Understanding skeletal diseases using human induced pluripotent stem cells

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

We have generated iPSCs from patients with two closely related rare skeletal dysplasias, multiple epiphyseal dysplasia and pseudoachondroplasia caused by mutations in matrix proteins, Matrilin3 (MATN3) or COMP. We have differentiated these through a mesenchymal stromal cell route to chondrocytes to produce growth plate-like cartilage pellets. Pellets from MATN3 mutant iPSCs are larger than related healthy pellets, with altered expression of cartilage matrix proteins, transcription factors (also for COMP mutants) and increased sensitivity to BMP2 . Building on this we will generate further MATN3 and COMP mutant iPSC lines, then using CRISPR-Cas9 gene editing correct and create the mutations in mutant and healthy iPSC lines respectively, to eliminate molecular changes due to other genetic factors. RNAseq will reveal aberrant molecular pathways of COMP and MATN3 mutant chondrocytes. Mutant cartilage pellets show regional differences from wt; the nature of which will be evaluated by laser capture-RNAseq, compared with cartilage and validated using qRT-PCR, RNAscope multiplex in situ hybridisation and immunocytochemistry (for protein). We will determine if the phenotype is caused by abnormal protein within the cell or abnormal matrix and signalling outside the cell by 1) BMP2 interaction analyses with mutated and wt MATN3, and COMP if implicated in BMP signalling; 2) determining the effect of mutant protein on wt cells by i) adding purified mutant MATN3 or COMP to wt cells, ii) mixing chondroprogenitors overexpressing mutant protein with wt cells carrying a BMP-Smad1(or other appropriate) reporter; 3) determining structural differences in the extracellular matrix by serial blockface-SEM imaging/ electron tomography. We will integrate all data, validating the role of key aberrant pathways using knock down and small molecule/antibody inhibition. We will determine similarities between MATN3 and COMP mutations and suggest new drug targets for alleviation of patient symptoms