Highly efficient genome editing in primary human bronchial epithelial cells differentiated at air–liquid interface

Rapiteanu, Radu; Karagyozova, Tina; Zimmermann, Natalie; Singh, Kanwaljeet; Wayne, Gareth; Martufi, Matteo; Belyaev, Nikolai N.; Hessel, Edith M.; Michalovich, David; Macarrόn, Ricardo; Rowan, Wendy C.; Cairns, William; Roger, Jan; Betts, Joanna; Beinke, Sören; Maratou, Klio

doi:10.1183/13993003.00950-2019

Cited by 15 publications

(19 citation statements)

References 50 publications

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“…CRISPR knockout mutations were generated as described ( 71 ) with slight modifications. Specifically, individual single guide RNAs (sgRNAs) and Cas9 Electroporation Enhancer (IDT, 1075916) were each resuspended at 100 μM in nuclease-free duplex buffer (IDT, 11-01-03-01).…”

Section: Methodsmentioning

confidence: 99%

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population

DePianto¹,

Heiden²,

Morshead³

et al. 2021

JCI Insight

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Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis associated interstitial lung disease ILD (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells, and interrogated these signatures in a single-cell RNA-seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence, and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.

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Section: Methodsmentioning

confidence: 99%

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population

DePianto¹,

Heiden²,

Morshead³

et al. 2021

JCI Insight

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“…Previous studies have reported the use of lipid-mediated reverse-transfection at the time of plating HBEs and before differentiation of ALIs (9), although there are reports showing resistance of ALI cultures to lipid-mediated small interfering RNA (siRNA) knockdown (10), whereas others have shown $30% siRNA knockdown in ALIs using nanoparticle-mediated delivery (11). In addition, CRISPR/Cas9 has been tested for gene silencing in HBEs before ALI cultures (12,13), but cost, target design, and experimental design limitations can pose problems for some investigators.…”

Section: Introductionmentioning

confidence: 99%

Passive siRNA transfection method for gene knockdown in air-liquid interface airway epithelial cell cultures

Bartman

Stelzig

Linden

et al. 2021

American Journal of Physiology-Lung Cellular and Molecular Phys

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Differentiation of human bronchial epithelial cells (HBEs) in air-liquid interface (ALI) cultures recapitulates organotypic modeling of the in vivo environment. Although ALI cultures are invaluable for studying the respiratory epithelial barrier, loss-of-function studies are limited by potentially cytotoxic reagents in classical transfection methods, the length of the differentiation protocol, and the number of primary epithelial cell passages. Here, we present the efficacy and utility of a simple method for siRNA transfection of HBEs in ALI cultures that does not require potentially cytotoxic transfection reagents and does not detrimentally alter the physiology of HBEs during the differentiation process. This transfection protocol introduces a reproducible and efficient method for loss-of-function studies in HBE ALI cultures that can be leveraged for modeling the respiratory system and airway diseases.

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“…Alternatively, gene editing can be performed once airway cells have differentiated in ALI cultures (105).…”

Section: Modelling Disease Initiation and Pathogenesismentioning

confidence: 99%

Stem Cell–derived Respiratory Epithelial Cell Cultures as Human Disease Models

Orr¹,

Hynds

2021

Am J Respir Cell Mol Biol

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Advances in stem cell biology and the understanding of factors that determine lung stem cell self-renewal have enabled long-term in vitro culture of human lung cells derived from airway basal and alveolar type II cells. Improved capability to expand and study primary cells long-term, including in clonal cultures that are recently derived from a single cell, will allow experiments that address fundamental questions about lung homeostasis and repair, as well as translational questions in asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and lung cancer research. Here, we provide a brief history of post-natal lung epithelial cell culture and describe recent methodological advances, including some culture systems that now permit clonal cell culture. We further discuss the applications of primary cultures in defining 'normal' epithelium, modelling lung disease and in future cell therapies.

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Highly efficient genome editing in primary human bronchial epithelial cells differentiated at air–liquid interface

Cited by 15 publications

References 50 publications

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population

Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population

Passive siRNA transfection method for gene knockdown in air-liquid interface airway epithelial cell cultures

Stem Cell–derived Respiratory Epithelial Cell Cultures as Human Disease Models

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