Award details

Challenging dogma: an alternative non-hierarchical, epigenetically regulated model of the urothelium.

Reference	BB/R009295/1
Principal Investigator / Supervisor	Dr Andrew Feber
Co-Investigators / Co-Supervisors
Institution	University College London
Department	Targeted Intervention
Funding type	Research
Value (£)	350,987
Status	Completed
Type	Research Grant
Start date	01/05/2018
End date	31/07/2020
Duration	27 months

Abstract

Differentiation of urothelial cells can be investigated in vitro as a time-resolved process where nuclear receptor activation initiates a downstream network of transcription factors (TFs) that engage the urothelial transcriptome. Recent work indicates a non-hierarchical relationship where, alongside chromatin reorganisation, some TFs act independently of others in the network. These relationships were construed using conflated whole populations, whereas differentiated urothelium is organised into basal, intermediate and superficial cell zones, arguing a need for spatial resolution of the regulatory networks. Study of separated basal versus suprabasal cells suggest that urothelial cell phenotype is plastic and determined by position or "niche". Rather than a staged hierarchical differentiation programme, we postulate individual urothelial cell phenotypes are modulated epigenetically in response to signalling from contextual cues within the tissue. We will exploit technical advances to develop maps incorporating transcriptome (RNA-Seq), epigenome (DNA methylation) and chromatin accessibility and occupancy (ATAC-Seq) datasets for each of the phenotypically-distinct urothelial cell subtypes, including the distinct pedicled c-Kit+ cell. Integration of these datasets will enable us to identify the molecular events underpinning urothelial regenerative capacity and differentiation potential. Ontological analysis of the transcriptome data will be used to infer subtype-specific marker sets, including regulatory genes, methylation-modifying enzymes, receptors and signal transduction network expression. We will validate and exploit these new data to explore the epigenetic regulation, plasticity and function of the different phenotypes that contribute histioarchitecturally to urinary barrier function and repair. These data will bring new understanding of urothelial tissue homeostasis that will challenge longstanding models of tissue biology.

Summary

Epithelial tissues occur at the interface of internal and external surfaces of the body, where they have evolved highly specialised forms and functions. For example, the skin has evolved for physical protection and to protect against dehydration, whereas the gut has specialised to digest food and absorb nutrients. Both these epithelial tissues show constant turnover, with specialised cells being replaced by others generated by proliferation and migration from a basal progenitor or stem cell pool. The hierarchical model of cells programmed to pursue a one-way journey from a stem cell to the specialised, differentiated cell is used to explain how tissues maintain a balance (homeostasis) between cell loss and gain, and even why cancer stem cells should be targeted in cancer therapy. This project challenges this generic model of epithelial regulation and differentiation, based on observations from a different epithelial tissue, the urothelium. Urothelium is the specialised epithelium which lines the bladder and ureters and functions as a tight urinary barrier. Unlike gut or skin, there is no constant cycle of renewal: cells of the urothelium are long-lived and mitotically-quiescent, but retain the capacity to contribute to efficient urinary barrier repair and regeneration by rapid re-entry into the mitotic cycle. No specific stem cell has ever been identified in human urothelium and dividing cells can be observed in any of the three layers. Phenotypically and positionally, urothelial cells adopt one of four distinct cell types (each of which we can isolate). Current molecular knowledge is based on homogenised urothelial preparations and therefore lacks sufficient subtype-specific detail. Our hypothesis is that rather than being part of a linear differentiation programme, urothelial cells display the appropriate phenotype in response to exogenous cues or their "niche" within the tissue, including adaptation to change (eg damage). Our hypothesis predicts an intimate relationship between epigenetic and signal transduction machineries to effect changes in cell phenotype. We will test our hypothesis using urothelium from the ureter and the bladder for comparison, as these are of different embryological derivations. From each source, we will isolate, to high purity, each of the four distinct urothelial cell phenotypes and perform an in depth characterisation of the transcriptome and epigenome of each subtype to determine differences and similarities. We will also analyse the separated cell types after adaptation to a non-permissive cell culture system where we predict that all cells will adopt a baseline or default reference squamous phenotype irrespective of derivation. We will use the cell culture system to investigate the signals of each "niche" and test how altering different regulatory pathways can modify cell phenotype, where we predict that the epigenetic machinery will play a hitherto unrecognised key role that can be manipulated and exploited. A deliverable from this project will be a data-rich, spatially-resolved urothelial map of the regulatory networks and machinery that defines the different urothelial subtypes. The outcome of this study will be important new understanding of urothelial tissue homeostasis that will challenge longstanding models of tissue biology and bring new perspectives to chronic diseases of ageing that affect the bladder, including future therapeutic opportunities in tissue engineering and regenerative medicine.

Impact Summary

Who could adopt or benefit from the research? The immediate beneficiaries of our research will be the academic community but, in the long term, it is the public who will benefit from our research, which we recognise will need industry engagement to help with translation. Urinary incontinence is a major problem associated with ageing and there are numerous, poorly understood chronic age-associated conditions affecting the urinary bladder where a better understanding of the basic biology of the tissue and how it is regulated will revolutionise treatment. These conditions not only affect quality of life, but have a significant negative impact on society and the economy. How will they benefit? The results of this basic biology project will provide important insight that could help develop better cell and tissue engineering solutions as well as opening doors to rejuvenation therapies using epigenetic modulators: 1) The existence or not of a hierarchical stem cell is important for understanding whether the stem cell should be the primary target for therapies. This includes not only cancer, but poorly-understood chronic benign inflammatory conditions, such as interstitial cystitis, wherein there is an underlying failure of urothelial homeostasis. 2) there is a recognised need for new "tissue-engineered" approaches for patients with end-stage bladder disease. The results of the project will help inform strategy, such as whether to selected particular cell sub-population(s), or how to direct differentiation of induced pluripotent stem cells for therapeutic use. 3) Discovering how urothelial cell phenotypes are epigenetically-regulated will enable us to understand how environmental epigenetic modifiers compromise urothelial health (eg by disrupting plasticity) and how to reverse this by therapy. This could impact on environmental and health & safety practices. 4) in seeking regenerative therapies aimed at the chronic diseases of ageing, it is possible that epi-modifiers or epi-modulators could be used to "re-set" the epigenetic state of a diseased urothelium to re-establish normal homeostasis and regeneration. The project is likely to generate knowledge and materials of interest to industry involved in delivering a) research tools to the life sciences and b) translation of regenerative medicine through to patient benefit. JS has substantial pharma industry contacts (including Astellas, Ono) with whom the potential of any research findings would be discussed under confidentiality agreement. Such an approach in the past has resulted in industry-funded research contracts and collaborations (eg through CASE studentships). Our tangible objective will be to actively engage with industry to generate a minimum of one CASE studentship or piece of contract research with industry directly from this research.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Regenerative Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/R006172/1 Challenging dogma: an alternative non-hierarchical, epigenetically regulated model of the urothelium.

BB/R009295/2 Challenging dogma: an alternative non-hierarchical, epigenetically regulated model of the urothelium.

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