Development of a sustainable primary human airway cell culture model for respiratory research

Lewis, A. (2018) Development of a sustainable primary human airway cell culture model for respiratory research. MPhil thesis, University of Nottingham.

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Abstract

The airway epithelium has an important protective role and is known to be abnormal in asthmatic individuals. However, epithelial cell research is restricted by the limited lifespan of primary human epithelial cells in vitro and therefore the cells isolated via bronchoscopy are exhausted after only a few experiments. This project aimed to extend cell lifespan by over-expressing Bmi1 in a controlled and temporal manner using an inducible Cumate Switch system.

Bmi1 has previously been shown to extend cell lifespan whilst maintaining plasticity. In this study, lentiviruses have been generated to deliver and over-express Bmi1 to primary human bronchial epithelial cells (HBECs) which contain the Cumate Switch System to control gene expression. Infected cells show evidence of fluorescent reporter proteins indicating successful gene delivery and expression. The effects of Bmi1 over-expression have been assessed through Electric Cell Substrate Impedance Sensing (ECIS) and also through differential gene expression (RNA-seq) using the data from a study published in Gene Expression Omnibus. Preliminary data show that barrier function differs in modified cells, but transcriptomic analysis does not identify differential expression of barrier function genes in correlation with elevated Bmi1. A large number of genes are identified as being differentially expressed (1024 by CuffDiff and 380 by Rank Prod), although this number is reduced when using multiple analyses, and amongst those genes consistently up-regulated with Bmi1 expression are genes involved in cell growth and migration.

The rationale behind this project is that Bmi1 over-expression can be returned to endogenous levels by removing gene induction. Initial data suggested that Bmi1 expression can be turned on and at least turned down in this system, but further work is needed.

Bmi1 has the potential to extend cell lifespan, providing the primary cells with a proliferative advantage to allow further expansion in culture than currently possible. This study has provided initial support that a Bmi1 switch system may be viable, but also demonstrated that Bmi1 induction may alter barrier properties and significantly influence the expression of many genes. The development of a Bmi1 switch system has the potential to significantly expand our ability to utilise primary cells from patients and provide an extended platform to study airway epithelial cell biology.

Item Type: Thesis (University of Nottingham only) (MPhil)
Supervisors: Sayers, I.
Keywords: Airway epithelium; Epithelial cells; Cell culture techniques; Bmi1; Cumate Switch system
Subjects: QS-QZ Preclinical sciences (NLM Classification) > QS Human anatomy
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 48854
Depositing User: Lewis, Amanda
Date Deposited: 10 May 2018 10:35
Last Modified: 10 May 2018 19:19
URI: http://eprints.nottingham.ac.uk/id/eprint/48854

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