Conditionally Reprogrammed Human Normal Airway Epithelial Cells at ALI: A Physiological Model for Emerging Viruses

Abstract

Cancer cell lines have been used widely in cancer biology, and as biological or functional cell systems in many biomedical research fields. These cells are usually defective for many normal activities or functions due to significant genetic and epigenetic changes. Normal primary cell yields and viability from any original tissue specimens are usually relatively low or highly variable. These normal cells cease after a few passages or population doublings due to very limited proliferative capacity. Animal models (ferret, mouse, etc.) are often used to study virus-host interaction. However, viruses usually need to be adapted to the animals by several passages due to tropism restrictions including viral receptors and intracellular restrictions. Here we summarize applications of conditionally reprogrammed cells (CRCs), long-term cultures of normal airway epithelial cells from human nose to lung generated by conditional cell reprogramming (CR) technology, as an ex vivo model in studies of emerging viruses. CR allows to robustly propagate cells from non-invasive or minimally invasive specimens, for example, nasal or endobronchial brushing. This process is rapid (2 days) and conditional. The CRCs maintain their differentiation potential and lineage functions, and have been used for studies of adenovirus, rhinovirus, respiratory syncytial virus, influenza viruses, parvovirus, and SARS-CoV. The CRCs can be easily used for airliquid interface (ALI) polarized 3D cultures, and these coupled CRC/ALI cultures mimic physiological conditions and are suitable for studies of viral entry including receptor binding and internalization, innate immune responses, viral replications, and drug discovery as an ex vivo model for emerging viruses. Keywords Normal cells Á Cell senescence Á Conditional reprogramming Á Physiological conditions Á Functional models Á Air-liquid interface (ALI) Á Emerging viruses Á SARS-CoVs Conventional Cell Line Models for Virus Studies Emerging and re-emerging viral infections are becoming severe global public health problems in the current century. In February 2003, an outbreak of severe acute respiratory syndrome (SARS), caused by SARS coronavirus (SARS-CoV), was reported in Guangdong, China (Ksiazek et al. 2003). The SARS-CoV infected 8096 cases and 774 deaths worldwide. In March 2009 a novel influenza virus (H1N1pdm) emerged in the United States and Mexico. H1N1pdm obtained the capacity to transmit in humans and quickly spread to more than 214 countries (Hendrickson and Matthay 2013). Thereafter the H1N1 infection became a seasonal virus circulating over the world (Dawood et al. 2009). Middle East respiratory syndrome (MERS) coronavirus was isolated from patients who developed acute & Xuefeng Liu

Serially passaged, conditionally reprogrammed nasal epithelial cells as a model to study epithelial functions and SARS-CoV-2 infection

“…It has been reported that cr increases the proliferative capacity of pAECs compared with conventional culture techniques ( 36 , 37 , 41 ). To confirm this effect in our experimental setup, population doublings were determined in pNECs cultured under conventional or cr conditions, respectively ( Fig.…”

“…Primary NECs (pNECs) can be obtained noninvasively from both healthy and diseased subjects for research purposes ( 32 – 35 ) and display a useful model to study the pathogenesis of infectious and inflammatory airway diseases. Recently, NEC cultures have been discussed as a suitable model to study aspects of SARS-CoV-2 infection ( 36 ). A major limitation for such studies is the relatively small cell number obtained from single donors via nasal brushings.…”

In Vitro and In Vivo Models for Studying SARS-CoV-2, the Etiological Agent Responsible for COVID-19 Pandemic

“…Upon isolation, these cells are cultured and differentiated on porous supports at an air-liquid interface (ALI), in which the apical side is in contact with air. HAE cells represent a complex tissue with differentiated ciliated, goblet and basal cells and retain the highest similarity to human airway epithelium physiological conditions found in vivo [34,35]. HAE cells were used to isolate and discover SARS-CoV-2 for the first time upon its emergence in Wuhan, China.…”

“…Cellular models based on HAE offer similarity to physiological conditions and maintain many of the important markers and functions from the donor tissue [35]. Despite the great advantages of using primary cells, mainly regarding more realistic virus-host interactions, models based on immortalized cell monolayers are easy to use, less time-consuming and bypass ethical concerns associated with the use of animal and human tissue [34].…”

Nasal Epithelial Cell-Based Models for Individualized Study in Cystic Fibrosis

“…This may reflect an inherent restriction of goblet cell hyperplasia to the lower airways in asthma but is an important consideration in terms of model selection. Nasal cell cultures have been used in the study of viral entry, receptors and internalization, viral replication and innate immune responses [ 89 ]. Studies using paired nasal and bronchial cell cultures have shown similar antiviral and viral induced pro-inflammatory responses between to the two tissue sources [ 90 ].…”