Generation of Multipotent Lung and Airway Progenitors from Mouse ESCs and Patient-Specific Cystic Fibrosis iPSCs

OBJECTIVES/SPECIFIC AIMS: Transforming growth factor-beta (TGFβ) is a genetic modifier of cystic fibrosis (CF) lung disease. TGFβ’s pulmonary levels in young CF patients and its mechanism of action in CF are unknown. We examined TGFβ levels in children with CF and investigated responses of human airway epithelial cells (AECs) and mice to TGFβ. METHODS/STUDY POPULATION: TGFβ levels in bronchoalveolar lavage fluid from CF patients (n=15) and non-CF control patients (n=21)<6 years old were determined by ELISA. CF mice and non-CF mice were intratracheally treated with an adenoviral TGFβ1 vector or PBS; lungs were collected for analysis at day 7. Human CF and non-CF AECs were treated with TGFβ or PBS for 24 hours then collected for analysis. RESULTS/ANTICIPATED RESULTS: Young CF patients had higher bronchoalveolar lavage fluid TGFβ than non-CF controls (p=0.03). Mouse lungs exposed to TGFβ demonstrated inflammation, goblet cell hyperplasia, and decreased CFTR expression. CF mice had greater TGFβ-induced lung mechanics abnormalities than controls; both CF human AECs and CF mice showed higher TGFβ induced MAPK and PI3K signaling compared with controls. DISCUSSION/SIGNIFICANCE OF IMPACT: For the first time, we show increased TGFβ levels very early in CF. TGFβ drives CF lung abnormalities in mouse and human models; CF models are more sensitive to TGFβ’s effects. Understanding the role of TGFβ in promoting CF lung disease is critical to developing patient specific treatments.

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ePS06.4 Patient-specific modeling of regional tobramycin concentration levels in airways of patients with cystic fibrosis: can we dose once daily?

Journal of Cystic Fibrosis ◽

10.1016/s1569-1993(16)30234-x ◽

2016 ◽

Vol 15 ◽

pp. S48-S49

Author(s):

A.C. Bos ◽

C. van Holsbeke ◽

J.W. de Backer ◽

H.M. Janssens ◽

W.G. Vos ◽

...

Keyword(s):

Cystic Fibrosis ◽

Patient Specific ◽

Once Daily ◽

Patient Specific Modeling ◽

Specific Modeling ◽

Concentration Levels

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Chronic Airway Colonization byAchromobacter xylosoxidansin Cystic Fibrosis Patients Is Not Sustained by Their Domestic Environment

Applied and Environmental Microbiology ◽

10.1128/aem.01739-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 4

Author(s):

Chloé Dupont ◽

Estelle Jumas-Bilak ◽

Clara Doisy ◽

Fabien Aujoulat ◽

Raphaël Chiron ◽

...

Keyword(s):

Cystic Fibrosis ◽

Respiratory Tract ◽

Antimicrobial Agents ◽

Patient Specific ◽

Achromobacter Xylosoxidans ◽

Opportunistic Pathogens ◽

Domestic Environment ◽

Content Type ◽

Domestic Environments ◽

Airway Colonization

ABSTRACTAchromobacterspp. are nonfermentative Gram-negative bacilli considered emergent pathogens in cystic fibrosis (CF). Although some cross-transmission events between CF patients have been described,Achromobacterstrains were mostly patient specific, suggesting sporadic acquisitions from nonhuman reservoirs. However, sources of these emergent CF pathogens remain unknown. A large collection of specimens (n= 273) was sampled in the homes of 3 CF patients chronically colonized byAchromobacter xylosoxidanswith the aim of evaluating the potential role of domestic reservoirs in sustaining airway colonization of the patients. Samples were screened for the presence ofAchromobacterby using genus-specific molecular detection. Species identification, multilocus genotypes, and antimicrobial susceptibility patterns observed for environmental isolates were compared with those of clinical strains. Patient homes hosted a high diversity ofAchromobacterspecies (n= 7), includingAchromobacter mucicolensandA. animicus, two species previously isolated from human samples only, and genotypes (n= 15), all showing an overall susceptibility to antimicrobial agents.Achromobacterstrains were mostly isolated from indoor moist environments and siphons, which are potential reservoirs for several CF emerging pathogens.A. xylosoxidans, the worldwide prevalent species colonizing CF patients, was not the majorAchromobacterspecies inhabiting domestic environments.A. xylosoxidansgenotypes chronically colonizing the patients were not detected in their household environments. These results support the notions that the domestic environment could not be incriminated in sustained patient colonization and that after initial colonization, the environmental survival ofA. xylosoxidansclones adapted to the CF airways is probably impaired.IMPORTANCEAchromobacterspp. are worldwide emerging opportunistic pathogens in CF patients, able to chronically colonize the respiratory tract. Apart from regular consultations at the hospital CF center, patients spend most of their time at home. Colonization from nonhuman sources has been suggested, but the presence ofAchromobacterspp. in CF patients' homes has not been explored. The domestic environments of CF patients chronically colonized byAchromobacter, especially wet environments, host several opportunistic pathogens, including a large diversity ofAchromobacterspecies and genotypes. However,Achromobactergenotypes colonizing the patients were not detected in their domestic environments, making it unlikely that a shuttle between environment and CF airways is involved in persisting colonization. This also suggests that once the bacteria have adapted to the respiratory tract, their survival in the domestic environment is presumably impaired. Nevertheless, measures for reducing domestic patient exposure should be targeted on evacuation drains, which are frequently contaminated by CF opportunistic pathogens.

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Long-Term Rasamsonia argillacea Complex Species Colonization Revealed by PCR Amplification of Repetitive DNA Sequences in Cystic Fibrosis Patients

Journal of Clinical Microbiology ◽

10.1128/jcm.01462-16 ◽

2016 ◽

Vol 54 (11) ◽

pp. 2804-2812 ◽

Cited By ~ 10

Author(s):

Abdelmounaim Mouhajir ◽

Olivier Matray ◽

Sandrine Giraud ◽

Laurent Mély ◽

Christophe Marguet ◽

...

Keyword(s):

Cystic Fibrosis ◽

Repetitive Dna ◽

Dna Sequences ◽

Species Complex ◽

Pcr Amplification ◽

Patient Specific ◽

Molecular Evidence ◽

Repetitive Dna Sequences ◽

Complex Species ◽

Content Type

The aim of this work was to document molecular epidemiology of Rasamsonia argillacea species complex isolates from cystic fibrosis (CF) patients. In this work, 116 isolates belonging to this species complex and collected from 26 CF patients and one patient with chronic granulomatous disease were characterized using PCR amplification assays of repetitive DNA sequences and electrophoretic separation of amplicons (rep-PCR). Data revealed a clustering consistent with molecular species identification. A single species was recovered from most patients. Rasamsonia aegroticola was the most common species, followed by R. argillacea sensu stricto and R. piperina , while R. eburnea was not identified. Of 29 genotypes, 7 were shared by distinct patients while 22 were patient specific. In each clinical sample, most isolates exhibited an identical genotype. Genotyping of isolates recovered from sequential samples from the same patient confirmed the capability of R. aegroticola and R. argillacea isolates to chronically colonize the airways. A unique genotype was recovered from two siblings during a 6-month period. In the other cases, a largely dominant genotype was detected. Present results which support the use of rep-PCR for both identification and genotyping for the R. argillacea species complex provide the first molecular evidence of chronic airway colonization by these fungi in CF patients.

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Patient-Specific Modeling of Regional Antibiotic Concentration Levels in Airways of Patients with Cystic Fibrosis: Are We Dosing High Enough?

PLoS ONE ◽

10.1371/journal.pone.0118454 ◽

2015 ◽

Vol 10 (3) ◽

pp. e0118454 ◽

Cited By ~ 26

Author(s):

Aukje C. Bos ◽

Cedric van Holsbeke ◽

Jan W. de Backer ◽

Mireille van Westreenen ◽

Hettie M. Janssens ◽

...

Keyword(s):

Cystic Fibrosis ◽

Patient Specific ◽

Antibiotic Concentration ◽

Patient Specific Modeling ◽

Specific Modeling ◽

Concentration Levels

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Bioorthogonal non-canonical amino acid tagging reveals translationally active subpopulations of the cystic fibrosis lung microbiota

10.1101/588640 ◽

2019 ◽

Author(s):

Talia D. Valentini ◽

Sarah K. Lucas ◽

Kelsey A. Binder ◽

Lydia C. Cameron ◽

Jason A. Motl ◽

...

Keyword(s):

Cystic Fibrosis ◽

Amino Acid ◽

Growth Strategy ◽

Fluorescent Imaging ◽

Cystic Fibrosis Lung ◽

Patient Specific ◽

Rrna Gene ◽

Metabolic Labeling ◽

Bacterial Cells ◽

Canonical Amino Acid

AbstractCulture-independent studies of cystic fibrosis lung microbiota have provided few mechanistic insights into the polymicrobial basis of disease. Deciphering the specific contributions of individual taxa to CF pathogenesis requires a comprehensive understanding of theirin situecophysiology. We applied bioorthogonal non-canonical amino acid tagging (BONCAT), a ‘click’ chemistry-based metabolic labeling approach, to quantify and visualize translational activity among CF microbiota. Using BONCAT-based fluorescent imaging on sputum collected from stable CF subjects, we reveal that only a subset of bacteria are translationally active. We also combined BONCAT with fluorescent activated cell sorting (FACS) and 16S rRNA gene sequencing to assign taxonomy to the active subpopulation and found that the most dominant taxa are indeed translationally active. On average, only ∼12-18% of bacterial cells were BONCAT labeled, suggesting a heterogeneous growth strategy widely employed by most airway microbiota. Differentiating translationally active populations from those that are dormant adds to our evolving understanding of the polymicrobial basis of CF lung disease and may help guide patient-specific therapeutic strategies targeting active bacterial populations that are most likely to be susceptible.

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A new platform for high-throughput therapy testing on iPSC-derived, immature airway from Cystic Fibrosis Patients

10.1101/2021.07.05.451013 ◽

2021 ◽

Author(s):

Jia Xin Jiang ◽

Leigh Wellhauser ◽

Onofrio Laselva ◽

Irina Utkina ◽

Zoltan Bozoky ◽

...

Keyword(s):

Cystic Fibrosis ◽

Stem Cell ◽

High Throughput ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Functional Responses ◽

Primary Defect ◽

Epithelial Cultures ◽

Therapy Development ◽

Induced Pluripotent

Induced pluripotent, stem cell (iPSC)-derived models of airway tissue have successfully modeled the primary defect in regulated chloride conductance caused by the major Cystic Fibrosis causing mutation, F508del. However, it remains unclear if iPSC-derived airway cultures can be used in high-throughput therapy development for F508del and rarer mutations. There is an urgent need for airway tissue models that reflect the variability of patient-specific responses and are scalable for therapy development. In the current work, we describe a robust, high-throughput fluorescence assay of mutant CFTR function in iPSCs differentiated to immature airway epithelium. This assay measures reproducible functional responses to modulators targeting either the major CF mutant F508del or the nonsense mutant: W1282X-CFTR. We show that the ranking of patient-specific responses to interventions in this stem-cell based model recapitulates the ranking observed in primary nasal epithelial cultures obtained from the same individuals. In summary, these proof-of-concept studies show that this novel platform has the potential to support therapy development and precision medicine for Cystic Fibrosis patients.

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Avatar acceptability: Views from the Australian Cystic Fibrosis community on the use of personalised organoid technology to guide treatment decisions

ERJ Open Research ◽

10.1183/23120541.00448-2020 ◽

2020 ◽

pp. 00448-2020

Author(s):

L.K. Fawcett ◽

C.E. Wakefield ◽

S. Sivam ◽

P.G. Middleton ◽

P. Wark ◽

...

Keyword(s):

Cystic Fibrosis ◽

Treatment Response ◽

Online Survey ◽

Treatment Decisions ◽

Turnaround Time ◽

Patient Specific ◽

Clinical Tool ◽

Cross Sectional ◽

Advantages And Disadvantages ◽

Recommended Treatment

BackgroundPatient-oriented research approaches that reflect the needs and priorities of those most affected by health research outcomes, improves translation of research findings into practice. Development of targeted therapies for Cystic Fibrosis (CF) is a viable treatment option now for some eligible individuals despite the heterogeneous patient-specific therapeutic response. This has necessitated development of a clinical tool that predicts treatment response for individual patients. Patient-derived mini-organs (organoids) have been at the forefront of this development. However, little is known about their acceptability in CF patients and members of the public.MethodsWe used a cross-sectional observational design to conduct an online survey in people with CF, their carers and community comparisons. Acceptability was examined in five domains; 1-willingness to use organoids, 2-perceived advantages and disadvantages of organoids, acceptable 3-out-of-pocket costs, 4-turnaround time, and 5-source of tissue.Results188 participants completed the questionnaire, including adults with CF and parents of children with CF (90(48%)), and adults without CF and parents of children without CF (98(52%)). Use of organoids to guide treatment decisions in CF was acceptable to 86(95%) CF participants and 98 (100%) community participants. The most important advantage was that organoids may improve treatment selection, improving the patient's quality of life and life expectancy. The most important disadvantage was that the organoid recommended treatment may be unavailable or too expensive.ConclusionsThese findings indicate acceptance of patient-derived organoids as a tool to predict treatment response by the majority of people surveyed. This may indicate successful future implementation into healthcare systems.

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A multimodal iPSC platform for cystic fibrosis drug testing

10.1101/2021.06.21.448578 ◽

2021 ◽

Author(s):

Andrew Berical ◽

Rhianna Lee ◽

Junjie Lu ◽

Mary Lou Beermann ◽

Jake LeSuer ◽

...

Keyword(s):

Cystic Fibrosis ◽

Drug Testing ◽

Rare Variants ◽

Anion Channel ◽

Airway Epithelial Cells ◽

In Vitro Assays ◽

Patient Specific ◽

Airway Epithelial ◽

Cftr Modulators

Cystic fibrosis (CF) is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane regulator (CFTR) anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of the CFTR channel using established animal and cell-based models led to the recent discovery of effective CFTR 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 assembled a panel of iPSCs derived from individuals with common or rare variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function in patient-specific iPSCs we adapted two established in vitro assays of CFTR function to iPSC-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 quantified CFTR baseline function and response to CFTR modulators and detected genotype-specific differences. Our results demonstrate the potential of the human iPSC platform as a research tool to study cystic fibrosis and in particular accelerate therapeutic development for CF caused by rare mutations.

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