ACE2 Expression in Organotypic Human Airway Epithelial Cultures and Airway Biopsies

Abstract

Coronavirus disease 2019 (COVID-19) caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an acute respiratory disease with systemic complications. Therapeutic strategies for COVID-19, including repurposing (partially) developed drugs are urgently needed, regardless of the increasingly successful vaccination outcomes. We characterized two-dimensional (2D) and three-dimensional models (3D) to establish a physiologically relevant airway epithelial model with potential for investigating SARS-CoV-2 therapeutics. Human airway basal epithelial cells maintained in submerged 2D culture were used at low passage to retain the capacity to differentiate into ciliated, club, and goblet cells in both air-liquid interface culture (ALI) and airway organoid cultures, which were then analyzed for cell phenotype makers. Airway biopsies from non-asthmatic and asthmatic donors enabled comparative evaluation of the level and distribution of immunoreactive angiotensin-converting enzyme 2 (ACE2). ACE2 and transmembrane serine proteinase 2 (TMPRSS2) mRNA were expressed in ALI and airway organoids at levels similar to those of native (i.e., non-cultured) human bronchial epithelial cells, whereas furin expression was more faithfully represented in ALI. ACE2 was mainly localized to ciliated and basal epithelial cells in human airway biopsies, ALI, and airway organoids. Cystic fibrosis appeared to have no influence on ACE2 gene expression. Neither asthma nor smoking status had consistent marked influence on the expression or distribution of ACE2 in airway biopsies. SARS-CoV-2 infection of ALI cultures did not increase the levels of selected cytokines. Organotypic, and particularly ALI airway cultures are useful and practical tools for investigation of SARS-CoV-2 infection and evaluating the clinical potential of therapeutics for COVID-19.

Human lung organoids as a model for respiratory virus replication and countermeasure performance in human hosts

“…Alternatively, ACE2, the receptor for SARS-CoV, SARS-CoV-2, and endemic hCoV NL63, is more prominently localized to the apical surface of ciliated cells, as well as with greater expression in the proximal vs distal airway. 52,[56][57][58][59] Importantly for organoid formation and differentiation, ACE2 is expressed in airway epithelium cultures differentiation from basal stem and progenitor cells, such that receptor expression is prevalent on fully differentiated organoids. 58 However, ACE2 is also expressed in AT2 cells, indicating that AT2 derived organoids serve as a strong ex vivo model to study these viruses and potential interventions.…”

“…52,[56][57][58][59] Importantly for organoid formation and differentiation, ACE2 is expressed in airway epithelium cultures differentiation from basal stem and progenitor cells, such that receptor expression is prevalent on fully differentiated organoids. 58 However, ACE2 is also expressed in AT2 cells, indicating that AT2 derived organoids serve as a strong ex vivo model to study these viruses and potential interventions. Lastly, expression of DPP4, the receptor for epidemic hCoV MERS-CoV, follows a more specific pattern and is rarely found on the surface epithelium of the proximal airway with a slight increase in expression in the distal airway.…”

Emerging toolset of three-dimensional pulmonary cell culture models for simulating lung pathophysiology towards mechanistic elucidation and therapeutic treatment of SARS-COV-2 infection

“…Since the proximal airway regions are the first target of SARS-CoV-2 infection, airway organoids are suitable for exploring the interaction of SARS-CoV-2 with proximal airway cells. To accurately mimic proximal airway physiological conditions during SARS-CoV-2 infection, ( Chen et al, 2022 ) used human airway basal cells to generate airway organoids, in which basal cells differentiate into ciliated cells, goblet cells, and club cells. Furthermore, they compared ALI cultures and airway organoid cultures and acknowledged that the latter expressed high levels of ACE2 and TMPRSS2, which are highly susceptible to SARS-CoV-2 infection and promote inflammatory cytokine responses ( Marescotti et al, 2019 ; Wang J. et al, 2020 ; Chen et al, 2022 ).…”

Biobanked tracheal basal cells retain the capacity to differentiate

“…In addition, the epithelium has a structure and function that is representative of the epithelium in vivo 16 . The utilization of ALI culturing of tracheal cells has been reported in the literature with a variety of animal and human models 17–27 . While utilization of pediatric cells in ALI culturing has been minimally studied when researching lung disorders such as cystic fibrosis and asthma, 28 pediatric patients with laryngotracheal pathology has not been studied in this way.…”

“… 16 The utilization of ALI culturing of tracheal cells has been reported in the literature with a variety of animal and human models. 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 While utilization of pediatric cells in ALI culturing has been minimally studied when researching lung disorders such as cystic fibrosis and asthma, 28 pediatric patients with laryngotracheal pathology has not been studied in this way. This culture process enables in vitro study of airway epithelial features, cell differentiation, and an evaluation of pathological changes in an injured epithelial layer.…”