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Developing pluripotent stem cell models of inherited retinal diseases


Faculty of Medicine and Health

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Prof Colin Johnson Applications accepted all year round Self-Funded PhD Students Only

About the Project

Background
Inherited retinal dystrophies are a leading cause of blindness and visual loss in the UK working age population. However, despite the widespread diagnostic use of next-generation sequencing, a molecular genetic diagnosis is unavailable for many patients world-wide. Understanding the genetic causes of these conditions enables accurate counselling of patients, improves knowledge of disease pathogenesis and catalyzes the development of novel treatments. To address the current limitations of genetics, this project will develop in vitro cellular modelling of inherited retinal disease in novel microfluidic devices, an approach that is currently under-studied and under-developed. This approach will hugely increase both throughput and the capacity to perform combinatorial assays because existing methods of retinal cell differentiation are costly, laborious and time-consuming.

Environment
We have an existing track-record in induced pluripotent stem cell (iPSC) characterization, differentiation into retinal cell types (retinal pigment epithelium and retinal organoids) and systematic characterization of normal and diseased cellular phenotypes [1]. We have also developed a range microfluidic devices [2], including for the rapid separation of mesenchymal stromal cells, with minimal manipulation, for autologous cell therapies [3].

Approach
The project will prototype initial PDMS (polydimethylsiloxane)-based “organ-on-a-chip” devices that enable wide-field microscopy in order to visualize cellular phenotypes. The iPSCs will be differentiated into retinal pigment epithelium following a standard protocol over 21 days [1]. The use of microfluidic devices will minimize culture volumes and cell manipulation, enabling higher throughput and potentially reducing the cost, labour and time that is required for assays of cellular phenotypes. Running several devices in parallel will enable the optimal design to be rapidly chosen for full-scale manufacture. In parallel, we will introduce mutations into normal control iPSCs using CRISPR-Cas9 gene-editing that will model RPE disease in patients. Once the basic design of the chip is optimized for RPE differentiation, we will manufacture multiplexed chips to enable matched sets of combinatorial assays to be performed in parallel (e.g. characterizing a range of cellular phenotypes using a panel of antibodies, reverse genetics screening with a panel of different CRISPR-Cas9 reagents to gene-edit different mutations, testing responses to a panel of drugs or small molecules). The incorporation of transparent indium tin oxide electrodes into the chip will enable transepithelial electrical resistance (TER) assays of cell monolayer integrity in mature RPE. In the longer term, we envisage culture of retinal organoids on chips, and their co-culture with RPE, will enable a more physiological tissue system to be developed. Furthermore, the manufacture of electrodes in the devices will enable us to assay physiological functions such as light-evoked responses.

Outcomes
The project develops methods for in vitro cellular modelling of inherited retinal disease in novel microfluidic devices. This is an approach that provides a more physiological model system for interrogating and understanding human genetic variation in health and disease. It provides unusually broad training research for an excellent graduate who is motivated and proactive in developing a novel area of multi-disciplinary biomedical research.

Techniques used in this project
Stem cell culture and differentiation, cellular function assays, confocal microscopy and live cell imaging, CRISPR-Cas9 gene editing (knock-out, knock-in and base editing), microfluidic chip design and manufacture.

Eligibility
You should hold a strong first degree equivalent to at least a UK upper second class honours degree in a relevant subject area.

The Faculty minimum requirements for candidates whose first language is not English are:

• British Council IELTS - score of 6.5 overall, with no element less than 6.0
• TOEFL iBT - overall score of 92 with the listening and reading element no less than 21, writing element no less than 22 and the speaking element no less than 23.

How to Apply
To apply for this scholarship applicants should complete a Faculty Scholarship Application form using the link below https://medicinehealth.leeds.ac.uk/downloads/download/129/faculty_graduate_school_-_application_form and send this alongside a full academic CV, degree certificates and transcripts (or marks so far if still studying) to the Faculty Graduate School [Email Address Removed]

We also require 2 academic references to support your application. Please ask your referees to send these references on your behalf, directly to [Email Address Removed]

If you have already applied for other scholarships using the Faculty Scholarship Application form this academic year you do not need to complete this form again. Instead you should email [Email Address Removed] to inform us you would like to be considered for this scholarship project.

Any queries regarding the application process should be directed to [Email Address Removed]

Funding Notes

This project is available for self funding applicants only.

References

1. Buskin A, Zhu L, Chichagova V, Basu B, Mozaffari-Jovin S, …33 others… Grellscheid S-N, Johnson CA, Lako M (2018). Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa. Nat Commun 9:4234.
2. Rimsa R, Smith AJ, Wälti C, Wood CD (2017). A planar surface acoustic wave micropump for closed-loop microfluidics. Appl Phys Lett 111:234102.
3. Smith AJ, O'Rorke RD, Kale A, Rimsa R, Tomlinson MJ, Kirkham J, Davies AG, Wälti C, Wood CD (2017). Rapid cell separation with minimal manipulation for autologous cell therapies. Sci Rep 7:41872.


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