Expansion of Human Airway Basal Stem Cells and Their Differentiation as 3D Tracheospheres

Although basal cells function as human airway epithelial stem cells, analysis of these cells is limited by in vitro culture techniques that permit only minimal cell growth and differentiation. Here, we report a protocol that dramatically increases the long-term expansion of primary human airway basal cells while maintaining their genomic stability using 3T3-J2 fibroblast coculture and ROCK inhibition. We also describe techniques for the differentiation and imaging of these expanded airway stem cells as three-dimensional tracheospheres containing basal, ciliated, and mucosecretory cells. These procedures allow investigation of the airway epithelium under more physiologically relevant conditions than those found in undifferentiated monolayer cultures. Together these methods represent a novel platform for improved airway stem cell growth and differentiation that is compatible with high-throughput, high-content translational lung research as well as human airway tissue engineering and clinical cellular therapy

Modeling pulmonary fibrosis: Impacts of glutaredoxin on differentiation in tracheosphere development

Idiopathic pulmonary fibrosis (IPF) is a deadly disease whose pathology involves improperly differentiated airway epithelia present in alveolar regions. Previous work from our group has shown decreased activity of the enzyme glutaredoxin (GLRX) in the lungs of IPF patients, and demonstrated reversal of lung fibrosis in mice via administration of exogenous GLRX protein. We modeled airway epithelial tissue using 3D cell culture of wild type and GLRX -/- airway epithelial basal cells, both in monoculture and in coculture with fibroblasts. We present data from qPCR and immunohistochemistry which suggest that GLRX is essential for the proper differentiation of airway epithelia in the presence of fibroblasts. These findings suggest a role for GLRX in the attenuation of fibrosis via the support of healthy epithelial differentiation

Characterisation of cultured airway basal cells to understand their role in human lung disease

Many studies in murine models have demonstrated the stem/progenitor cell potential of basal epithelial cells in the tracheal epithelium. However, significant differences exist between the respiratory epithelium in rodents and in man. As such, novel methodologies to study respiratory epithelial cells in vitro are in demand. Here, methods to expand primary human airway epithelial cells from living patients were explored. The field’s ‘gold standard’ medium for the expansion of these cells was poorly suited to initiating cultures from small endobronchial biopsy samples as proliferation of these cells was time-limited and after a short period of time in culture the cells became senescent and were unable to regenerate a mucociliary epithelium in organotypic models. As such, an alternative epithelial culture strategy involving the co-culture of human airway epithelial cells with 3T3-J2 fibroblast feeder cells in medium containing a small molecule Rho-associated protein kinase (ROCK) inhibitor was assessed. This method greatly improved both the yield and the longevity of human basal cell cultures and allowed multipotent airway differentiation in organotypic assays after longer culture periods than conventional techniques. Finally, the epithelial-stromal cell crosstalk between epithelial cells and feeder cells in co-culture was investigated, revealing a novel signalling pathway involving phosphorylation of the transcription factor signal transducer and activator of transcription 6 (STAT6) by hepatocyte growth factor (HGF) signalling

Expansion of airway basal epithelial cells from primary human non-small cell lung cancer tumors

Pre-clinical non-small cell lung cancer (NSCLC) models are poorly representative of the considerable inter- and intra-tumor heterogeneity of the disease in patients. Primary cell-based in vitro models of NSCLC are therefore desirable for novel therapy development and personalized cancer medicine. Methods have been described to generate rapidly proliferating epithelial cell cultures from multiple human epithelia using 3T3-J2 feeder cell culture in the presence of Y-27632, a RHO-associated protein kinase (ROCK) inhibitor, in what are known as "conditional reprograming conditions" (CRC) or 3T3+Y. In some cancer studies, variations of this methodology have allowed primary tumor cell expansion across a number of cancer types but other studies have demonstrated the preferential expansion of normal epithelial cells from tumors in such conditions. Here, we report our experience regarding the derivation of primary NSCLC cell cultures from 12 lung adenocarcinoma patients enrolled in the Tracking Cancer Evolution through Therapy (TRACERx) clinical study and discuss these in the context of improving the success rate for in vitro cultivation of cells from NSCLC tumors. This article is protected by copyright. All rights reserved

Notch-Dependent Differentiation of Adult Airway Basal Stem Cells

The epithelium lining the airways of the adult human lung is composed of ciliated and secretory cells together with undifferentiated basal cells (BCs). The composition and organization of this epithelium is severely disrupted in many respiratory diseases. However, little is known about the mechanisms controlling airway homeostasis and repair after epithelial damage. Here, we exploit the mouse tracheobronchial epithelium, in which BCs function as resident stem cells, as a genetically tractable model of human small airways. Using a reporter allele we show that the low level of Notch signaling at steady state is greatly enhanced during repair and the generation of luminal progenitors. Loss-of-function experiments show that Notch signaling is required for the differentiation, but not self-renewal, of BCs. Moreover, sustained Notch activation in BCs promotes their luminal differentiation, primarily towards secretory lineages. We also provide evidence that this function of Notch signaling is conserved in BCs from human airways

Optimized isolation and expansion of human airway epithelial basal cells from endobronchial biopsy samples

Autologous airway epithelial cells have been used in clinical tissue-engineered airway transplantation procedures with a view to assisting mucosal regeneration and restoring mucociliary escalator function. However, limited time is available for epithelial cell expansion due to the urgent nature of these interventions and slow epithelial regeneration has been observed in patients. Human airway epithelial cells can be expanded from small biopsies or brushings taken during bronchoscopy procedures but the optimal mode of tissue acquisition from patients has not been investigated. Here, we compare endobronchial brushing and endobronchial biopsy samples in terms of their cell number and their ability to initiate basal epithelial stem cell cultures. We found that direct co-culture of samples with 3T3-J2 feeder cells in culture medium containing a Rho-associated protein kinase (ROCK) inhibitor, Y-27632, led to the selective expansion of greater numbers of basal epithelial stem cells during the critical early stages of culture than traditional techniques. Additionally, we established the benefit of initiating cell cultures from cell suspensions, either using brushing samples or through enzymatic digestion of biopsies, over explant culture. Primary epithelial cell cultures were initiated from endobronchial biopsy samples that had been cryopreserved prior to the initiation of cell cultures, suggesting that cryopreservation could eliminate the requirement for close proximity between the clinical facility in which biopsy samples are taken and the specialist laboratory in which epithelial cells are cultured. Overall, our results suggest ways to expedite epithelial cell preparation in future airway cell therapy or bioengineered airway transplantation procedures

Ciliated Epithelial Cell Differentiation at Air-Liquid Interface Using Commercially Available Culture Media

The human nasal epithelium contains basal stem/progenitor cells that produce differentiated multiciliated and mucosecretory progeny. Basal epithelial cells can be expanded in cell culture and instructed to differentiate at an air-liquid interface using transwell membranes and differentiation media. For basal cell expansion, we have used 3T3-J2 co-culture in epithelial culture medium containing EGF, insulin, and a RHO-associated protein kinase (ROCK) inhibitor, Y-27632 (3T3 + Y). Here we describe our protocols for ciliated differentiation of these cultures at air-liquid interface and compare four commercially available differentiation media, across nine donor cell cultures (six healthy, two patients with chronic obstructive pulmonary disease (COPD), and one with primary ciliary dyskinesia (PCD)). Bright-field and immunofluorescence imaging suggested broad similarity between differentiation protocols. Subtle differences were seen in transepithelial electrical resistance (TEER), ciliary beat frequency, mucus production, and the extent to which basal cells are retained in differentiated cultures. Overall, the specific differentiation medium used in our air-liquid interface culture protocol was not a major determinant of ciliation, and our data suggest that the differentiation potential of basal cells at the outset is a more critical factor in air-liquid interface culture outcome. Detailed information on the constituents of the differentiation media was only available from one of the four manufacturers, a factor that may have profound implications in the interpretation of some research studies

Innovative three-dimensional models for understanding mechanisms underlying lung diseases: powerful tools for translational research

Chronic lung diseases result from alteration and/or destruction of lung tissue, inevitably causing decreased breathing capacity and quality of life for patients. While animal models have paved the way for our understanding of pathobiology and the development of therapeutic strategies for disease management, their translational capacity is limited. There is, therefore, a well-recognised need for innovative in vitro models to reflect chronic lung diseases, which will facilitate mechanism investigation and the advancement of new treatment strategies. In the last decades, lungs have been modelled in healthy and diseased conditions using precision-cut lung slices, organoids, extracellular matrix-derived hydrogels and lung-on-chip systems. These three-dimensional models together provide a wide spectrum of applicability and mimicry of the lung microenvironment. While each system has its own limitations, their advantages over traditional two-dimensional culture systems, or even over animal models, increases the value of in vitro models. Generating new and advanced models with increased translational capacity will not only benefit our understanding of the pathobiology of lung diseases but should also shorten the timelines required for discovery and generation of new therapeutics. This article summarises and provides an outline of the European Respiratory Society research seminar "Innovative 3D models for understanding mechanisms underlying lung diseases: powerful tools for translational research", held in Lisbon, Portugal, in April 2022. Current in vitro models developed for recapitulating healthy and diseased lungs are outlined and discussed with respect to the challenges associated with them, efforts to develop best practices for model generation, characterisation and utilisation of models and state-of-the-art translational potential. </p

Cell Stem Cell

Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/\u3b2-catenin within ABSCs was essential for proliferation post-injury in\ua0vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFR\u3b1| lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of \u3b2-catenin in young mice induced their formation in\ua0vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging.T32 GM008042/GM/NIGMS NIH HHSUnited States/R01 CA208303/CA/NCI NIH HHSUnited States/UL1 TR000124/TR/NCATS NIH HHSUnited States/R25 GM055052/GM/NIGMS NIH HHSUnited States/UL1 TR001881/TR/NCATS NIH HHSUnited States/F31 CA239655/CA/NCI NIH HHSUnited States/T34 GM008563/GM/NIGMS NIH HHSUnited States/T32 CA009056/CA/NCI NIH HHSUnited States/U66 IP000597/IP/NCIRD CDC HHSUnited States/2021-09-03T00:00:00Z32721381PMC74840541025