Understanding skeletal diseases using human induced pluripotent stem cells
Lead Research Organisation:
University of Manchester
Department Name: School of Biological Sciences
Abstract
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.
Technical Summary
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
Planned Impact
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.
Publications
Cain SA
(2022)
ADAMTS6 cleaves the large latent TGFß complex and increases the mechanotension of cells to activate TGFß.
in Matrix biology : journal of the International Society for Matrix Biology
De Kinderen P
(2022)
Differentiation of Induced Pluripotent Stem Cells Into Chondrocytes: Methods and Applications for Disease Modeling and Drug Discovery.
in Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Ferreira MJS
(2022)
Pluripotent stem cells for skeletal tissue engineering.
in Critical reviews in biotechnology
Fonseca AC
(2020)
Emulating Human Tissues and Organs: A Bioprinting Perspective Toward Personalized Medicine.
in Chemical reviews
Griffiths R
(2020)
The Transcription Factor-microRNA Regulatory Network during hESC-chondrogenesis.
in Scientific reports
Hopton C
(2022)
Characterization of the mechanism by which a nonsense variant in RYR2 leads to disordered calcium handling.
in Physiological reports
Humphreys PA
(2020)
Optogenetic Control of the BMP Signaling Pathway.
in ACS synthetic biology
Humphreys PA
(2022)
Developmental principles informing human pluripotent stem cell differentiation to cartilage and bone.
in Seminars in cell & developmental biology
Humphreys PEA
(2023)
Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs.
in Cell reports
Description | 21EBTA Driving Pluripotent Stem Cell Osteogenesis with Light for Tissue Engineering |
Amount | £355,915 (GBP) |
Funding ID | BB/W013940/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 02/2022 |
End | 01/2024 |
Description | MRC Confidence in Concept |
Amount | £58,510 (GBP) |
Organisation | University of Manchester |
Sector | Academic/University |
Country | United Kingdom |
Start | 11/2018 |
End | 07/2019 |
Description | Understanding Acrodysostosis type 1 and 2 through a pluripotent stem cell-disease model. |
Amount | £720,311 (GBP) |
Funding ID | MR/X002020/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2022 |
End | 03/2026 |
Description | Understanding an endogenous mechanism that protects against osteoarthritis; towards a new paradigm for disease management |
Amount | £1,315,313 (GBP) |
Funding ID | 22277 |
Organisation | Versus Arthritis |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 01/2020 |
End | 04/2024 |
Title | Constructs for light driven differention |
Description | Light driven BMPR1a and 1b constructs for driving human stem cell development |
Type Of Material | Technology assay or reagent |
Year Produced | 2024 |
Provided To Others? | Yes |
Impact | Too early |
Description | Sarah Teichman |
Organisation | The Wellcome Trust Sanger Institute |
Department | Human Genetics |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We collaborate with the Sanger over single cell RNAseq comparison between our protocols for chondrogenesis and fetal limb cells |
Collaborator Contribution | Singel cell RNAseq and bioinformatic expertise. Single cell fetal limb dataa |
Impact | Paper under review |
Start Year | 2020 |
Description | comparing hPSC-cartilage to limb cartilage |
Organisation | The Wellcome Trust Sanger Institute |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | Materials protocols data |
Collaborator Contribution | Protocols data |
Impact | Data and a paper will result |
Start Year | 2020 |
Description | Faraday Lecture Cambridge |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Faraday Public Lecture |
Year(s) Of Engagement Activity | 2019 |
Description | Knutsford Sci bar |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Presentation and discussion on developments an uses in Pluripotent stem cell research. Discussed therpeutic uses in e,g, drug development anf testing and cell based therapy. |
Year(s) Of Engagement Activity | 2023 |
Description | Liverpool Medical Institue Symaposium on Stem Cell Regeneration and Osteoarthritic Tissue |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | Talk on use of stem cells in orthopedics by Dr Chris Smith (Pdra) |
Year(s) Of Engagement Activity | 2019 |
Description | Patient information talk Manchester |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Patients, carers and/or patient groups |
Results and Impact | Informationa dn exchange talk with teh RUG group associated with a osteo and rheumatoid arthritis in Central Manchester Trust |
Year(s) Of Engagement Activity | 2019 |
Description | School 6th form talk 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Atalk given to 5, 6th form groups about stem cells and medicine |
Year(s) Of Engagement Activity | 2019 |
Description | School 6th form talk 2019 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Talk about Stem Cells and regenerative medicine to 5 6th form groups at local school |
Year(s) Of Engagement Activity | 2019 |
Description | Scibar Disbury |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Talk about the uses and developments in pluripotent stem cell biology anf the potential for drug testing and cell therapy |
Year(s) Of Engagement Activity | 2023 |
Description | South Africa public engagement |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Series of talks in cities and a rural community in South Africa to community, university and church groups |
Year(s) Of Engagement Activity | 2020 |
Description | Stem cells magic for future medicine U3A |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | U3A and other organisations in area Public engagement and discussion about future medicine |
Year(s) Of Engagement Activity | 2019 |