Single-Cell RNA Sequencing Reveals New Basic and Translational Insights in the Cystic Fibrosis Lung

Abstract

Cystic fibrosis (CF) is a multisystemic, autosomal recessive disorder caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, with the majority of morbidity and mortality extending from lung disease. Single-cell RNA sequencing (scRNA-seq) has been leveraged in the lung and elsewhere in the body to articulate discrete cell populations, describing cell types, states, and lineages as well as their roles in health and disease. In this translational review, we provide an overview of the current applications of scRNA-seq to the study of the normal and CF lungs, allowing the beginning of a new cellular and molecular narrative of CF lung disease, and we highlight some of the future opportunities to further leverage scRNA-seq and complementary single-cell technologies in the study of CF as we bridge from scientific understanding to clinical application.

Cystic fibrosis (CF) is a multisystemic, autosomal recessive disorder caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. The CFTR gene encodes a membrane protein and anion channel of the same name that is found in various epithelia throughout the body, including the respiratory tract, gastrointestinal tract, pancreas, hepatobiliary system, urogenital tract, and sweat gland (Figure 1). Defective or deficient CFTR protein leads to the common manifestations of CF, including obstructive lung disease, pancreatic insufficiency, and male infertility. Lung disease remains the primary cause of morbidity and mortality in people with cystic fibrosis (pwCF).

Figure 1.

The organ systems most commonly affected in cystic fibrosis with representative small airway respiratory and epididymis epithelial cell types, including CFTR-expressing cell types. ASL = airway surface liquid; CFTR = cystic fibrosis transmembrane conductance regulator; NE = pulmonary neuroendocrine cell. Images reproduced and modified from Mount Sinai Health System with permission.

Lung health is dependent on effective mucociliary clearance (1). Mucociliary clearance may be impaired by changes in the composition or hydration of the airway surface liquid, encompassing the mucus and periciliary layers, or by abnormalities of ciliary motion (2). In the respiratory epithelium, defective or deficient CFTR protein, as seen in CF, disrupts airway surface liquid homeostasis, resulting in dehydrated and hyperconcentrated mucus and ineffective mucociliary clearance (3). Advanced cystic fibrosis lung disease (ACFLD) is clinically characterized by mucus airway obstruction, chronic bacterial airway infection, neutrophil-predominant airway inflammation, and progressive airway damage with the development of bronchiectasis.

The application of single-cell RNA sequencing (scRNA-seq) to the study of the CF lung offers the opportunity to define the cellular basis of CF lung disease at previously unprecedented resolution. scRNA-seq not only allows the identification of discrete cell populations with regard to their distinctive gene expression profiles, developmental trajectories, physiologic states, regulatory networks, and specified locations but also provides an opportunity to characterize the cellular and molecular alterations that accompany the disease state (4).

In this translational review, we provide an overview of the current applications of scRNA-seq to the study of the normal and CF lungs, allowing the beginning of a new cellular and molecular narrative of CF lung disease, and we highlight some of the future opportunities to further leverage scRNA-seq and complementary single-cell technologies in the study of CF as we bridge from scientific understanding to clinical application.

The Normal and CF Lungs

Unique in form and function, the lung is composed of a series of branching airways that become shorter, narrower, and more numerous with distal progression, ultimately ending in alveoli. CF has long been touted as a disease of the small airways with the early development of small airway obstruction, although large airway and alveolar diseases have also been described (5–9). Generally defined as noncartilaginous airways with an internal diameter of less than 2 mm, the small airways start with the bronchioles of the conducting zone and extend distally. The superficial bronchiolar respiratory epithelium is composed primarily of basal, multiciliated, and secretory cells, although rarer cells, including brush cells, pulmonary ionocytes, and pulmonary neuroendocrine cells, are also present (Figure 1) (10–15). Although canonical gene markers of expected superficial respiratory epithelial cells are often used to identify cell clusters present in scRNA-seq data, there are notable challenges particular to the use of current single-cell technologies (16). For instance, the identified superficial respiratory epithelial cell types are not consistent across scRNA-seq studies, with some studies specifically identifying club and goblet cells and other studies using the more general secretory cell to include both of the aforementioned. In addition, many scRNA-seq studies identify cell clusters with transcriptional profiles unique from previously described superficial respiratory epithelial cell types that then obtain a new name, such as the “undefined rare cell,” necessitating corroboration through additional experimentation (14, 16). With the continued and increasing use of scRNA-seq, we must pay particular attention to controlled vocabularies, common ontologies, and defined canonical gene markers as our data sets expand, lest we lose the ability to meaningfully communicate with one another. Indeed, the success of reference single-cell atlases will be predicated on their ability to embed new data points in a stable reference framework, allowing variations in data resolution (4, 17–19).

Although the small airways have indeed been a location of immense focus in the study of the CF lung, the airway submucosal glands (SMGs) in the larger, cartilaginous airways must also be considered, because SMGs become hyperplastic with mucus ductal occlusion early in CF lung disease. Lying beneath the superficial respiratory epithelium, the SMGs consist of numerous branching secretory tubules that join to form the main collecting duct, leading to the ciliated duct and ultimately the airway surface (20). Composed primarily of mucous and serous cells, the cells of the SMGs exhibit their own unique transcriptional profiles distinct from the secretory cells of the superficial respiratory epithelium (12–14, 20–22). The structure of the SMGs, along with their location in the airway submucosa, provides a challenge to direct study, limiting our knowledge of their cellular architecture and molecular composition. That being said, three groups have now leveraged scRNA-seq to better understand the cellular and molecular basis of SMG function in health and disease, although only one study used human samples (23–25). Importantly, all three studies required tracheal harvest, necessitating, in the case of humans, donor lungs. Notably, however, several studies applying scRNA-seq to endobronchial forceps biopsies obtained during flexible bronchoscopy were able to capture SMG cells, suggesting that optimal minimally invasive biopsies, either with forceps or the cryoprobe, may be sufficient for future studies of SMGs (12, 14).

Indeed, there are many methods by which to sample the airway for scRNA-seq studies (Figure 2). The least invasive method targets the nose with either nasal brush or forceps biopsies. Special attention must be paid to the study objectives, however, because nasal superficial respiratory epithelial cell types demonstrate distinct gene expression profiles from their corresponding tracheobronchial cell types (14). Alternatively, flexible bronchoscopy provides a minimally invasive method to sample the tracheobronchial airways with either BAL, endobronchial brush or forceps biopsies, or transbronchial forceps or cryobiopsies (Figure 2). Although BAL fluid (BALF) contains superficial respiratory epithelial cells, the concordance of superficial respiratory epithelial cell populations obtained via BAL versus endobronchial brush or forceps biopsies has yet to be evaluated. With regard to endobronchial brush and forceps biopsies, brush biopsies should enable the collection of all of the superficial respiratory epithelial cell types, although basal cells may be underrepresented because of their position in the epithelium, whereas forceps biopsies may enable the collection of all of the superficial respiratory epithelial cell types as well as SMG and mesenchymal cell types (Figure 2) (14). Designed to fit down a 1.2-mm bronchoscope channel, both endobronchial brush and forceps biopsies should enable the sampling of both the large and small airways, at least in adults. Although transbronchial forceps or cryobiopsies have yet to be used in scRNA-seq studies, either should enable the collection of all of the superficial respiratory epithelial cell types as well as SMG and mesenchymal cell types, the latter technique without crush artifact. Notably, the recent introduction of the 1.1-mm flexible cryoprobe makes transbronchial cryobiopsies an intriguing sample source, particularly for studies leveraging single-cell spatial transcriptomics. Finally, resected or donor lung tissue provides an opportunity to sample the entire lung from trachea to alveoli, including the superficial respiratory epithelial, SMG, endothelial, and mesenchymal cell types (Figure 2). Epithelial stripping should enable the collection of all of the superficial respiratory epithelial cell types, although basal cells may again be underrepresented, whereas tissue mincing should enable the collection of the entire spectrum of lung cell types. Importantly, any of the aforementioned methods will also enable the collection of airway immune cell populations, although with expected differences in cell type proportions, depending on sampling method.

Figure 2.

Sample sites and methods for obtaining respiratory epithelial cells. Images reproduced and modified from Mount Sinai Health System with permission.

CFTR Gene Expression in the Normal and CF Lungs

After the identification of the CFTR gene in 1989, focus shifted to the structure, function, and distribution of the resulting CFTR protein (26–28). Located at the apical surface of epithelial cells, the CFTR protein regulates ion transport and fluid homeostasis (29). Initial quantitative studies aimed at defining the distribution and regulation of CFTR gene expression in the normal lung demonstrated overall low levels of CFTR gene transcripts and protein in the respiratory epithelium from nose to alveolus (30–33). Importantly, CFTR gene expression was noted to be heterogeneous with increased expression in a small minority, roughly 1–10%, of uncharacterized superficial respiratory epithelial cells as well as in SMGs (33–35). As technologies advanced, histological studies began identifying specific CFTR-expressing cell types in the superficial respiratory epithelium and SMGs with a particular focus on the abundant multiciliated cell and the serous cell, respectively (33, 34, 36–39).

The application of scRNA-seq to the study of the normal lung fostered a paradigm shift. Indeed, the characterization of the pulmonary ionocyte, defined in part by the increased cell type–specific expression of CFTR gene transcripts, in the superficial respiratory epithelium by scRNA-seq in 2018 raised significant questions regarding the distribution and regulation of CFTR gene expression (10, 11). Initially proposed to be the major source of CFTR gene transcripts in the superficial respiratory epithelium, the role of the pulmonary ionocyte has since been challenged with the prevailing belief now ascribing this role to the more abundant secretory cell (10, 11, 14, 15, 21, 25, 40, 41). Notably, many cells in the superficial respiratory epithelium express CFTR gene transcripts, albeit at varying levels, including basal and multiciliated cells in addition to secretory cells and pulmonary ionocytes (Table 1) (14, 15, 21, 25, 40). Although only one study to date has applied scRNA-seq to the study of the CF lung, the CF lung had overall increased CFTR gene expression as compared with the normal lung with increased CFTR gene expression in pulmonary ionocytes and subsets of basal and secretory cells in particular (21). This may indeed indicate an adaptive response to defective or deficient CFTR protein in the CF lung.

Table 1.

Characteristics of Seven Single-Cell RNA Sequencing Data Sets from the Human Lung Describing the Cell Type–Specific Expression of CFTR Gene Transcripts

Authors	Data Set	Sampling Site	Sampling Method	Sequencing Technology	Reference
Montoro et al.	78,217 cells from one previously healthy donor	Large airways	—	10× Genomics	10
Deprez et al.	18,191 cells from seven healthy people	Nose	Brush and forceps biopsies	10× Genomics	14
	41,134 cells from nine healthy people	Large airways	Forceps biopsy	10× Genomics
	18,644 cells from nine healthy people	Small airways	Brush biopsy	10× Genomics
Okuda et al.	11,688 cells from eight previously healthy donors	Large airways	Epithelial stripping	10× Genomics	15
	4,955 cells from eight previously healthy donors	Small airways	Tissue mincing	10× Genomics
	16,488 cells from four healthy people	Large airways	Brush biopsy	Drop-seq
	9,831 cells from three previously healthy donors	Small airways	Tissue dissection	Drop-seq
Carraro et al.	Ten donors with CF and 11 previously healthy donors	Large airways	Epithelial stripping and mincing	10× Genomics	21
	Nine donors with CF and eight previously healthy donors	Large airways	Epithelial stripping	Drop-seq
Goldfarbmuren et al.	36,248 cells from 15 donors, including six never-smokers and six heavy smokers	Large airways	Epithelial stripping	10× Genomics	25
Habermann et al.	114,396 cells from 20 donors with pulmonary fibrosis and 10 previously healthy donors	Lung parenchyma	—	10× Genomics	40
Miller et al.	6,548 cells from two fetuses at 15–21 wk of gestation	Large airways	Epithelial stripping	10× Genomics	41
	11,829 cells from three fetuses at 11.5–18 wk of gestation	Small airways	Tissue mincing	10× Genomics
	18,430 cells from three fetuses at 11.5–18 wk of gestation	Lung parenchyma	Tissue mincing	10× Genomics

Definition of abbreviations: CF = cystic fibrosis; Drop-seq = droplet sequencing.

Although scRNA-seq data regarding the distribution and regulation of CFTR gene expression in SMGs are not yet available in humans, scRNA-seq data from porcine models notably ascribe equal CFTR gene expression to both mucous and serous cells, suggesting comparable contributions and, once again, challenging prevailing belief (23).

The application of scRNA-seq to the study of the normal lung has enabled the general characterization of CFTR-expressing cell types in the respiratory epithelium. It is unlikely, however, that the functional role of the CFTR protein is uniform between cell types or along the tracheobronchial tree. Although certain cell type–specific functionalities have been proposed, many others have yet to be elucidated (15). Indeed, further investigations are needed to define the physiologic functions of CFTR-expressing cell types, the role of the CFTR protein in these functions, and the contribution of the loss of these functions to the CF disease state. The answers to the aforementioned will be of great importance to the identification of targets for CFTR gene correction.

Disease-related Changes in the CF Lung

In 2021, Carraro and colleagues published the first and currently the only article to leverage scRNA-seq in the study of the CF lung (Table 2) (21). Comparing the large airways of pwCF undergoing lung transplant for ACFLD with those of previously healthy lung donors, Carraro and colleagues sought to define disease-related changes in the superficial respiratory epithelium of CF large airways through the application of scRNA-seq (21). Disease-related changes were reported in specific subsets of secretory, multiciliated, and basal cells within the CF large airways, including an increase in secretory cell function, an increase in superficial respiratory epithelial cells transitioning to specialized secretory and multiciliated cell subsets with increased secretory-to-multiciliated cell and direct basal-to-multiciliated cell transitioning, and a decrease in proliferating basal cells, respectively (21). Notably, the latter challenges prior histological studies, also performed in CF large airways, that reported an increase in proliferating basal cells (42, 43). This discrepancy undoubtedly merits further investigation because the basal cell is the main respiratory epithelial progenitor cell in the large airways.

Table 2.

Characteristics of Five Single-Cell RNA-Sequencing Data Sets in Study of Cystic Fibrosis Lung

Authors	Species and Source	Data Set	Sampling Site	Sampling Method	Sequencing Technology	Reference
Carraro et al.	Human: superficial respiratory epithelial cells	Ten donors with CF and 11 previously healthy lung donors	Large airways	Epithelial stripping and tissue mincing	10× Genomics	21
		Nine donors with CF and eight previously healthy lung donors	Large airways	Epithelial stripping	Drop-seq
Yu et al.	Pig: SMGs	14,561 cells from four CFTR−/− and four wild-type pigs	Large airways	Tissue dissection	10× Genomics	23
Schupp et al.	Human: sputum	12,494 cells from nine pwCF	Spontaneously expectorated sputum	Filtering	10× Genomics	44
		7,601 cells from five healthy people	Induced sputum	Filtering	10× Genomics
Li et al.	Human: BALF	113,213 cells from three pwCF and four healthy people	BALF	Filtering	10× Genomics	46
Thurman et al.	Pig: lung	8,928 cells from five CFTR−/− and five wild-type pigs	Large airways	Epithelial stripping	10× Genomics	67
		17,773 cells from three CFTR−/− and four wild-type pigs	Small airways	Tissue dissection	10× Genomics

Definition of abbreviations: BALF = BAL fluid; pwCF = people with cystic fibrosis; SMG = submucosal gland.

Carraro and colleagues provided the first report of superficial tracheobronchial respiratory epithelial cell types and states in ACFLD. Although they provided key insights into the pathophysiology of CF lung disease, further investigations leveraging scRNA-seq should target more distal airways, including the small airways, and SMGs as well as pwCF with different genotypes, degrees of disease severity, and treatment regimens, including the use of CFTR protein modulator therapies. Indeed, we can envision the utility of repeated minimally invasive airway sampling via flexible bronchoscopy in conjunction with scRNA-seq to define the progression of disease-related changes in the CF lung and, in doing so, to identify targets for CFTR gene correction as well as to monitor an individual person’s disease course and response to therapy.

In 2020, Schupp and colleagues published the first and currently the only article to leverage scRNA-seq in the study of CF sputum samples (Table 2) (44). Although not intrinsic to the CF lung, neutrophil-predominant airway inflammation is a hallmark of CF lung disease, and Schupp and colleagues thus sought to characterize airway immune cell populations from pwCF (44). A disease-related shift in the airway immune cell repertoire was appreciated, moving from alveolar macrophages in induced sputum from otherwise healthy people to a predominance of recruited monocytes and neutrophils in spontaneously expectorated sputum from pwCF (44, 45). Disease-related changes were reported in specific populations of airway immune cells within CF sputum samples, including a predominance of activated proinflammatory monocytes and heat shock–activated monocytes within recruited lung mononuclear phagocytes and a shift in polymorphonuclear neutrophils toward an immature and proinflammatory phenotype (44).

In 2022, Li and colleagues published the first and currently the only article to leverage scRNA-seq in the study of CF BALF (Table 2) (46). Similarly to Schupp and colleagues, Li and colleagues sought to characterize airway immune cell populations from pwCF, choosing, however, to focus on pwCF with normal lung function and minimal lung inflammation. Interestingly, only a minor disease-related shift in the airway immune cell repertoire was appreciated with a predominance of alveolar macrophages in both pwCF and otherwise healthy people (46). Although many studies have pointed to impaired innate immune cell function in the CF lung, the presence of only a minor disease-related shift in the airway immune cell repertoire in this subset of pwCF suggests that lung disease may not be a predetermined outcome in CF (46–52). Indeed, it will be critical to include infants and young children in future investigations, monitoring disease-related changes in the respiratory epithelium of the large and small airways and in airway immune cell populations over time and with disease progression. It may be that correcting the defective or deficient CFTR protein and preventing cycles of infection and inflammation might delay or prevent the development of CF lung disease.

Animal Models of CF Lung Disease

Multiple animal models for CF have been generated to better understand the pathophysiology of disease and facilitate the development and testing of potential therapies. The mouse is the most commonly employed experimental animal, and murine scRNA-seq studies were indeed critical to the characterization of the pulmonary ionocyte (10, 11). Although several CFTR-deficient murine lines have been developed, CFTR-deficient mice do not readily recapitulate the CF lung disease seen in humans, despite evidence of other CF-specific features, such as intestinal obstruction and impaired growth (53–55). Despite the limitations of murine models, particularly for CF lung disease, we expect to see continued and increasing use of scRNA-seq in mice, particularly with the current global initiatives driving the development of reference human and murine cell atlases (4, 17, 18).

The ferret and the pig are frequently employed experimental animal models for human respiratory diseases, including CF (56, 57). Contrary to mice, CFTR-deficient ferrets and pigs readily recapitulate the CF lung disease seen in humans, likely in part because of greater similarities in lung anatomy with robust expression of SMGs throughout the cartilaginous airways as well as in CFTR protein structure and bioelectric properties (54, 57–65). Although no scRNA-seq studies in ferret lung have been published to date, we expect these studies in the near future. Indeed, scRNA-seq has been employed to characterize the airway immune cell repertoire of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected ferrets from BALF samples (66). Two studies to date have leveraged scRNA-seq in the study of normal and CFTR-deficient porcine lungs, one focusing on the superficial respiratory epithelium of the large and small airways and the other on SMGs (Table 2) (23, 67). Very few disease-related changes were reported in the superficial respiratory epithelium of the large and small airways of CFTR-deficient pigs at birth, suggesting that chronic airway infection and inflammation, and not cell-intrinsic abnormalities, drive the disease-related changes noted in ACFLD, providing further evidence that correcting the defective or deficient CFTR protein and preventing cycles of infection and inflammation may delay or prevent the development of CF lung disease (67). Interestingly, the superficial respiratory epithelial cell types in the large airways had gene expression profiles distinct from their corresponding cell types in the small airways, with location-specific differences in secreted mucins and innate antimicrobial molecules (67). Given the importance of the small airways in the pathogenesis of CF lung disease, the presence of basal cells at the apical surface of the small airways with a suggestion of participation in transepithelial ion transport is significant for both CFTR gene correction and our understanding of CF pathophysiology (67). Indeed, differences in ion and fluid transport between the large and small airways require further investigation and consideration when modeling and interpreting the pathophysiology of CF lung disease (67).

Although the application of scRNA-seq to the study of animal models of CF lung disease will undoubtedly help inform the pathophysiology of CF lung disease and facilitate the development and testing of potential therapies, Pennitz and colleagues offer an additional utility (68). Through a workflow for interspecies scRNA-seq data integration using a single unified gene nomenclature, Pennitz and colleagues identified marker genes for each of six species, including mouse, pig, and human (68). Such comparisons have the potential to systematically identify the opportunities and limitations of generated animal models for CF, allowing the selection of appropriate animal models for future studies of CF lung disease.

Future Opportunities for scRNA-seq and Complementary Single-Cell Technologies in the Study of CF

Single-Cell Spatial Transcriptomics in the CF Lung

The application of single-cell spatial transcriptomics affords the opportunity to capture the transcriptional landscape of a native histological tissue section at the single-cell scale, allowing the localization of specific cell populations and informing local networks of intercellular communication in situ (69). The majority of scRNA-seq studies in normal and diseased lung to date have relied on the dissociation of lung tissue or biopsies, which inherently lack detailed spatial resolution and may not represent the entirety of the lung (10–13, 15, 21, 22, 40, 70). Multiple studies have now leveraged single-cell spatial transcriptomics in the study of the lung, including two in humans (71–77). It should be noted, however, that although single-cell spatial transcriptomic technologies are rapidly advancing, the resolution of the currently available technologies has not yet reached the single-cell scale, instead averaging roughly 1–10 cells per spot (76, 78–80).

Informed by matched scRNA-seq data, the application of single-cell spatial transcriptomics to the study of the lung holds the potential to provide a detailed description of the location of and physiologic interactions between lung cell populations, including respiratory epithelial and airway immune cell populations, as well as dynamic changes in the aforementioned in the CF lung. Indeed, the interaction between respiratory epithelial and airway immune cell populations is essential for the maintenance of tissue homeostasis, the initiation of an effective immune response, and the promotion of tissue repair. Elucidation of these context-dependent interactions through single-cell spatial transcriptomics in the normal lung as well as derangements of such interactions in the CF lung may provide insight into the cycles of infection and inflammation characteristic of CF lung disease and their impact on the architecture of the CF lung.

Single-Cell Multiomics in the CF Lung

The application of scRNA-seq to the study of the CF lung has allowed the articulation of discrete respiratory epithelial and airway immune cell populations, informing the cellular alterations that accompany CF lung disease (21, 44). Because of the limitations of scRNA-seq, including variations in resolution, scRNA-seq fails to capture the full spectrum of molecular processes that influence CFTR gene expression. Complementary single-cell technologies that allow the simultaneous assessment of gene expression through transcriptome profiling in addition to genome, epigenome, and/or proteome profiling have been exploited, albeit outside of the study of CF (19, 81).

As an example, it is well known that specific cis-regulatory elements (CREs) play critical roles in the cell type–specific regulation of CFTR gene expression (82). Despite the fact that CREs drive spatiotemporal patterns of gene expression through associations with specific transcription factors and enable cell type–specific responses to intra- and extracellular signals, such as inflammation, there is a paucity of single-cell data focused on mapping CREs in the human genome that are active in specific respiratory epithelial cell populations (83, 84). Given that accessible or open chromatin is a hallmark of CREs, the single-cell sequencing assay for transposase-accessible chromatin may be used to map the epigenome and gene regulatory programs for specific respiratory epithelial cell populations and, when used in conjunction with scRNA-seq data, to further inform the cell type–specific regulation of gene expression, including CFTR gene expression.

As another example, in 2022, Xu and colleagues performed a single-cell multiomic study in the murine lung, leveraging scRNA-seq, single-cell sequencing assay for transposase-accessible chromatin, and single-cell spatial transcriptomics to profile tissue-resident T cells (76). Interestingly, Xu and colleagues demonstrated a dynamic change in the location of T helper cells and their corresponding chemokines after Klebsiella pneumoniae immunization and rechallenge, demonstrating the potential of single-cell multiomic studies to characterize spatially dependent and cell type–dependent mechanisms of lung immunity (76).

Although single-cell multiomic technologies are rapidly evolving, the available technologies are restricted by several technical and computational limitations that are outside the scope of this translational review. We fully expect, however, that continued advancements in single-cell multiomic technologies will provide the opportunity to investigate not only the cell type–specific regulation of CFTR gene expression but also the dynamic interactions between the respiratory epithelium and the immune microenvironment.

scRNA-seq in the CF Lung and in Other Affected Organs

The application of scRNA-seq to the study of CF has been largely relegated to the lung, the primary site of morbidity and mortality in pwCF. CF, however, is a multisystemic disorder, and the application of scRNA-seq to the various affected epithelia may allow an enhanced understanding of the location, distribution, and function of the CFTR protein in health and disease. Notably, Paranjapye and colleagues have begun this work, comparing the superficial respiratory epithelium from the nose and bronchus with the superficial epididymal epithelium (85). Interestingly, the pulmonary ionocyte and epididymis clear cell, both of which demonstrate increased cell type–specific expression of CFTR gene transcripts, show a strongly conserved identity (Figure 1) (10, 11, 85, 86). Whether these two highly specialized cell types serve the same function in their respective epithelia has yet to be fully elucidated, although the suggestion is certainly present.

scRNA-seq studies have now been performed in multiple epithelia throughout the body, including the gastrointestinal tract, pancreas, and urogenital tract in addition to the respiratory tract, defining, in part, the distribution and regulation of CFTR gene expression (86–89). Comparative scRNA-seq data from epithelia throughout the body may serve to better inform the function of the CFTR protein as well as the functional relationship of CFTR gene expression to other cellular and intercellular processes, some of which are likely to be cell type and tissue specific.

Conclusions

The application of scRNA-seq to the study of CF, and the CF lung in particular, has allowed the beginning of a new cellular and molecular narrative of CF lung disease. Such insight may prove important to the clinical care of pwCF from the development of new therapeutic and curative technologies to the personalized monitoring of disease progression and response to intervention. Importantly, the advancement of complementary single-cell technologies is expected to further delineate the cell type–specific regulation of CFTR gene expression and, in doing so, to identify targets for CFTR gene correction. Because concepts arising from studies leveraging scRNA-seq and complementary single-cell technologies are actively evolving, corroborative studies must be undertaken to improve our understanding of the pathophysiology of CF.