Stability of gene expression by primary bronchial epithelial cells over increasing passage number

Lucille M. Rich,Kaitlyn A. Barrow,Stephen R. Reeves,Jason S. Debley,Maria P. White,Maryam Naushab

doi:10.1186/s12890-018-0652-2

Lucille M. Rich, Kaitlyn A. Barrow + Show 4 more

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https://doi.org/10.1186/s12890-018-0652-2

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Abstract

BackgroundAn increasing number of studies using primary human bronchial epithelial cells (BECs) have reported intrinsic differences in the expression of several genes between cells from asthmatic and non-asthmatic donors. The stability of gene expression by primary BECs with increasing cell passage number has not been well characterized.MethodsTo determine if expression by primary BECs from asthmatic and non-asthmatic children of selected genes associated with airway remodeling, innate immune response, immunomodulatory factors, and markers of differentiated airway epithelium, are stable over increasing cell passage number, we studied gene expression patterns in passages 1, 2, 3, 4, and 5 BECs from asthmatic (n = 6) and healthy (n = 6) subjects that were differentiated at an air-liquid interface. RNA was harvested from BECs and RT-PCR was performed for TGFβ1, TGFβ2, activin A, FSTL3, MUC5AC, TSLP, IL-33, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1.ResultsExpression of TGFβ1, TGFβ2, activin A, FSTL3, MUC5AC, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1 by primary BECs from asthmatic and healthy children was stable with no significant differences between passages 1, 2 and 3; however, gene expression at cell passages 4 and 5 was significantly greater and more variable compared to passage 1 BECs for many of these genes. IL-33 and FOXJ1 expression was also stable between passages 1 through 3, however, expression at passages 4 and 5 was significantly lower than by passage 1 BECs. TSLP, p63, and KRT5 expression was stable across BEC passages 1 through 5 for both asthmatic and healthy BECs.ConclusionsThese observations illustrate the importance of using BECs from passage ≤3 when studying gene expression by asthmatic and non-asthmatic primary BECs and characterizing the expression pattern across increasing cell passage number for each new gene studied, as beyond passage 3 genes expressed by primary BECs appear to less accurately model in vivo airway epithelial gene expression.

Highlights

An increasing number of studies using primary human bronchial epithelial cells (BECs) have reported intrinsic differences in the expression of several genes between cells from asthmatic and non-asthmatic donors
In the present study, we have demonstrated that primary differentiated BECs obtained from children with or without atopic asthma maintain stable expression of a panel of genes related to airway remodeling, innate immunity, immunomodulation, epithelial differentiation, and epithelial basal cells through passage 3 in ex vivo air-liquid interface (ALI) cell cultures
While ex vivo primary BECs differentiated at an ALI represent one of the best available models to study the role of the airway epithelium in disease processes such as asthma in children, care must be taken to ensure that cell phenotype and gene expression patterns are preserved such that ex vivo studies reflect the in vivo condition as closely as possible

Summary

Introduction

An increasing number of studies using primary human bronchial epithelial cells (BECs) have reported intrinsic differences in the expression of several genes between cells from asthmatic and non-asthmatic donors. The stability of gene expression by primary BECs with increasing cell passage number has not been well characterized. Our understanding of asthma pathogenesis has grown to include a central role for bronchial epithelial cells (BECs) in the establishment and maintenance of asthmatic airway disease [2]. Understanding of the importance of BECs in immune surveillance and coordination of the immune response to infections and environmental antigens has grown to include an important role for BEC-derived cytokines and direct cell-to-cell communication beyond their role in barrier function and innate immunity [3]. BECs have become the focus of many recent studies aimed to elucidate mechanisms underlying asthma pathogenesis in children [4]. Unlike studies in adults, obtaining cells via bronchoscopic airway biopsy

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