Developing and testing a humanised mouse model of fibrosis
- Project No: #OxKEN-2023/11
- Intake: OxKEN 2023
Project overview
Fibrosis is a final common pathway of disease in many organ system, and chronic fibroproliferative diseases are estimated to be responsible for 45% of all natural deaths in economically developed countries. However, there are no widely available therapeutics that target fibrosis, partly due to a poor understanding of the pathological process across different organs, and crucially a lack of animal models that accurately recapitulate human disease. In particular, there is a complex interaction between fibrotic tissue foci and the immune system that has not been previously modelled.
In this project, the student will use the combined expertise of the supervisory team to develop and test new humanised mouse models of two common fibrotic conditions – Dupuytren Disease and Keloid Scarring. There will be an opportunity to be trained in a very wide variety of laboratory techniques, and advanced data analytics.
Organ specific fibroses cause both morbidity and mortality. Dupuytren disease (DD) is a progressive fibroproliferative disease of the palmar fascia of the hand affecting 1-5% of adults. It causes flexion contractures of the involved digits, functional impairment and reduced quality of life. Keloid scarring (KS) is a fibroproliferative disorder of the skin, characterised by excessive, invasive scar formation after skin injury. It is more common in darkly pigmented skin, and in certain anatomical locations.
The student will develop and characterise a new model of DD and KS, using subcutaneous placement of excised tissue from patients in a humanised immune system (HIS) mice. They will implant a 5mm3 piece of DD nodule under the under the flank, and harvest the tissue at 1, 3, and 6 weeks (n=3 per timepoint). They will analyse harvested DD tissues for engraftment, looking for vascular ingrowth and cell survival using qPCR and immunofluorescence microscopy from. They will then repeat the time-course using DD tissue and control tissue after re-constituting the immune system of the HIS mice with peripheral blood mononuclear cells (PBMCs) from the donor patient (n=6 disease and control per timepoint). They will determine immune infiltration using lymphocyte (CD3, CD4, CD8, FOXP3, CD19) and monocyte (CD14, CD16) markers by immunohistochemistry as well as examining general tissue architecture. They will use single cell RNA sequencing of the engrafted tissue, comparing it to the tissue processed directly from patients to demonstrate the survival of each of the cellular components of the tissues from the model with and without reconstitution of mice with donor PBMCs. Phenotypic and transcriptomic data will be compared to published datasets of fibrotic disease to determine overlap with human pathology. Further detailed characterisation of the immune cell components of the humanised mouse model and freshly harvested human samples will be undertaken. Finally, they will culture cells from engrafted tissue and perform functional assays, such as comparing cellular contraction, migration assays, and wound healing assays. Establishment of this model will provide a useful platform for the assessment of anti-fibrotic therapies which may also be possible in the course of this project.
Keywords
Fibrosis, Humanised Mouse Model, Single Cell Sequencing
training opportunities
The Botnar Research Centre plays host to the University of Oxford's Institute of Musculoskeletal Sciences, which enables and encourages research and education into the causes of musculoskeletal disease and their treatment. A core curriculum of lectures will be taken in the first term to provide a solid foundation in a broad range of subjects including musculoskeletal biology, inflammation, epigenetics, translational immunology, data analysis and the microbiome. All students are required to attend a 2 - day Statistical and Experimental Design course at NDORMS. The student will attend regular seminars within the department and those relevant in the wider University.
The student will receive training in relevant related research methodologies including cell culture, in vivo techniques (including humanised mouse systems and surgical techniques), immunohistochemistry, molecular techniques, flow cytometry, and the handling and analysis of single cell sequencing datasets, and cross species analysis.
Additional on the job training opportunities will arise, and the supervisors will encourage the student to pursue such opportunities. Attendance at formal training courses will be encouraged. In addition, courses from the Oxford Learning Institute and the Oxford University Computer Sciences on generic skills for scientific research will be available and encouraged. Students will be expected to present data regularly in the departmental PGR seminars, Furniss group meetings, and to attend external conferences to present their research globally.
key publications
- Layton TB, Williams L, McCann F, Zhang M, Fritzsche M, Colin-York H, Cabrita M, Ng MTH, Feldmann M, Sansom SN, Furniss D, Xie W, Nanchahal J. Cellular census of human fibrosis defines functionally distinct stromal cell types and states. Nat Commun. 2020 Jun 2;11(1):2768. doi: 10.1038/s41467-020-16264-y.PMID: 32488016
- Ng M, Thakkar D, Southam L, Werker P, Ophoff R, Becker K, Nothnagel M, Franke A, Nürnberg P, Espirito-Santo AI, Izadi D, Hennies HC, Nanchahal J, Zeggini E, Furniss D. A Genome-wide Association Study of Dupuytren Disease Reveals 17 Additional Variants Implicated in Fibrosis. Am J Hum Genet. 2017 Sep 7;101(3):417-427.
- Adigbli G, Hua P, Uchiyama M, Roberts I, Hester J, Watt SM, Issa F. Development of LT-HSC-Reconstituted Non-Irradiated NBSGW Mice for the Study of Human Hematopoiesis In Vivo. Front Immunol, 2021. doi: 10.3389.fimmu.2021.642198.
- Adigbli G, Menoret S, Cross AR, Hester J, Issa F, Anegon I. Humanization of Immunodeficient Animals for the Modeling of Transplantation, Graft Versus Host Disease, and Regenerative Medicine. Transplantation, 2020. doi: 10.1097/TP.0000000000003177.
- Allen RJ, Guillen-Guio B, Oldham JM, Ma SF, Dressen A, Paynton ML, Kraven LM, Obeidat M, Li X, Ng M, Braybrooke R, Molina-Molina M, Hobbs BD, Putman RK, Sakornsakolpat P, Booth HL, Fahy WA, Hart SP, Hill MR, Hirani N, Hubbard RB, McAnulty RJ, Millar AB, Navaratnam V, Oballa E, Parfrey H, Saini G, Whyte MKB, Zhang Y, Kaminski N, Adegunsoye A, Strek ME, Neighbors M, Sheng XR, Gudmundsson G, Gudnason V, Hatabu H, Lederer DJ, Manichaikul A, Newell JD Jr, O'Connor GT, Ortega VE, Xu H, Fingerlin TE, Bossé Y, Hao K, Joubert P, Nickle DC, Sin DD, Timens W, Furniss D, Morris AP, Zondervan K, Hall IP, Sayers I, Tobin MD, Maher TM, Cho MH, Hunninghake GM, Schwartz DA, Yaspan BL, Molyneaux PL, Flores C, Noth I, Jenkins RG, Wain LV. Genome-Wide Association Study of Susceptibility to Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med. 2019 Nov 11.