Newcastle Stem Cell Biology Fund PhD Scheme 2024

Project outline

Background: The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. Most drug studies are performed in vivo in experimental animals, but this approach is far from optimal because there are fundamental, structural, and functional differences between the rodent and human retina. The advent of organoid technology has made possible the generation of three-dimensional (3D) self-organised laminated and light-responsive retinal organoids from pluripotent stem cells (PSCs), a powerful tool for disease modelling and drug discovery. However the lack of microglia and vasculature currently limits their utility in two ways: firstly, it is known that microglia and vasculature play an important role in retinal development and disease, and secondly, the vascular system is necessary to prevent formation of necrosis in tissues that grow beyond 500µm diameter because of impaired diffusion of oxygen and nutrients. The vascular cells in the retina interact closely with the retinal neurons and glial cells, forming the retinal neurovascular unit (NVU), which together with the retinal pigment epithelium (RPE) form the core of the blood retina barrier (BRB). The BRB mediates highly selective diffusion of molecules from the blood to the retina and maintains retinal homeostasis. Alterations of the BRB play a crucial role in the development of retinal diseases, thus dynamic modelling of human BRB in highly desirable.

Aims and Objectives: Current conventional co-culture models of retinal organoids with endothelial and microglia cells bypass the developmental processes that lead to formation of spatially organised retinal NVU and BRB. Furthermore, the mesodermal origin of microglia and vascular cells, and ectodermal origin of neural retina, provide a significant barrier for the generation of vascularised immunocompetent retinal organoids incorporating NVU/BRB. Our aim is to reach beyond the state of the art to combine multiple organoid types in 3D culture to generate vascularised immunocompetent retinal assembloids that combine the neural and mesodermal cells and provide all the components of retinal NVU/BRB.

Methods: We will generate PSC-vascular organoids that contain endothelial cells, pericytes, smooth muscle and microglia and assemble those with cardiac and retinal organoids to make vascular immune competent functional retinal assembloids. We will employ a combination of molecular (immunofluorescence confocal microscopy, single cell RNA-Seq) and functional (retina light response, assembloids perfusion, BRB permeability, microglia activation) to fully characterize the assembloids.

Potential impact: Combining cardiac contractility with blood-life flow can provide significant improvements to the development and function of retinal assembloids in a tractable fashion with the potential to reduce animal usage and increase their use in drug discovery and disease modelling.

Supervisory team: The supervisory team has a strong expertise in generation of all three types of organoids required for the retinal assembloids delivery. Each of the supervisors has a strong research and PhD supervision record. The student will benefit from regular exchange visits between Universities of Newcastle and Liverpool where the supervisor’s research teams are based.

Supervisors

1. Prof. Majlinda Lako (majlinda.lako@ncl.ac.uk)
2. Prof. Reinhold Medina (r.medina-benavente@liverpool.ac.uk)
3. Prof. Evelyne Sernagor (evelyne.sernagor@ncl.ac.uk)
4. Prof. Lyle Armstrong (lyle.armstrong@ncl.ac.uk)
5. Dr. Gerrit Hilgen (gerrit.hilgen@northumbria.ac.uk)

Project outline

Epithelial stem cells play a fundamental role in homeostasis and repair of stratified epithelial tissues. For example, transplantation of cultured keratinocyte autografts provides a landmark example of successful cellular therapies by restoring durable integrity in stratified epithelia lost to devastating tissue conditions. A further example is the expansion and transplant of limbal epithelial stem cells from the boundary between the cornea and the conjunctiva to treat total limbal stem cell deficiency – a serious condition which leads to sight loss and significant pain and irritation in the affected eye.   Despite the overall success of such procedures, failures still occur in case of paucity of cultured stem cells in therapeutic grafts. Strategies aiming at a further amplification of stem cells during keratinocyte ex vivo expansion may thus extend the applicability of these treatments to subjects in which endogenous stem cells pools are depauperated by ageing, trauma, or disease. The ageing status of the stem cell pool is of particular interest since many of the therapeutic procedures alluded to above will need to be performed on older patients thus there is a currently unmet need to understand how epithelial stem cell functionality changes with donor age.  Furthermore, the basis of most of the above therapeutic procedures involves the in vitro expansion of epithelial stem cells harvested from the patient, many of whom are elderly individuals therefore it is important to understand the impact of such extended proliferation on a group of cells who may already have been exposed to significant age-related decline.  Primary epithelial stem cells harvested from patients of any age are only capable of limited numbers of population doublings in vitro therefore methods have been developed to induce a state of so-called “conditional immortalization” by exposing the cells to small molecule inhibitors of Rho-kinase activity and feeder layers such as those comprising mitotically inactivated 3T3 cells. These methods are distinct from “traditional” cell immortalization procedures which utilize stable transfection with genes such as TERT, which are capable of indefinite numbers of population doublings during in vitro culture without the need for additives to the culture media. Ageing is a complex multifactorial process in vivo that is accompanied by loss of tissue integrity and organismal homeostasis.  At a cellular level, ageing is associated with altered gene expression and a perturbed epigenome in addition to telomere attrition and accumulation of misfolded proteins. The outcomes of these changes include perturbed nutrient sensing, mitochondrial dysfunction and cellular senescence which impacts overall cell function and intercellular communication, causing exhaustion of stem cell reservoirs and tissue dysfunction. 

Data from several groups suggests that epigenetic changes play a significant role in age related cell dysfunction. Changes in DNA methylation are well correlated with ageing which has led to the development of so-called “epigenetic clocks” that are thought to reflect chronological ageing. The accuracy of such clocks is still an open debate but recent studies of tissue specific stem cells at a single cell level have revealed an association between age related DNA methylation changes and increasing transcriptomic heterogeneity and corresponding degradation of coherent transcriptional networks and stem cell functional decline. These studies imply that epithelial stem cells from older patients may be less effective at tissue repair than those from younger donors; however, it has been noted that similar DNA methylation changes occur during long term in vitro cell culture to those observed during organismal ageing so it is conceivable that additional cell proliferation required to deliver therapeutically useful numbers of epithelial stem cells may result in further epigenetic changes that compromise the ability of these cells to effect repair and regeneration of the target epithelia. An observation that supports this possibility is the continuing ablation of DNA methylation in fibroblasts immortalized by ectopic expression of the telomerase reverse transcriptase subunit, hTERT, until a severely hypomethylated equilibrium state is reached.

In view of this the overarching aims of this project are twofold

1. An understanding of the baseline ageing state of primary donor epithelial stem cells. What are the DNA methylation and histone modification profiles of keratinocyte stem cells and limbal epithelial stem cells harvested from young vs old donors? 
2. An understanding of the changes occurring in the epigenome of keratinocyte stem cells and limbal epithelial stem cells harvested from young vs old donors when subjected to extended proliferation after conditional immortalization induced by the Rho-kinase inhibitor, Y27632 and a 3T3 feeder cell layer. In addition, we will correlate the epigenetic changes with biomarkers such as nutrient sensing, telomere lengths, telomerase expression, genomic instability, mitochondrial activity and overall transcriptomic changes as a function of (a) initial donor age, (b) number of population doublings undergone during conditional immortalization.

We anticipate that a successful outcome from this project will elucidate the changes occurring during extended culture of epithelial cells using conditional immortalization techniques and how this may affect the utility of such cells for transplantation-based therapies. A secondary but perhaps no less important outcome will be an increased understanding of the molecular mechanisms contributing to epigenetic in vitro ageing and the development of strategies to overcome these changes which may be useful in the search for anti-ageing therapies.  If a successful strategy against ageing is to be found, then distinct and parallel aging mechanisms must be addressed, for example, by the removal of senescent cells, together with the retardation of epigenetic aging.

Supervisors

1. Lyle Armstrong (lyle.armstrong@ncl.ac.uk)
2. Majlinda Lako (majlinda.lako@ncl.ac.uk)

Project outline

Osteoarthritis is a common debilitating musculoskeletal disease that affects over 9 million people in the UK, yet there are no drugs that slow disease progression. The disease is typified by loss of cartilage, the tissue which allows joints friction-free movement. Genetic risk loci, along with epigenetic changes, for Osteoarthritis occur predominately in non-coding regulatory regions of the genome, which control target gene transcription. Many of these risk loci are also linked to skeletal developmental and are associated with adult height and joint shape. However, we still need to identify the target genes of these osteoarthritis-associated regulatory regions to know how these contribute to joint formation and to increase our understanding of osteoarthritis susceptibility.

Human pluripotent stem cells (hPSCs) can now be selectively differentiated in a process that recapitulates the developmental system of limb-bud formation to produce accurate articular pre-chondrocytes, cartilage cells. Importantly, hPSCs are amenable to genome-editing (CRISPR) and CRISPR-mediated gene regulation.

In collaboration with colleagues at the University of Manchester, the student will establish hPSC differentiation protocols and use these cells to generate a 3D genomic interaction map to utilise with established CRISPR-editing, CRISPR-gene-activator and CRISPR-gene-inhibitor systems to functionally define important osteoarthritis regulatory regions and effector genes.
This is an opportunity to work on a highly interdisciplinary project where a successful PhD student will learn fundamental molecular, cell biology and bioinformatic skills. Importantly, they will receive training in stem cell biology with a strong focus on aiming to improve clinical outcomes for patients with common and rarer skeletal diseases.

Supervisors

1. David Young (david.young@ncl.ac.uk)
2. Louise Reynard (louise.reynard@ncl.ac.uk)

We will fund up to three PhDs studentships provided suitably qualified candidates apply.

You will not be judged for having been out of academia, whether it is for work, caring duties, illness or anything else. Like everyone else, you will need a degree – however, there is no time limit on when this was awarded. We appreciate that experiences outside of academia can be a rich source of key skills that you would need for a PhD. Be sure to think about skills this experience has given you and make sure you tell us about them. It is likely that the supervisor or interview panel might want to know what drew you back to academia. Use this time to show how passionate you are about research.

You will not be penalised for not having a master’s degree. PhD studentships are highly competitive, and most successful applicants will have a master’s qualification. This is because of the experience a master’s degree provides rather than the certificate. However, experience can equally come from many other sources, such as work, both academic and non-academic.

If you are offered an interview, a standard email will be sent from the Centre team to your referees requesting a reference before your interview. We would advise that you contact your referees to tell them that they may receive a reference request.

A regular CV should be approximately 1-2 pages depending on how much experience you have (but please make sure to note all your experience).

The cover letter should explain your interest in your chosen projects and should include any additional information you feel is important to your application. You may wish to add why you are choosing Newcastle University. There are no formal word limits for your CV or cover letter, but we recommend you keep them concise.

Each student has a different set of strengths and weaknesses. That said, a passion for the project is an essential part of being a successful PhD student. It is a basic requirement that any supervisor will look for in selecting a student. Do remember that there may be some (but not endless) flexibility in what you actually do within the PhD project.

This will be dependent on the project supervisor. Our funding does not dictate any work schedule. It does ask that any difference from standard working patterns be agreed with your supervisor. It would be sensible to discuss this with them before you apply. Most supervisors will support a student's requirements (for example, to accommodate caring responsibilities), but the project may have specific requirements. E.g., where a particular type of lab work is necessary to complete the project.

Students may take on teaching or demonstration work, where this is compatible with their training in addition to a full-time studentship. This needs to be approved by their supervisors, in addition to a full-time studentship. Other paid work would need the consent of the supervisor and should not delay or interfere with your research training. You may ask primary supervisors about flexibility of the PhD; this varies depending on the PhD project. Part time study is usually available; we advise that you discuss this further with the project supervisor.