Viral airway injury promotes cell engraftment in an in vitro model of cystic fibrosis cell therapy.

Cell therapy is a potential treatment for cystic fibrosis (CF). However, cell engraftment into the airway epithelium is challenging. Here, we model cell engraftment in vitro using the air-liquid interface (ALI) culture system by injuring well-differentiated CF ALI cultures and delivering non-CF cells at the time of peak injury. Engraftment efficiency was quantified by measuring chimerism by droplet digital PCR and functional ion transport in Ussing chambers. Using this model, we found that human bronchial epithelial cells (HBECs) engraft more efficiently when they are cultured by conditionally reprogrammed cell (CRC) culture methods. Cell engraftment into the airway epithelium requires airway injury, but the extent of injury needed is unknown. We compared three injury models and determined that severe injury with partial epithelial denudation facilitates long-term cell engraftment and functional CFTR recovery up to 20% of wildtype function. The airway epithelium promptly regenerates in response to injury, creating competition for space and posing a barrier to effective engraftment. We examined competition dynamics by time-lapse confocal imaging and found that delivered cells accelerate airway regeneration by incorporating into the epithelium. Irradiating the repairing epithelium granted engrafting cells a competitive advantage by diminishing resident stem cell proliferation. Intentionally, causing severe injury to the lungs of people with CF would be dangerous. However, naturally occurring events like viral infection can induce similar epithelial damage with patches of denuded epithelium. We found that viral preconditioning promoted effective engraftment of cells primed for viral resistance.NEW & NOTEWORTHY Cell therapy is a potential treatment for cystic fibrosis (CF). Here, we model cell engraftment by injuring CF air-liquid interface cultures and delivering non-CF cells. Successful engraftment required severe epithelial injury. Intentionally injuring the lungs to this extent would be dangerous. However, naturally occurring events like viral infection induce similar epithelial damage. We found that viral preconditioning promoted the engraftment of cells primed for viral resistance leading to CFTR functional recovery to 20% of the wildtype.

Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection.

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy.

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

Hospital-level variation in practices and outcomes for patients with severe acute exacerbations of idiopathic pulmonary fibrosis: a retrospective multicentre cohort study.

BACKGROUND: In the absence of evidence-based strategies to improve patient outcomes, the management of patients with severe idiopathic pulmonary fibrosis (IPF) exacerbations may vary widely across centres. We assessed between-hospital variation in practices and mortality for patients with severe IPF exacerbations. METHODS: Using the Premier Healthcare Database from 1 October 2015 to 31 December 2020, we identified patients admitted to intensive care unit (ICU) or intermediate care unit with an IPF exacerbation. We assessed idiosyncratic, between-hospital variation in ICU practices (invasive mechanical ventilation (IMV), non-invasive mechanical ventilation (NIMV), corticosteroid use, and immunosuppressive and/or antioxidant use) and hospital mortality by determining median risk-adjusted hospital rates and intraclass correlation coefficients (ICCs) from hierarchical multivariable regression models. A priori, an ICC>15% was deemed 'high variation'. RESULTS: We identified 5256 critically ill patients with a severe IPF exacerbation at 385 US hospitals. Hospital median risk-adjusted rates of practices were: IMV (14% (IQR: 8.3%-26%)), NIMV (42% (31%-54%)), corticosteroid use (89% (84%-93%)), and immunosuppressive and/or antioxidant use (3.3% (1.9%-5.8%)). Model ICCs were: IMV (19% (95% CI: 18% to 21%)), NIMV (15% (13% to 16%)), corticosteroid use (9.8% (8.3% to 11%)), and immunosuppressive and/or antioxidant use (8.5% (7.1% to 9.9%)). The median risk-adjusted hospital mortality was 16% (IQR: 11%-24%) with an ICC of 7.5% (95% CI: 6.2% to 8.9%). INTERPRETATION: We observed high variation in the use of IMV and NIMV, and less variation in corticosteroid and immunosuppressant and/or antioxidant use among patients hospitalised with severe IPF exacerbations. Further research is needed to guide the decisions surrounding initiation of IMV and role of NIMV and to understand the effectiveness of corticosteroids among patients with severe IPF exacerbations.

Airway stem cell reconstitution by the transplantation of primary or pluripotent stem cell-derived basal cells.

Life-long reconstitution of a tissue's resident stem cell compartment with engrafted cells has the potential to durably replenish organ function. Here, we demonstrate the engraftment of the airway epithelial stem cell compartment via intra-airway transplantation of mouse or human primary and pluripotent stem cell (PSC)-derived airway basal cells (BCs). Murine primary or PSC-derived BCs transplanted into polidocanol-injured syngeneic recipients give rise for at least two years to progeny that stably display the morphologic, molecular, and functional phenotypes of airway epithelia. The engrafted basal-like cells retain extensive self-renewal potential, evident by the capacity to reconstitute the tracheal epithelium through seven generations of secondary transplantation. Using the same approach, human primary or PSC-derived BCs transplanted into NOD scid gamma (NSG) recipient mice similarly display multilineage airway epithelial differentiation in vivo. Our results may provide a step toward potential future syngeneic cell-based therapy for patients with diseases resulting from airway epithelial cell damage or dysfunction.

Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

A multimodal iPSC platform for cystic fibrosis drug testing.

Cystic fibrosis is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of CFTR using established animal and cell-based models led to the recent discovery of effective modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we generate a panel of airway epithelial cells using induced pluripotent stem cells from individuals with common or rare CFTR variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function we adapt two established in vitro assays for use in induced pluripotent stem cell-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we detect genotype-specific differences in CFTR baseline function and response to CFTR modulators. These results demonstrate the potential of the human induced pluripotent stem cell platform as a research tool to study CF and in particular accelerate therapeutic development for CF caused by rare variants.

Differentiation of human pluripotent stem cells into functional airway basal stem cells.

Airway basal cells play an essential role in the maintenance of the airway epithelium. Here, we provide a detailed directed differentiation protocol to generate ''induced basal cells (iBCs)'' from human pluripotent stem cells. iBCs recapitulate biological and functional properties of airway basal cells including mucociliary differentiation in vitro or in vivo in tracheal xenografts, facilitating the study of inherited and acquired diseases of the airway, as well as potential use in regenerative medicine. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2021).

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells.

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

Correction of Airway Stem Cells: Genome Editing Approaches for the Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although CF affects multiple organs, the primary cause of mortality is respiratory failure resulting from poor clearance of hyperviscous secretions and subsequent airway infection. Recently developed CFTR modulators provide significant therapeutic benefit to the majority of CF individuals. However, treatments directed at the underlying cause are needed for the ∼7% of CF patients who are not expected to be responsive to these modulators. Genome editing can restore the native CFTR genetic sequence and function to mutant cells, representing an approach to establish durable physiologic CFTR correction. Although editing the CFTR gene in various airway cell types may transiently restore CFTR activity, effort is focused on editing airway basal stem/progenitor cells, since their correction would allow appropriate and durable expression of CFTR in stem cell-derived epithelial cell types. Substantial progress has been made to directly correct airway basal cells in vitro, theoretically enabling transplantation of autologous corrected cells to regenerate an airway with CFTR functional cells. Another approach to create autologous, gene-edited airway basal cells is derivation of CF donor-specific induced pluripotent stem cells, correction of the CFTR gene, and subsequent directed differentiation to airway basal cells. Further work is needed to translate these advances by developing effective transplantation methods. Alternatively, gene editing in vivo may enable CFTR correction. However, this approach will require robust delivery methods ensuring that basal cells are efficiently targeted and corrected. Recent advances in gene editing-based therapies provide hope that the genetic underpinning of CF can be durably corrected in airway epithelial stem cells, thereby preventing or treating lung disease in all people with CF.

Pulmonary Ionocytes Challenge the Paradigm in Cystic Fibrosis.

Two recent studies have identified novel airway cells termed pulmonary ionocytes that express higher levels of CFTR than other airway cells express. These findings raise new questions in the evolving debate about the physiological role of CFTR in lung epithelia and its importance in the pathogenesis of cystic fibrosis (CF).

Medical and surgical management of chylothorax and associated outcomes.

Chylothorax is an uncommon form of pleural effusion that can be associated with traumatic and nontraumatic causes. Optimal management and outcome for patients with chylothorax remain unclear. This retrospective single-center study assessed the modes of management for chylothorax in 74 adult patients (> or =18 years old) and associated outcomes. The role of lymphangiographic imaging was also evaluated. Initial treatment approach was nonsurgical in 57 patients (77%) but a surgical procedure (pleurodesis, thoracic duct ligation, and/or surgical repair) was eventually performed in 44 patients (59%). The rate of resolution with initial treatment measures was significantly worse for patients with nontraumatic chylothorax compared with those with traumatic causes (27% versus 50%, P = 0.048). Even after additional therapeutic maneuvers including surgery, chylous effusion recurred more commonly in nontraumatic chylothorax when compared with the traumatic group (50% versus 13%, respectively, P < 0.001). Lymphatic imaging did not seem to materially influence management. Nonsurgical approaches may lead to resolution of the chylothorax in nearly one half of patients with traumatic chylothorax but in only a minority of those with nontraumatic chylothorax. The majority of patients with nontraumatic chylothorax will eventually require surgical maneuvers, but one third of such patients still fail to resolve their chylothorax.

Pleural fluid characteristics of chylothorax.

OBJECTIVE: To determine the biochemical parameters of chylous pleural fluids and better inform current clinical practice in the diagnosis of chylothorax. PATIENTS AND METHODS: We retrospectively reviewed 74 patients with chylothorax (defined by the presence of chylomicrons) who underwent evaluation during a 10-year period from January 1, 1997, through December 31, 2006. The biochemical parameters and appearance of the fluid assessed during diagnostic evaluation were analyzed. RESULTS: The study consisted of 37 men (50%) and 37 women (50%), with a median age of 61.5 years (range, 20-93 years). Chylothorax was caused by surgical procedures in 51%. The chylous pleural fluid appeared milky in only 44%. Pleural effusion was exudative in 64 patients (86%) and transudative in 10 patients (14%). However, pleural fluid protein and lactate dehydrogenase levels varied widely. Transudative chylothorax was present in all 4 patients with cirrhosis but was also seen with other causes. The mean +/- SD triglyceride level was 728+/-797 mg/dL, and the mean +/- SD cholesterol value was 66+/-30 mg/dL. The pleural fluid triglyceride value was less than 110 mg/dL in 10 patients (14%) with chylothorax, 2 of whom had a triglyceride value lower than 50 mg/dL. CONCLUSION: Chylothoraces may present with variable pleural fluid appearance and biochemical characteristics. Nonmilky appearance is common. Chylous effusions can be transudative, most commonly in patients with cirrhosis. Traditional triglyceride cutoff values used in excluding the presence of chylothorax may miss the diagnosis in fasting patients, particularly in the postoperative state.