRNA:
-
single guide RNA
- SOX2:
-
SRY-box transcription factor 2
- STAP1:
-
signal transducing adaptor family member 1
- TP63:
-
tumour protein p63
- WT:
-
wild-type
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Acknowledgements
We thank UTHEALTH and Jed Mahoney, Megan Peasley and the scientists at the Cystic Fibrosis Foundation for the gift of the CF17/NKX2.1-GFP human hiPSC line and Scott H. Randell of the University of North Carolina for his advice on ALI cultures. We are also thankful to Anna Osnato, from the Cambridge Stem Cell Institute, for her input in cell sorting experiments.
‘This research was funded in whole, or in part, by the Wellcome Trust [203151/Z/16/Z] and the UKRI Medical Research Council [MC_PC_17230]. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.’
Funding
This work was supported by the Wellcome Trust Sir Henry Wellcome Postdoctoral Fellowship (218663/Z/19/Z to MVG), the UK Cystic Fibrosis Trust (IH001 to MVG, RAF; SRC 016 to LPi, RF, PC; SRC 021 to DNS; VIA 028 to HK, ELR; RM, SLH), the EU ITN PhyMot (to EC), the European Research Council Grant New-Chol (ERC: 741707 to LV), the MRC (MR/P009581/1 to HK, ELR), the Medical Research Foundation Fellowship (to MA-T, AG), the NC3Rs (Training Fellowship NC/R001987/1 to CMM; Project Grant NC/S001204/1 to LPo, WG, FM), the Roy Castle Lung Cancer Foundation grant (2015/10/McCaughan to LPo, WG, FM), the Biotechnology and Biological Science Research Council (BB/W014564/1 to FM and WG), the Cystic Fibrosis Foundation (to RM, SLH) and the core support grant from the Wellcome Trust (203151/Z/16/Z) and the UKRI Medical Research Council (MC_PC_17230) for the Wellcome MRC – Cambridge Stem Cell Institute. This paper presents independent research supported by the NIHR Cambridge BRC. The NIHR Cambridge Biomedical Research Centre (BRC) is a partnership between Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge, funded by the National Institute for Health Research (NIHR). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.
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MVG: Study design, experiment execution, data analysis and interpretation, manuscript writing; LPi: Experiment execution, data analysis and interpretation; RF: Experiment execution, code development, data analysis; EC: Experiment execution, code development, data analysis; HK: In vivo experiment execution, data analysis; MR: Experiment design, experiment execution, data analysis and interpretation; CMM: Generation of GFP hiPSC reporter line; MAT: Experiment execution; LPo: Experiment execution; WG: Experiment execution; RM: Experiment execution; SLH: Experiment design; FM: Experiment design; AG: Experiment design; DNS: Experiment design, data analysis and interpretation, manuscript revision; RAF: Experiment design; ELR: In vivo experiment design and execution, data analysis, manuscript revision; PC: Code development, experiment design; LV: Study design, data analysis, manuscript writing; All authors: reviewing and approval of the final version of the manuscript.
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All animal experiments were approved by local ethical review committees and conducted according to Home Office project license PPL PEEE9B8E4 (Emma L. Rawlins, University of Cambridge).
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Vilà-González, M., Pinte, L., Fradique, R. et al. In vitro platform to model the function of ionocytes in the human airway epithelium. Respir Res 25, 180 (2024). https://0-doi-org.brum.beds.ac.uk/10.1186/s12931-024-02800-7
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DOI: https://0-doi-org.brum.beds.ac.uk/10.1186/s12931-024-02800-7