- Pluripotent stem cells (PSCs) are cells with the ability to become any cell type of the adult body.
- By studying the early stages of embryo development, Dr Jennifer Zenker at the Australian Regenerative Medicine Institute, Monash University, improves our understanding of how these cells form in real time.
- Zenker and colleagues applied light-activatable molecules called photostatins to control microtubule growth, a crucial process of PSC development.
- Such new techniques might be applied to regenerative medicines involving PSCs.
A human being is made of many different cell types, ranging from hair cells to those which make up our complex immune system. All the different cell types arise from unspecialised pluripotent cells, which hold the potential to become any type of cell in the adult body.
Dr Jennifer Zenker is a group leader at the Australian Regenerative Medicine Institute, Monash University, where her group uses new imaging methods in a live embryo to reveal cellular structures and the basis of what makes cells pluripotent.
The cellular microtubule ‘motorway’
Microtubules make the scaffolding of a cell, ensuring that functions like cellular transport and cell division can take place. Imagine this network like the motorways of cities that enable people and cargo to move from one location to another using cars, bikes, trucks, buses, trains, or taxis. If there are issues with the roads, traffic jams form and the city may even come to a standstill. Similarly, if microtubules don’t form – or don’t form properly – the communication and transport system of a cell is compromised, and the cell will have widespread functional problems.
The ‘motorway bridges’ of embryonic cell division
In most animal cells, the main focal point where microtubules originate from (known as a microtubule-organising centre, or MTOC) is the centrosome. However, the centrosome is not fully functional, or even present in the cell, during the first few days until the embryo attaches to the womb, a period called preimplantation. Shortly after the embryo’s implantation, the cells lose their pluripotency (ability to become any cell type) and take on specific roles. How these processes are controlled inside each cell via the microtubule network remained unclear.
Normally during cell division the microtubules form bridges to hold the two dividing cells (known as sister cells) together until they split apart. Zenker’s work in 2017 showed that, unlike in the adult cell, the sister cells remain connected via a web of microtubule filaments (called the interphase bridge) in the early stages of embryo development, even after division has occurred. The interphase bridge structures don’t necessarily appear from the centre of the cell either, as previously thought. Instead, Zenker’s study showed that these connections between sister cells arise in a non-symmetrical way within the embryonic cell, just like different cities are linked on a motorway system.
Through live cell imaging, Zenker was able to determine that a protein called calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) builds at the interphase bridge between sister cells in early development. When this protein is absent, the interphase bridges are smaller in size and show little microtubule growth. This led Zenker to believe that the CAMSAP3 protein transforms the bridges into non-centrosomal MTOCs, making interphase bridge formation reliant on CAMSPA3 as a location for microtubular stable growth and expansion.
Photostatins provide the chance to manipulate microtubule filament formation with high precision in real-time
within a cell.
Zenker and colleagues showed that if the bridges were broken using laser techniques, the cells became much more spherical and did not contribute to the pluripotent inner mass of an embryo. This suggests that the mechanical coupling between the cells could drive pluripotency during the early stages of embryo development and coordinate the later stages. In doing so, the connections between early embryonic cells could determine cell fate and shape as the embryo grows.
New traffic light system to guide cell development
Anticancer drugs which work by targeting microtubules to slow cell growth and replication, can be used as tools to study these processes in a research setting. Previously, drugs like nocodazole were used to stop microtubules from expanding (polymerising). However, because of the widespread importance of microtubules in many cell functions, from protein transport to cell division, impacting microtubules has many side effects for the cell.
The same principles from experiments by Zenker and others have the potential to be used across the medical field.
Zenker applied a group of reversibly light-switchable drugs called photostatins (PSTs). The PSTs are not active in the dark, but when they are lit up in UV light they can cause a chemical reaction, blocking the expansion of microtubules within the cell. PSTs provide the chance to manipulate microtubule filament formation with high precision in real-time within a cell. This is because their effects on blocking microtubule polymerisation can be reversed by washing off the PSTs or by applying green light. This huge discovery allowed Zenker and colleagues to inhibit microtubule polymerisation in a much more controlled, targeted manner than current anticancer drugs by using directed light. Zenker and colleagues were the first to apply this methodology using PSTs to manipulate the microtubule cytoskeleton in a living, three-dimensional subject – ie, the mouse embryo – as outlined in their 2017 and 2021 publications.
The future for photostatins
PSTs are a powerful tool for research that enable real-time manipulation of microtubules – the motorways of cells – both in time and space. Use of PSTs in research could minimise the side effects on microtubule function in embryonic, pluripotent and pluripotent-like cells, compared to the drugs that were previously used. Using PSTs and a 3D live embryo mouse model allows Zenker and colleagues to study cell development during early-stage embryo development. This work can improve our understanding on how the microtubule networks form and function in a cell.
The same principles from experiments by Zenker and others have the potential to be used across the medical field. The possibilities range from regenerative medicines to cancer treatments, in addition to studying healthy cells. By improving tools for visualising how cells develop and maintain function, cell developmental biology could be considered the missing piece of the puzzle for understanding cell pluripotency.
How do you see the research progressing?
Light-controllable drugs, also called phototherapeutics, is an up-and-coming era of manipulating the function of a cell non-invasively. Light has been predominantly used for visualisation using microscopes, in research and clinics. Together with the constant improvement of cutting-edge microscope technologies, the benefits of using light as a guide to control cell function and behaviour will come into the spotlight across a wide range of research fields. Furthermore, the microtubule cytoskeleton is not the only cellular structure that light-activatable drugs can be developed for, and the diversity of targets will be continuously increasing, including, for instance receptors, organelles, or hormones.
How do your findings impact the medical field within regenerative medicine?
Regenerative medicine means repairing or replacing damaged or diseased tissues by using the body’s own capability of ‘re-creation’. To do so, it is fundamental to understand how new life was created in the first place – and that is what my team and I are aiming to uncover from a cell biological perspective. Using innovative live imaging of the preimplantation mouse embryo, an excellent system for studying how pluripotent cells organise dynamically into increasingly complex structures within their physiological 3D environment, allows us to leverage our current static view to sub-cellular real-time processes of these cells. By unravelling the secrets on the inner structural aspects of pluripotency using live imaging of the living mammalian embryo, we aim to repurpose those same principles for the application of induced pluripotent stem cells in regenerative medicine.
What next step in this research are you looking forward to?
The interest and potential to translate the work on light-activatable drugs to clinical applications is undeniable. However, I am also incredibly keen to apply them to advance our fundamental knowledge about the origin of new life. It all starts with a single cell – the fertilised egg – but how the different components inside a cell, its cytoskeleton and organelles, move around, and how they communicate with each other to form a human being requiring about 30 trillion different cells, still remains elusive. Innovative tools, like the photostatins, enable us to synchronously visualise and modulate such processes in real-time.
What’s the most impactful part of this research within your career?
When I started, our knowledge of pluripotent cells and early embryonic development was limited to static snapshots of genetic, epigenetic, and metabolic requirements. My research is the first to conclusively demonstrate a pluripotent, cell-specific, microtubule cytoskeleton (Zenker et al, 2017): the roadmap to regulate intracellular trafficking, which was still widely regarded as disorganised at that point, and its contribution to cell fate specification had been largely ignored.
Through my research, we have provided fundamental insights into the structural logic that governs early mammalian development, lineage plasticity, pluripotent identity, and stem cell self-renewal (Hawdon et al, 2021).Therefore, it is of ultimate significance for far-reaching fields, including developmental biology, cell and stem cell biology, tissue engineering, pharmacology, and medicine. Understanding the construction of a pluripotent cell is fundamental to identify unhealthy cells, and informs how to produce new ones to treat diseases. Thus, this exciting field has the potential to further advance regenerative and assisted reproductive medicine, including applications for safer and more efficient therapeutic use of induced pluripotent stem cells for the survival rate and early detection of cellular abnormalities of in vitro fertilised human embryos and livestock improvements.
