Conditional reprogramming of pediatric airway epithelial cells: A new human model to investigate early‐life respiratory disorders

Abstract

Background Airway epithelial cells (AEC) are quite difficult to access in newborns and infants. It is critically important to develop robust life-extended models to conduct translational studies in this age group. We propose the use of a recently described cell-culture technology (conditionally reprogrammed cells - CRC) to generate continuous primary cell cultures from nasal and bronchial AEC of young children. Methods We collected nasal and/or bronchial AEC from a total of 23 subjects of different ages including newborns/infants/toddlers (0–2 years; N=9), school age children (4–11 years; N=6), and a group of adolescent/adult donors (N=8). For CRC generation, we used conditioned medium from mitotically inactivated 3T3 fibroblasts and Rho-associated kinase (ROCK) inhibitor (Y-27632). Antiviral immune responses were studied using 25 key antiviral genes and protein production of type III epithelial interferon (IFN λ1) after double stranded (ds) RNA exposure. Results CRC derived from primary AEC of neonates/infants and young children exhibited: 1) augmented proliferative capacity and life-extension, 2) preserved airway epithelial phenotype after multiple passages, 3) robust immune responses characterized by expression of innate antiviral genes and parallel nasal/bronchial production of IFN λ1 after exposure to dsRNA, and 4) induction of airway epithelial inflammatory and remodeling responses to dsRNA (e.g. CXCL8 and MMP9). Conclusion Conditional reprogramming of AEC from young children is a feasible and powerful translational approach to investigate early-life airway epithelial immune responses in humans.

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics

“…The concept of co-culturing multiple cell types together to improve proliferation is relative commonplace 12 , 13 . Fibroblast-epithelial co-culture models include those from the nose 14 , 15 , cornea 16 , particular tumours 17 , 18 and skin keratinocytes 19 . However, this cell co-culture method has not yet been applied for hard to obtain tissues, including pAECs from children.…”

Innate IFN‐lambda responses to dsRNA in the human infant airway epithelium and clinical regulatory factors during viral respiratory infections in early life

“…A myriad of pro‐inflammatory mediators can induce canonical NF‐kB signalling activation in the AECs during viral respiratory infections 37 . Nonetheless, the role of these molecules in the production of IFN lambda in the human AEC is still unclear.…”

“…Nasal AEC brushing cultures and conditionally reprogrammed cells (CRC) were generated as described 37 (Online Repository). CRC methods were used to optimize initial proliferation and survival of neonatal/infant AEC cultures, 35 and to life‐extend primary AEC to perform and replicate mechanistic experiments.…”

“…Accordingly, the central goal of this study was to better understand the regulation of innate IFN‐lambda responses driven by viral PAMPs recognition in primary human infant AEC cultures and to examine the clinical factors linked to the in vivo production of IFN lambda in nasal aspirates from young children hospitalized due to viral respiratory infections. We specifically aimed to: (a) define the mechanistic contribution of NF‐kB and MAPKs signalling activation in the poly(IC)‐induced production of IFN lambda using an in vitro model of the human infant airway epithelium 37 ; (b) characterize the airway production of IFN lambda during naturally occurring viral respiratory infections in young children according to viral pathogen and individual characteristics; and (c) correlate airway IFN‐lambda production with acute respiratory disease parameters and recurrence of respiratory symptoms.…”

Characterisation of morphological differences in well-differentiated nasal epithelial cell cultures from preterm and term infants at birth and one-year

“…As a possible strategy to deal with this challenge, Wolf et al described the generation of conditionally reprogrammed cells (CRC) from harvested infant AECs with enhanced proliferative and survival capacity. (11) However, generating CRCs requires alteration of primary AECs and it remains unclear what impact this might have on AEC innate immune responses.…”