In vitro modelling of craniofacial disorders using iPS cells

Background
Collectively, up to 35% of all birth defects are craniofacial. A subgroup of these patients have syndromic craniosynostosis, which is characterised by the premature fusion of cranial sutures [1]. Although mutations in the FGFR2 gene have been identified in many of these patients, major questions remain unanswered about the molecular and developmental pathogenesis. In particular, it is still not known how dysregulated Fibroblast Growth Factor (FGF) signalling leads to the skeletal defects that characterise these syndromes.

Work in our laboratory has focussed on a mouse model for Crouzon syndrome [2] that carries a missense mutation (p.C342Y) in the gene encoding FGFR2. We have found skeletal dysmorphology of cartilage and bone in the C342Y mutant that mimics the human condition, including craniosynostosis of the coronal suture and cleft palate [3]. Specifically, mutant skulls display cartilage hyperplasia as well as bone hypoplasia. A cell fate switch may underpin the pathogenesis of the disorder.

Stem cells have been identified in the sutural mesenchyme as well as the intramembranous periosteum of the skull [4,5]. Periosteal stem cells are capable of forming mesenchymal cartilage and bone precursors. Our hypothesis is that FGF signalling is essential for the correct differentiation of these stem cells. Misregulation of the FGF signalling pathway by way of FGF receptor perturbation will lead to abnormal mesenchymal differentiation and skeletal defects.

Aims/Objectives
To test our hypothesis we aim to perform a number of experiments as part of this PhD project:

• Using an established human induced pluripotent stem (iPS) cell line and CRISPR-Cas9 gene editing we will introduce FGFR2 mutations associated with craniosynostosis. These cell lines will be induced to form either bone or cartilage in vitro, and differentiation assays will be used to determine the impact of the mutation on their osteogenic and chondrogenic potential. Analysing gene expression profiles at different stages of differentiation, with or without mutation, using (single-cell) RNAseq can identify specific genes or pathways that play a major role during these processes.
• Alongside, we will extract mouse fibroblasts from our FGFR2-C342Y mouse model and convert them into iPS cells. Similar to above, we can differentiate these into bone and/or cartilage and analyse their respective gene expression profiles.
• Findings from both these experiments will be validated in vivo using our mouse model of craniosynostosis. Genes or pathways identified above will be tested for their role during embryonic formation of bone and cartilage in the skull as well as during the postnatal period when suture homeostasis is critical to keep the skull able to accommodate brain growth.
• Additional ex vivo techniques involve extracting mouse osteo/chondroprogenitor mesenchymal cells and culture these in vitro. Using these cells will allow us to assess the impact of small molecule pathway inhibitors for their therapeutic potential.

Ultimately, we hope that this project will make a significant contribution towards the improved understanding of the molecular and cellular events leading to craniofacial dysmorphology in patients and can develop stem cell-based clinical interventions to prevent and/or treat craniosynostosis and associated skeletal defects.

References
1. Wilkie, A.O., et al., Prevalence and complications of single-gene and chromosomal disorders in craniosynostosis. Pediatrics, 2010. 126(2): p. e391-e400.
2. Lee KKL, Stanier P, Pauws E. Mouse Models of Syndromic Craniosynostosis. Mol Syndromol, 2019 Feb;10(1-2):58-73.
3. Peskett, E., et al., Analysis of the Fgfr2C342Y mouse model shows condensation defects due to misregulation of Sox9 expression in prechondrocytic mesenchyme. Biol. Open, 2017. 6(2): p. 223-231.
4. Zhao H, et al., The suture provides a niche for mesenchymal stem cells of craniofacial bones. Nat Cell Biol, 2015Apr;17(4):386-96.
5. Debnath S, et al., Discovery of a periosteal stem cell mediating intramembranous bone formation. Nature, 2018 Oct;562(7725):133-139.