Developmental engineering models of skeletal ciliopathies.

Project No: #OxKEN-2023/23
Intake: OxKEN 2023

project overview

Mutations in genes associated with the primary cilium underlie the congenital ciliopathies. An expanding collective of mutations have been associated with skeletal ciliopathies [1]. For example, mutations in IFT80, a protein component of the intraflagellar transport (IFT) system cause Jeune asphyixiating thoracic dystrophy (JATD) [2] and short rib polydactyly (SRP) type III. Both diseases are autosomal recessive chondrodysplasia and share clinical and radiological similarities. These include long bone shortening and constriction of the thoracic cage, the result of impaired endochondral ossification. Murine [3] and cell models have begun to build our molecular and cellular understanding of these disorders, but many open questions remain.

As part of a programme seeking to understand the biology of limb morphogenesis [4] and pathology, the Wann group is already exploiting a developmental engineering approach to model endochondral ossification. The project will exploit a validated chondro-osseous, scaffold supported organoid model [5], seeded with mouse and human mesenchymal stem cells carrying CRISPR edits (disease-causing mutations) and/or patient-derived iPSC. The model transitions from stem-cartilage-bone enabling a qualitative and quantitative tracking and assessment of molecular, cellular and matrix changes associated with mineralisation. It will be used here to model skeletal disorders such as JATD and SRP to understand disease mechanisms and, in the longer term, offer platforms for therapeutic screening. This work will run in parallel to collaborative analysis of IFT80 mouse models (UCL Cilia disorders laboratories). Other synergistic projects use the model to understand fundamental limb mechanobiology and other chondropathies including osteoarthritis.

The project will exploit the model to test the hypothesis that chondro-hypertrophic-bone transition mechanisms regulated by primary cilia underpin faulty endochondral ossification in JATD/SRP type III

Using this in vitro model of endochondral transitions this studentship will:

1 Generate CRISPR mutant (IFT80) stem cells/ explore iPSC use.
2 Validate in vitro tissue model of skeletal ciliopathies, comparing with in vivo mouse models.
3 Explore the molecular, cellular and matrisomal phenomena associated with impaired endochondral transitions, modelling JATD/SRP type III.
4 Trial gene therapy/small molecule interventions in growth plate on a chip model (With Emulate centre).

keywords

Organoid, Chondrodysplasia, Mineralisation, Ciliopathy, Organ-on-Chip

Figure 1. Stem cells seeded with GelMA scaffold (left). Middle images: Buoyancy established gradient and (Right) histological section of chondro-osseous model [5].

training opportunities

The successful candidate will be embedded within the Centre for OA Pathogenesis Versus Arthritis at the Kennedy Institute of Rheumatology, directed by Professor Vincent. They will benefit from supervision by an experienced team of clinician and basic scientists interested in the cell biology of MSK disease. They will work closely with genetics and clinical (ciliopathy) expertise at UCL, Cilia disorders laboratories and collaborate with Emulate Organ-on-a-chip predictive models centre.

You will be based in the laboratories of the Kennedy Institute of Rheumatology, a world-leading centre in the fields of tissue biology, inflammation, and repair, with a strong emphasis on clinical translation. The project will use a combination of human stem cells carrying disease-causing mutations and an in vitro developmental engineering model. There is support available from post-doctoral scientists and laboratory managers in our groups. In summary, you will be working within:

Cutting-edge cell and tissue biology, imaging and next generation sequencing techniques available in-house, including 3D tissue culture, multi-channel immunohistochemistry analysed using world-class imaging facilities including light sheet microscopy and single cell RNA-sequencing analysis
Strong translational environment.
Well-established DPhil programme with defined milestones, ample training opportunities within the University and Department, and access to university/department-wide seminars by world-leading scientists
Highly collaborative local environment and opportunities to participate in several other clinical and bioengineering collaborations with UCL and Emulate centre for predictive models at QMUL.

key publications

Huber, C. and V. Cormier-Daire, Ciliary disorder of the skeleton. Am J Med Genet C Semin Med Genet, 2012. 160C(3): p. 165-74.
Beales, P.L., et al., IFT80, which encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic dystrophy. Nat Genet, 2007. 39(6): p. 727-9.
Rix, S., et al., An Ift80 mouse model of short rib polydactyly syndromes shows defects in hedgehog signalling without loss or malformation of cilia. Hum Mol Genet, 2011. 20(7): p. 1306-14.
Coveney, C.R., et al., Ciliary IFT88 Protects Coordinated Adolescent Growth Plate Ossification From Disruptive Physiological Mechanical Forces. J Bone Miner Res, 2022.
Li, C., et al., Buoyancy-Driven Gradients for Biomaterial Fabrication and Tissue Engineering. Adv Mater, 2019. 31(17): p. e1900291.