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

Lead Research Organisation: University of York
Department Name: Biology

Abstract

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.

Technical Summary

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.

Planned Impact

As expanded in the Academic Beneficiaries section, our research will be of interest to Academics from a broad range of fields including epigenetics, epithelial cell & tissue biology and stem cells. In addition, by developing epigenetic approaches to manipulate cell fate and phenotype as new avenues for translation and application, our research will be important to the Life Sciences industry.

The UK economy will benefit through the patent-protection of any IP generated during the project, which we will be vigilant in identifying, protecting and exploiting through our close partnership with Business Development Managers in the University of York/UCL Research and Industry Offices and through the Investigators' own involvement in commercial enterprise and industry collaborations (eg through BBSRC CASE studentships).

The project will be of immediate and substantial benefit to the cell biology and bioinformatics postdoctoral researchers supported on the project through the experience of research collaboration and the combining of skills and opportunity to learn from each other's ideas and approaches. Regardless of whether they pursue careers in industry or academia, these highly-skilled individuals will emerge in a strong position to contribute to the UK Biosciences.

The wider public will benefit via active engagement of the investigators and researchers in outreach, including school and charity events in which both investigators actively engage.

We can also consider the longer term impact of our research for health and well-being. The distinction between urothelium as a stem cell-regulated tissue undergoing a hierarchical programme of differentiation versus a tissue composed of cells whose phenotype is epigenetically-regulated and responsive to external cues holds major implications for understanding urothelial health and disease and for the future development of new therapies:
> In areas allied to Urology, researchers, clinicians, pathologists and pharmaceutical companies will have interest in the tissue-specific urothelial detail uncovered and the possibility of translating this into benefit for patients. This will include potential regenerative therapies for tissue reconstruction, differentiation and/or homeostasis-modifying therapies for benign chronic disorders. The existence or not of a hierarchical stem cell is also important for understanding whether the stem cell is the primary target for therapy. This obviously includes cancer, as well as chronic benign inflammatory conditions, such as interstitial cystitis, where the disease states can be described as an underlying failure of urothelial homeostasis.
> Moderate to severe urinary incontinence is found in 22.6% of women and 8.8% of men aged 40 years and 33.4% women and 23.5% men aged over 80 years. We anticipate our work will impact on the quality of life and health of the ageing population by contributing to the availability of effective regenerative and other therapies.
> Discovering how urothelial cell phenotypes are epigenetically-regulated will allow us to understand the role of environmental epigenetic modifiers in urothelial disease (eg by disrupting plasticity) and how to moderate or reverse this for therapy. This is likely to carry implications for the Health and Safety Executive and for Environmental Agencies who need to understand and limit the impact of epigenotoxins on human health.

Publications

10 25 50
 
Description Biomedical Vacation Scholarship
Amount £5,536 (GBP)
Funding ID 216869/Z/19/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2019 
End 09/2019
 
Description Fetal development of the differentiated barrier phenotype by human urothelium 
Organisation St James's University Hospital (Jimmy's)
Department Pathology Unit Jimmys
Country United Kingdom 
Sector Public 
PI Contribution Project submitted for use of HDBR resource
Collaborator Contribution Registered by HDBR for supply of fetal tissue to our study
Impact None yet.
Start Year 2018
 
Description Fetal development of the differentiated barrier phenotype by human urothelium 
Organisation University College London
Department MRC/Wellcome Trust Human Developmental Biology Resource
Country United Kingdom 
Sector Private 
PI Contribution Project submitted for use of HDBR resource
Collaborator Contribution Registered by HDBR for supply of fetal tissue to our study
Impact None yet.
Start Year 2018
 
Description Provision of urological tissues 
Organisation St James's University Hospital (Jimmy's)
Country United Kingdom 
Sector Hospitals 
PI Contribution Intellectual involvement through workshops and presentations at multidisciplinary meetings and audits.
Collaborator Contribution Urological tissues supplied to our research project
Impact NA
Start Year 2018
 
Title An informatics pipeline for annotating features from metabolomics and lipidomics data 
Description A lack of automated informatics tools is the major bottleneck for analysing lipidomic pathways and metabolites in plants and animal cells. Current analysis workflows require each feature extracted by mass spectrometry to be identified individually, involving manual searches of multiple databases with different data input and output formats. This leads to a frustrating lack of confidence in accurate metabolite calling and pathway dissection. This project developed an automated analysis pipeline that can be applied with confidence against database(s) of choice to aid in accurate metabolite identification and define lipid pathways. 
Type Of Technology Software 
Year Produced 2020 
Impact The programme will enhance the capability of the Biology Technology Facility in metabolomics and lipidomics and enable it to take a more leading role in this emergent area 
 
Description Interview for That's TV York 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Interview of clinical fellow on our biomaterial
Year(s) Of Engagement Activity 2019
URL https://www.york.ac.uk/biology/jack-birch-unit/news-and-events/2019/naturalbiomaterialsuccessfulinsu...
 
Description Living With and Beyond Cancer Event. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Took part in a celebration day at York Hospital to demonstrate the work of various groups in support of people living with and beyond cancer.
Year(s) Of Engagement Activity 2018
 
Description Presentation to HYMS MedTech Society & York Surgical Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact "Fit-for-purpose Tissue Engineering of the Urinary Bladder" invited presentation to HYMS MedTech Society & York Surgical Society (recorded).
Year(s) Of Engagement Activity 2018
 
Description Public engagement at charity event (presentation of work at YAC annual meeting) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Our PDRA, Dr Sreemoti Banerjee presented her project to a charity Annual General Meeting
Year(s) Of Engagement Activity 2019
 
Description Talk and visit to laboratories 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact York Festival of Ideas. Talk and lab tour of the Jack Birch Unit enabling the public to find out more about 'how science works'.
Year(s) Of Engagement Activity 2018
 
Description Urothelial cross-disciplinary research meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Researchers met clinical professionals (pathology, paediatric urology, transplantation, adult urology and NHS R&D) for workshop to discuss ongoing bladder research and opportunities for engagement and translation. The activity sparked enthusiastic debate and identified minuted actions for individuals, with two follow on studies initiated.
Year(s) Of Engagement Activity 2019
 
Description YAC AGM July 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact York Against Cancer Annual General Meeting, including networking of research team, fundraisers and committee members of York Against Cancer.
Year(s) Of Engagement Activity 2018