Understanding skeletal diseases using human induced pluripotent stem cells

Multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH) are clinically similar skeletal diseases, involving short stature and osteoarthritis at a young age. MED and PSACH can be caused by mutations in two extracellular matrix proteins matrilin-3 and cartilage oligomeric matrix protein (COMP). We made stem cells by reprogramming blood cells from patients with MED and PSACH, and developed a method to produce cartilage from these stem cells in a dish. Thus we produced a tractable model for analysis of the mechanisms underlying the pathology in these diseases which can be compared with cartilage from people with other skeletal diseases or osteoarthritis. We have found that the chondrocytes (cells within cartilage) produced from patients with skeletal disease respond differently to those produced from healthy people. Our aim is to now build on these data to understand the molecular mechanisms underlying these skeletal diseases and to identify potential therapeutic drugs. In this project, we will generate additional stem cells [known as induced pluripotent stem cells (iPSCs)] from other patients with MED and PSACH and as controls their healthy relatives. We need to look at several different families to make sure the symptoms are not caused by other genetic factors than the diagnosed mutation. We will also use a technique called gene editing to generate the same disease causing mutations in healthy iPSCs also allowing us to determine that the differences we see are caused by the mutation. Further, we will correct mutations in disease iPSCs, after which the formation of cartilage by those cells should be the same as cartilage from healthy stem cells. We will then perform a deep analysis of the molecules in chondrocytes and cartilage produced by both mutant and healthy stem cells. This will include looking at all the RNA molecules that code for protein (by a technique called RNA-Seq) and evaluating different regions of the cartilage pellets from the disease iPSC-chondrocytes, and comparing these to healthy cartilage produced in our culture system and to adult cartilage. Advanced ultrastructural analysis will be used to reveal structural changes in cartilage proteins contributing to the differences seen in the patients. We will also identify and quantify how protein interactions, particularly that of a small protein which promotes cartilage formation (BMP-2), differ between healthy and mutant Matrilin-3, for which we have preliminary data and find out if this is also the case for COMP mutation. Based on our findings, we will select drugs to correct the disease phenotype of the mutant cells and experimentally induce it in healthy stem cell derived cartilage to confirm the identified mechanism(s) of pathology. This structural and mechanistic analysis will increase general understanding of cartilage development and disease, and help identify new drug targets for MED and PSACH and potentially for a subset of patients with osteoarthritis.

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

This research will impact both clinicians and academic researchers in the areas of RSDs and osteoarthritis (OA). It will impact those working in the field of regenerative medicine, particularly in musculosketal diseases, a declared MRC priority. By developing this human in vitro model for cartilage formation from iPSCs it will help to reduce animal usage as researchers are able to move away from transgenic animals and adopt a human culture system for studies involving formation of cartilage. Our work will have impact on individuals with skeletal conditions which predispose to joint diseases like OA and the clinicians treating them: OA presents a significant clinical challenge with important socioeconomic costs, its incidence is increasing and it is estimated to cost the NHS £850 million annually. Our focus on clinically similar diseases of known genetic origin; multiple epiphyseal dysplasia (MED: prevalence 1 in 20,000) and pseudoachodroplasia (PSACH: prevalence 1 in 30,000), which cause early onset OA, will inform research into those specific diseases, but the methods and the data should also be more widely applicable to research on other rare skeletal dysplasias. We anticipate our research will suggest new treatments for those suffering from MED and PSACH. The validated new human culture models will also facilitate the development of new treatments for other RSDs with the assistance of biopharma and clinical collaborations and interactions. We will publish our research in high impact, open access journals and present our findings in international and national conferences. We will utilise our University of Manchester Research Business Managers and Intellectual Property Company to enable tech transfer in relation to possible therapeutic interventions or other translatable findings, making early contact with current and potential business partners in the first year with follow up in later years as the research progresses. Engagement with the public will span the age range with inclusion of school students though hosting year 3-13 students at different events and giving talks to adults at large and small events including patient groups. We will utilise our Media Relations Officer for dissemination of key findings through press releases or similar outward media reporting.