Investigation of neonatal mouse kidney and endometrial cell populations in explant culture combined with an in silico approach to the endometrium

Mathew, David
Investigation of neonatal mouse kidney and endometrial cell populations in explant culture combined with an in silico approach to the endometrium. Master of Philosophy thesis, University of Liverpool.

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Background The endometrium is a highly dynamic tissue undergoing up to 400 cycles of proliferation, differentiation and breakdown in an average woman’s reproductive life. The mechanisms of this proliferative potential are the subject of active study and one candidate is Adult Stem Cells (ASCs). Endometriosis is a benign proliferative endometrial disease resulting from ectopic growth of endometrial tissue comprising glands and stroma with unknown pathogenesis. ASCs have also been implicated in the pathogenesis of endometriosis and attempts have been made in recent years to characterise and isolate this cell population within the endometrium. A great deal of investigation into cellular and molecular processes governing this disease has been undertaken with conflicting results. A useful tool to guide future investigation is the emerging discipline of bioinformatics. Objectives: One of the difficulties with investigating endometriosis and endometrial ASCs is the lack of suitable in-vitro models to study the benign yet abnormal growth of endometrial epithelial cells in culture. Ex-vivo culture models using a multicellular culture system are highly desirable for future work and the present study aimed to investigate two of the features associated with ASCs: proliferation and differentiation potential. A crudely fractionated epithelial population of endometrial cells in a neonatal kidney explant model was used to assess this. The kidney is an ideal tissue for use in co-culture with endometrial cells as the endometrium and the kidney both embryologically derive from the intermediate mesoderm. Furthermore, it has been shown previously that human endometrial cells can generate endometrial-like tissue, including glandular structures, when transplanted into the kidneys of immune-compromised adult mice. To investigate whether an in vitro model using neonatal kidney cells could support the growth and differentiation of endometrial cells, endometrial epithelial cells were grown in a chimeric explant model using kidney tissue isolated from CD1 neonatal mice. Chimeras were generated using a recently developed reaggregation protocol. Controls comprised explants of reaggregated kidneys that did not contain any endometrial cells. Explants were cultured at an air-medium interface for 2 hours (0 Days), 3 Days and 7 Days respectively before being fixed, processed and analysed with immunofluorescence and RT-qPCR to detect the presence of endometrial-derived glandular structures, and endometrial differentiation markers. Markers of interest for analysis of the chimeras included: Laminin, Progesterone Receptor and Mucin1. A human-specific cytoplasmic antibody was used to identify human cells in the chimeras. This work was further extended to assess nephrogenesis and endothelial cell survival in the neonatal kidney explants. In the study of the developmental processes governing the mammalian kidney progress has been achieved through the use of embryo explant models. These models are useful for studying early developmental processes such as reciprocal induction but are less suitable for studying later maturation events (e.g. foot process formation in podocytes), due to the fact that the endothelial cell population dies. Podocytes depend upon interactions with the glomerular basement membrane (GBM) to achieve maturity and the GBM is known to be formed, at least in part, from factors secreted by endothelial cells. If an explant model could be established with an intact endothelial population there is potential to study these maturation processes. Markers of interest for neonatal kidney explants included: WT1, Pecam, Synaptopodin, Laminin and Calbindin. Bioinformatics tools were employed to examine existing microarray data sets of normal endometrium during various stages of the menstrual cycle with the aim of identifying molecular pathways of interest and generating suitable hypotheses. The same process was carried out for endometrium from patients with endometriosis with the future goal of comparing these datasets. Results: Analysis of chimeric explants indicated that a large proportion of the endometrial cells died in culture with only a small population present by Day 7 that showed no evidence of forming glandular structures. A degree of nephrogenesis appeared to have occurred in the neonatal kidney explant model with evidence of endothelial cells surviving and forming cord like structures in culture. Initial investigation of the transcriptomic datasets associated with normal endometrium and patients with endometriosis was submitted to Ingenuity Pathways Analysis (IPA) and Cytoscape. A large amount of output was obtained and specific pathways suitable for further investigation were identified including: Super Oxide Radicals Degradation, Tryptophan Degradation to 2-amino-3 Semi Aldehyde and Wnt/β Catenin Pathway. Conclusions: The three main conclusions were as follows. 1 Human endometrial cells were not able to survive in the chimeric model of ex-vivo culture, indicating that this is unlikely to be a suitable model system for studying the formation of endometrial cell-derived glandular structures. 2 Nephrogenesis may be occurring in the reaggreagted neonatal explants, and the endothelial population appeared to have survived and formed capillary like structures in vitro. This is an important observation because in explants of embryonic kidneys, the endothelial cells do not survive. 3. Investigation of the molecular processes at play in the normal endometrium and endometriosis has generated some interesting hypotheses that will be further investigated using ‘wet’ laboratory techniques.

Item Type: Thesis (Master of Philosophy)
Additional Information: Date: 2013-08 (completed)
Subjects: ?? RG ??
Depositing User: Symplectic Admin
Date Deposited: 31 Jul 2014 09:53
Last Modified: 16 Dec 2022 04:41
DOI: 10.17638/00017073