Effect of the topography of the human epidermal-dermal junction in influencing stem cell behaviour

The epidermis is the outer covering of the skin and plays an essential role in protecting our bodies from bacteria and other pathogens. It is made up of multiple layers of cells that are stacked on top of one another. The deepest cell layer, furthest from the skin surface, contains stem cells. Their role is to divide throughout our lives to make more cells that subsequently mature as they move through the upper cell layers. The most mature cells are specialised to form a protective barrier on the skin surface. If a tiny piece of skin is removed from the body and taken to the laboratory, it is possible to grow sheets of epidermis that have similar properties to normal epidermis and indeed these sheets can be used to repair burns and other types of skin wound.

One interesting feature of human epidermis is that the stem cells are clustered in specific positions in the basal cell layer, which correlate with natural undulations in the boundary between the epidermis and the underlying connective tissue. As we age the undulations become much less pronounced, but in contrast in some common skin diseases such as psoriasis the undulations become more prominent. We would like to understand why the stem cells are clustered in this way and whether the size of the undulations affects their behaviour. To investigate this we will grow human epidermal cells on special surfaces made out of artificial materials, such as the rubbery substance used as bath sealant, that recreate the undulations. We will measure whether cells in different positions on these surfaces are more likely to remain as stem cells or to leave the basal layer and mature. We will investigate whether on substrates that resemble aged skin the cells show an altered ability to divide and mature. We will discover how the substrates direct the stem cells to behave by identifying the signalling events that take place inside the stem cells. We will also find out whether we can over-ride the effects of the substrates by artificially stimulating changes in gene expression inside the cells.

The outcome of the project will be to explain, for the first time, why stem cells lie in specific locations in human epidermis and whether the information provided by their location contributes to the changes in the epidermis that are linked to skin ageing.

The goal of the project is to examine how the topography of the interface between the epidermis and dermis in human skin influences the location and properties of stem cells and the signalling pathways that control the onset of terminal differentiation. The first objective is to recreate the undulations of the epidermal-dermal interface in young and aged skin using two different bioengineering strategies. In one, based on PDMS substrates, the topography is created prior to seeding human keratinocytes. In the second - dynamic substrates - cells are initially seeded on a flat substrate and the undulating topography is subsequently generated by suction or magnetic force. Secondly, we will investigate how different topographies influence stem cell patterning, self-renewal and terminal differentiation, using a combination of end-point assays involving antibody-based detection of cell subpopulations and live cell imaging of cell fate decisions with GFP reporters. Thirdly, we will determine the relative contributions of different signalling pathways to triggering differentiation in different positions relative to epidermal-dermal topography. Previous studies have implicated actin-regulated activation of SRF dependent transcription and SRF independent integrin-mediated mechano-sensing. In addition, YAP signalling will be investigated because it is known to regulate differentiation in response to intercellular signalling. We will examine the relative importance of these pathways at different positions on the substrates, using both antibody-based approaches in fixed cells and FRET probes in living cells. Finally, we will genetically activate Notch and Wnt signalling within reconstituted epidermis to discover how this influences responsiveness to extrinsic, topographical cues. The proposed experiments will provide new information about how topographical information provided by the dermal-epidermal boundary influences the location and fate of human epidermal stem cells.

Who will benefit from this research? The immediate and direct academic beneficiaries include all investigators who participate directly in the project: the PI, the research assistant (RA) and postdoctoral scientist (PDRA) employed on the grant, and the collaborators who have provided letters of support. The benefit will extend to the diverse regenerative medicine, developmental biology, cell biology and bioengineering communities in the UK and internationally. Dermatologists will find the work relevant to the clinical challenges of skin repair, ageing and hyperproliferative conditions such as psoriasis. The results of the research will benefit the commercial sector, including companies that are interested in regenerative medicine to repair or replace damaged skin, the cosmetics industry and companies who perform toxicology and drug screens on cultured human skin. Members of the lay public, both healthy individuals and patients, will be indirect beneficiaries. UK government agencies that contribute funds to the UK Regenerative Medicine Platform (UKRMP) will benefit. The benefits will not be restricted to the UK but will have an impact globally.
How will they benefit from this research? Academics will benefit by increased knowledge of how the topography of the epidermal-dermal interface influences epidermal function, through obtaining data to facilitate further funding applications and publications, through career progression and, particularly in the case of the staff employed directly on the grant, training opportunities that enable them to pursue an independent research career or transfer to other employment sectors (timescale: 3-5 years). Although the research has no immediate clinical applications, the changes in epidermal topography under study were first noticed by Dermatologists many decades ago and are a routine score of severity in psoriasis. They will benefit from the research by finding out whether altered topography is purely symptomatic of changes with age and disease, or whether modifying it, for example pharmacologically, could improves skin function (timescale: 3-8 years). The commercial sector will benefit by having new knowledge on which to develop new therapeutics (cell or drug based) for skin indications, new platforms for screening compounds for potential toxicity, potential to commercialise new epidermal culture substrates, and the ability to sell materials, such as antibodies, to researchers funded by the grant (timescale: 1-10 years). Patients will benefit if Dermatologists use the research to devise new interventions in skin disease and if the commercial sector adopts the findings for toxicology testing or drug screens (time scale: 8-15 years). Healthy individuals will benefit by potential developments in skin cosmetic applications (timescale 10-15 years) and increased understanding of research (timescale 3 years). The UK government (timescale: 1-15 years) will benefit by generation of intellectual property, leverage of ongoing investment in the UKRMP and commercialisation of products arising from the research. The stature of the UK internationally will increase, because the proposed research will be widely disseminated to the global regenerative medicine community.