20 April, 2022
“So, what is your PhD on?”
I often get asked this question when people find out that I am a doctoral student. I tell them I am investigating the function of a group of enzymes called protein tyrosine phosphatases (PTPs, for short) and that I’ll be using the skin as a model system to study these PTPs. Having always been interested in skincare, I carried out a year in industry where I learnt how to make skin care formulations and screened proteins for their use in skin care products, and pondering how skin can heal at different rates between individuals and locations within the body, I thought this PhD project was perfect for me. Below, I will discuss my opportunity to do a short placement at the University of Edinburgh, where I learnt how to generate lab-grown skin tissue (so-called skin organoids), a technique which will be used to facilitate my PhD project at the Institute.
Fluorescent images of a skin organoid. Each colour stain indicates
a different protein found within the skin. Bottom right (blue) = nucleus.
Top right and bottom left (pink and green) = cell-adhesion proteins.
Top left is a merge of all three images.
My project in a nutshell
PTPs catalyse the removal of phosphate groups from phosphorylated tyrosine residues on proteins. This is known as a dephosphorylation event. Our lab has shown that certain PTPs dephosphorylate proteins involved in cell adhesion. Cell adhesion is the process by which cells interact and attach to neighbouring cells through specialised molecules on the cell surface. A great model to look at cell adhesion is the skin; numerous layers of cells are held together with the help of numerous protein complexes to form this stable, yet dynamic unit. Fundamentally, the organisation of this structure is maintained and monitored in a highly controlled fashion, giving the skin its regular ordered appearance. Without this, our skin would simply float off our bodies. Changes to the steady state of the skin caused by tissue damage and ageing often give rise to impaired functioning of the skin. I am interested in what the role of these phosphatases, particularly PTPRF and PTPRK, are in skin repair and barrier integrity.
From light discussions to plan of action
Talks of this collaboration began before I joined the lab between Dr Hayley Sharpe, my current supervisor, and Professor Sara Brown at a Wellcome Trust meeting when they were discussing PTPs as a hit in Sara’s skin phosphoproteomics data. Phosphoproteomics is a technique used to identify changes in protein phosphorylation levels within a cell that has been exposed to different conditions. The data suggested that PTPs may have a role in the skin, and remained to be investigate. Sara’s research focuses on understanding how a person’s genetic make-up may predispose them to inflammatory skin conditions, particularly that of atopic eczema. Her lab specifically uses human skin cells to create a 3D skin organoid model which can be precisely manipulated to investigate specific molecular genetic effects. Organoids are complex collections of cells grown in a 3D culturing medium that recapitulate many of the physiological and genetic features of various tissues or organs. Hayley wanted to follow up on the proteomics observation made by Sara and so there was discussion of a potential collaboration to explore this further. I think it is incredible how small discussions can lead to large project collaborations!
Mid-way through 2021, I had a meeting with Sara over video call about organising a trip to Edinburgh so that I could learn how to generate the skin organoids, and we decided October would be an ideal time. I was there for six weeks, which gave me ample time to learn the 3D organoid culture technique. The idea was for me to learn the technique and bring the knowledge back with me to Cambridge. Alongside being a Principle Investigator at the University of Edinburgh, Sara is an excellent clinical academic dermatologist, so she was often in the clinic. Therefore, I was under the supervision of Dr Martina Elias, a post-doc in the lab.
Prior to the placement, I had already optimised the experimental treatment conditions, where I used siRNA gene silencing to lower the expressions of PTPRF and PTPRK in immortalised keratinocytes (skin cells that have been specifically cultured for use in laboratories).
What I didn’t realise was that I’d become a registered research student at Edinburgh, and so had access to all the libraries and student associations (I’ll get onto exploring Edinburgh later on…). Martina had prepared a thorough schedule for me, including plenty of repeat experiments from different skin donors. The organoids are derived from primary human skin keratinocytes and fibroblasts isolated from patients that have undergone surgery. I was able to observe Martina preparing and extracting cells from the skin, which had just come fresh from the hospital the day before. Skin contains three layers. The epidermis, the outermost layer of skin, that provides a protective and waterproof barrier. Next is the dermis, found under the epidermis, contains extracellular matrix connective tissue, hair follicles, and sweat glands. Finally, the hypodermis or subcutaneous layer, is made of adipose (fat) and connective tissue. It was as if I was a junior doctor observing an intense procedure as I watched Martina slice her way through the adipose tissue to leave behind only the dermis and epidermis.
Skin organoids in a 6-well plate containing growth media.
Organoids are just that little bit closer to reality
A schematic of the skin organoid culture method (adapted from Elias MS et al., 2019).
There have been debates between whether 2D or 3D cultures are best. Could a single layer of cells (2D culture) really be reputable and allow us to draw direct conclusions for what would happen in a 3D organ in the body? Yes, no and maybe? Still, it offers a great starting point and basis for further elaboration. Building organs outside of the body, that can survive for months, have been incrementally successful over the past decade. Adding a third-dimension to cell culture will allow the ability to view special and temporal changes to the cell. For skin organoids, this is achieved by the generation of dermis mimicking gels composed of extracellular matrix and fibroblasts. Keratinocytes treated with siRNA are layered on top and form the epidermis. The epidermis of the skin consists of stratified squamous epithelia. Although, I did not touch the organoid with my bare hands, through my gloves I would describe the texture as “baby skin-like” (smooth and no wrinkles). When harvesting the epidermis for analysis, I simply peeled it away from the dermis with tweezers. Much like peeling off real skin but a slightly more extreme version.
After several days and a few media changes, the organoid is raised and exposed to an air-liquid interface, by placing it onto a level metal grid. Usually in single-layered cell cultures, the cells are submerged in a liquid, known as growth media. This contains all the vital nutrients required for cell function and growth. Similar to how our bodies need food and water to live, our cells also need to obtain nutrients to survive. Allowing the cells to be in contact with air stimulates them to grow and differentiate where they become specialised and start dividing upwards to form the many layers of skin. The organoids develop for a further week until they are harvested and evaluated. In terms of how can the organoids be analysed, the possibilities are endless. The main ones I focused on whilst in Edinburgh were Trans-Epidermal Water Loss (TEWL) and capacitance to assess skin hydration levels and barrier function. Further, I also looked at changes to the appearance of the organoids through imaging analyses.
Exploring a new city
Tiffany standing outside of the IGC labs.
As I am sure this is the case for many of you, lockdown and the pandemic put my travel plans to a halt for a couple of years. I had never been to Scotland before, let alone Edinburgh so I was not entirely sure what to expect. Hands down, it was the highlight of my year in 2021. I didn’t think I could adore a city this much, after only living there for less than two months! From hearing stories told by my friends, Edinburgh was a lovely place and the people there are incredibly friendly. They were completely correct on those points. My accommodation was only a 25-minute walk to the lab, which was the Institute of Genetics and Cancer (IGC), and only a 15-minute walk to the beach. IGC was closed on weekends so I was able to do a lot of voyaging of the main city, touring through coffee shops and restaurants and even visited Glasgow too.
Daily walk views in the evening from the lab to my accommodation.
I am incredibly thankful to Sara and her lab members, Martina, Luke and Mona for sharing their protocols with me and teaching me this intricate technique; I look forwards to utilising it back at the Babraham Institute. With this model system, I hope to further elucidate the functions of PTPs and dive deeper into establishing their roles in cell-adhesion. For the future, a couple of things I’d be interested in seeing is how long the organoids can grow for and if this could somehow mirror oxidation and ageing of the skin. As of current, I believe there are no labs which culture 3D skin organoids here in Cambridge, so it is excellent to be able to bring the expertise back to the Institute!
20 April 2022
By Tiffany Lai