Stem Cells: Developing pluripotent stem cell models of inherited retinal diseases

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.

<h3 style="margin-left: 2.85pt;">Approach:&nbsp;</h3> <p style="margin-left: 2.85pt;">The project will prototype initial PDMS (polydimethylsiloxane)-based &ldquo;organ-on-a-chip&rdquo; 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.</p> <h3 style="margin-left: 2.85pt;">Outcomes:</h3> <p style="margin-left:2.85pt;">The project develops methods for <em>in vitro</em> 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.</p> <h3 style="margin-left: 2.85pt;">Techniques used in this project:</h3> <p style="margin-left:2.85pt;">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.</p> <h3 style="margin-left: 2.85pt;">Environment:<strong> </strong></h3> <p style="margin-left:2.85pt;">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].</p> <h3 style="margin-left: 2.85pt;">References:</h3> <ul> <li style="margin-left: 2.85pt;">Buskin A, Zhu L, Chichagova V, Basu B, Mozaffari-Jovin S, &hellip;33 others&hellip; Grellscheid S-N, <span class="underline_text">Johnson CA</span>, Lako M (2018). Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa. <em>Nat Commun</em> 9:4234.</li> <li style="margin-left: 2.85pt;">Rimsa R, Smith AJ, <span class="underline_text">W&auml;lti C</span>, Wood CD (2017). A planar surface acoustic wave micropump for closed-loop microfluidics. <em>Appl Phys Lett</em> 111:234102.</li> <li style="margin-left: 2.85pt;">Smith AJ, O&#39;Rorke RD, Kale A, Rimsa R, Tomlinson MJ, Kirkham J, Davies AG, <span class="underline_text">W&auml;lti C</span>, Wood CD (2017). Rapid cell separation with minimal manipulation for autologous&nbsp; cell therapies. <em>Sci Rep</em> 7:41872.</li> </ul> <p style="margin-left: 2.85pt;">This project is available as part of the <a href="https://medicinehealth.leeds.ac.uk/leeds-institute-research-st-james/doc/international-phd-academy-medical-research">International PhD Academy: Medical Research</a></p> <p style="margin-left: 2.85pt;"><strong>In line with the bespoke nature of our International PhD Academy a modified PhD project can be proposed dependent on students interests and background.</strong></p>

<p>Please note these are not standalone projects and applicants must apply to the PhD academy directly.</p> <p>Applications can be made at any time. You should complete an <a href="https://medicinehealth.leeds.ac.uk/faculty-graduate-school/doc/apply-2">online application form</a> and attach the following documentation to support your application.&nbsp;</p> <ul> <li>a full academic CV</li> <li>degree certificate and transcripts of marks (or marks so far if still studying)</li> <li>Evidence that you meet the programme&rsquo;s minimum English language requirements (if applicable, see requirement below)</li> <li>Evidence of funding to support your studies</li> </ul> <p>To help us identify that you are applying for this project please ensure you provide the following information on your application form;</p> <ul> <li>Select PhD in Medical Research as your programme of study</li> <li>Give the full project title and name the supervisors listed in this advert</li> </ul>

You should hold a first degree equivalent to at least a UK upper second class honours degree in a relevant subject. A Masters degree in a relevant subject may also be required in some areas of the Faculty. For entry requirements for all other research degrees we offer, please contact us.

Applicants whose first language is not English must provide evidence that their English language is sufficient to meet the specific demands of their study. The minimum requirements for this programme in IELTS and TOEFL tests are: &bull; British Council IELTS - score of 7.0 overall, with no element less than 6.5 &bull; TOEFL iBT - overall score of 100 with the listening and reading element no less than 22, writing element no less than 23 and the speaking element no less than 24.

<p>Informal enquires about regarding the bespoke taught first year of the PhD programme and research projects can be made by contacting LIMRPhD@leeds.ac.uk.</p> <p>Enquiries regarding the application process should be directed to the Faculty of Medicine and Health Graduate School e: <a href="mailto:fmhpgradmissions@leeds.ac.uk">fmhpgradmissions@leeds.ac.uk</a></p>