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Transcriptomic dissection of complex stratified epithelia and functional characterisation of epithelial barrier formation

Principal Supervisor: Dr Malgorzata Wiench

Secondary Supervisor(s): Professor Jo Parish

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Systems Biology)

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Deadline: 4 January, 2024


Project Outline

Skin and mucous membranes form the body's largest organ, making up around 16% of body mass. The uppermost part of skin and mucous membranes is formed by stratified squamous epithelium, within which the epithelial cells (keratinocytes) undergo a tightly controlled differentiation process leading to the establishment of a protective barrier against the environment. This is essential to maintain functional, healthy tissues. The multilayered structure reflects tissue maturation, originating from basal layer with active cell proliferation to support constant tissue renewal, through spinous and granular layers formed of differentiating keratinocytes that produce essential substrates for the impermeable barrier, up to the stratum corneum, which contains terminally differentiated, protein- and lipid-rich cells. Any deviation in the process of epidermal differentiation is associated with disorders, including malignancies, and can result from bacterial or viral infections.

The overall aim of the project is to unravel how physiological and pathogen-disrupted epithelial maturation processes are regulated on transcriptional level, including protein-coding and long non-coding RNAs. To study epithelial differentiation and barrier function, it is crucial to use specific models that can mimic the various layers of the epithelium. These typically involve keratinocyte culture at an air-liquid interface (ALI) on top of a fibroblast-containing matrix, usually collagen, to replicate connective tissue. In this project, we will utilize the Buoyant Epithelial Culture Devices (BECDs) recently developed and validated in our lab [1] to establish tissue mimics using primary oral keratinocytes. The tissue mimics will be challenged by pathogens affecting either basal part of epithelium (viruses) or acting from the surface (bacteria), and the changes in transcriptome across the different layers will be recorded using single cell RNA sequencing (scRNA-seq). This will lead to in silico models of epithelial differentiation and selection of major genetic players in the process of barrier formation, for further functional validation screen using CRISPR-Cas9 and various methods of epithelia barrier assessment.

Several innovative approaches will be used to achieve the aim: (1) advanced 3D-printed models of epithelial tissue mimics, including high-throughput systems, currently under development in our lab; (2) well-established models of viral (HPV, [2]) and bacterial (heat killed Porphyromonas gingivalis) infections; (3) epidermal differentiation-related RNAs (ED-RNAs) will be identified using single-cell RNA sequencing. More specifically, we aim to produce and analyze scRNA-seq data to provide epithelial strata separation in silico, and compare cell subsets of the stratified epithelium. In addition, we will apply strand-specific total RNA-seq with Ribo-Zero kits to preserve both polyadenylated and non-polyadenylated RNA species; (4) selected ED-RNAs will be screened through CRISPR-Cas9 gene knockdown to perform loss-of-function studies using tissue mimics within a semi-high throughput system. The knock down effects on various aspects of epithelial differentiation and barrier formation will be investigated through Light Sheet Microscopy, immunohistochemistry, transepithelial electrical resistance and tracer flux assay.

Interdisciplinary supervision and the wide range of state-of-the-art methods will form basis to a robust post-doctoral training programme. The acquired knowledge will provide unique transcriptomic datasets and aid our understanding of the formation and maintenance of epithelial tissues, ultimately leading to improvements in diagnosis and treatment of wide range of epithelial disorders

References

[1] Hewitt B, at al. A 3D Printed Device for In Vitro Generation of Stratified Epithelia at the Air-Liquid Interface. Tissue Eng Part C Methods. 2022 Nov;28(11):599-609. doi: 10.1089/ten.TEC.2022.0130. Describes validation of the BECD model that will be used in the proposed project (Wiench, main supervisor)

[2] Roberts S, et al., Modelling human papillomavirus biology in oropharyngeal keratinocytes. Philos Trans R Soc Lond B Biol Sci. 2019 May 27;374(1773):20180289. doi: 10.1098/ rstb.2018.0289. Describes HPV infection models that will be used in the project (Parish, supervisor)

Techniques

  • 2D and 3D tissue culture
  • 3D printing
  • Single cell RNA-sequencing
  • CRISPR-Cas9 genome editing
  • Immunohistochemistry
  • Confocal microscopy
  • Light sheet microscopy
  • Tracer flux assay
  • Transepithelial electrical resistance assay