The Role of Cervical Reserve Cells in Epithelial Regeneration and Homeostasis in the Murine and Human Cervix
How cervical epithelium maintains homeostasis and repair, and how the high-risk human papilloma viruses (HR-HPV) drive neoplasia at the cervical transformation zone (TZ) has been poorly researched. The cervical TZ is thought to be a stem cell niche as it harbours a unique cell population called reserve cells. Reserve cells (marked by KRT17) are believed to have a dual fate ability depending on local microenvironment, in a process of metaplasia (1). However, this belief was based on observational studies. Metaplasia is thought to be the way that the cervical epithelium recovers after an injury (2). Developing an in vitro model that is truly representative of normal cervical epithelium is crucial to address basic questions about homeostasis mechanism and the role of reserve cells in HR-HPV infections. In recent years, advances in modelling the human tissue in 3D manner using organoids have been made (3). In this thesis, a 3D murine cervical organoid model was established. Two distinct microenvironments for the growth of endocervix and ectocervix is defined. Generated cultures retain their proliferative capacity over several months and are representative of the epithelial population in vivo. Furthermore, steps were taken towards optimising human cervix organoids. The established in vitro system was exploited along with in vivo samples to further to uncover specific signalling pathways that regulate the different cervical lineages (endo and ectocervix) using Affymetrix microarray and immunofluorescence techniques. Wnt signalling was found to regulate the endocervical lineage, whilst Notch pathway was active in the ectocervix. Moreover, a novel P75 NGF receptor was found to be expressed in the basal squamous epithelium and in the reserve cells. Additionally, the established organoids were utilised together with lineage tracing techniques to investigate the dual-fate potential of KRT17 cells and their contribution in cervical TZ formation. Two transgenic mice strains were evaluated Krt17tm1(cre,Cerulean)Murr;ROSA26(CAG-tdTomato) and Krt17CreERT2; ROSA26 (CAG-ZsGreen1). The inducible regulation via tamoxifen in Krt17CreERT2 was found to have a confined KRT17 expression compared to the Cre/loxp only (Krt17tm1(cre,Cerulean)Murr) strain. (1) Martens JE, Arends J. Cytokeratin 17 and p63 are Markers of the HPV Target Cell, the Cervical Stem Cell. Anticancer Res. 2004;6. (2) Giroux V, Rustgi AK. Metaplasia: tissue injury adaptation and a precursor to the dysplasia–cancer sequence. Nat Rev Cancer. 2017 Oct;17(10):594–604. (3) Lancaster MA, Huch M. Disease modelling in human organoids. Dis Model Mech. 2019 Jul 1;12(7).
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