Rheumatology and Metabolic Medicine

Rheumatology and Metabolic Medicine - Research

RHEUMATOLOGY RESEARCH

Members of the Research Team:

Dr Oksana Kehoe (Lead), Mr Andrei Stefan, Miss Rebecca Morgan and Miss Mairead Hyland

Clinical Support: Dr Roshan Amarasena (Lead), Dr Rob Butler and Dr Ayman Askari, Professor Jim Middleton (Honorary Clinical Scientist)

Our team has continued to be active this year carrying out basic science work into understanding mechanisms of rheumatoid arthritis (RA) progression and possible treatments.

We have been publishing our findings in Scientific Reports, Stem Cells International and the Journal of Immunology.  We have presented our work at several national and international meetings such as FIRM Symposium, Girona (Spain), September 2017; 11th UK MSC Meeting, Chester, September 2017; 11th Oswestry Cartilage Symposium, October 2017; British Society for Immunology Congress, Brighton, December 2017 and the Annual Research day at the RJAH, April 2018.

The projects which we are currently working are explained below.

Stem Cell Conditioned Medium as a Therapeutic Agent in Arthritis

Alasdair Kay, Andrei Stefan, Jim Middleton (Bristol University), Oksana Kehoe

Funded by Orthopaedic Institute Ltd, Dowager Countess Eleanor Peel Trust, and Oswestry Rheumatology Association

Alasdair completed work on understanding the therapeutic potential of mesenchymal stem cell conditioned medium (CM-MSC) and published a paper in Scientific Reports (Figure 1). The key message is conditioned medium may provide an effective cell-free therapy for inflammatory arthritis.

 Figure 1. Recently published paper.

Evaluation of Mesenchymal Stem cell-derived extracellular vesicles as a therapy for Rheumatoid Arthritis

Alasdair Kay, Rebecca Morgan, Andrei Stefan, Oksana Kehoe

Funded by the Orthopaedic Institute Ltd, RJAH Orthopaedic Hospital Charity and Oswestry Rheumatology Association  

Rheumatoid arthritis is a painful autoimmune disorder characterised by synovial inflammation that leads to progressive destruction of articular cartilage.  There is no cure for RA and life expectancy of sufferers may be reduced by up to 18 years. For that reason, it is vital to develop a new and more effective therapy for RA. Mesenchymal stem cells (MSCs) possess anti-inflammatory and immunosuppressive properties and function predominantly through paracrine mechanisms, via growth factors, cytokines, chemokines and bioactive, membrane-bound extracellular vesicles (EVs) found in mesenchymal stem cell conditioned medium. Our previous project demonstrated that CM-MSC reduces swelling and cartilage destruction through modulation of the immune response in a murine model of inflammatory arthritis. This current study is assessing the contribution of extracellular vesicles to this therapeutic outcome, and examining how the culture microenvironment of MSCs can be manipulated to enhance their therapeutic effectiveness. Extracellular vesicles are applied as a treatment in our pre-clinical model of experimental arthritis, looking at their effects on inflammation, histological outcomes of disease progression and T cell polarisation towards either pro- or anti-inflammatory cell types.

Stem cells isolated from human bone marrow aspirate were used to derive extracellular vesicles. Following the application of vesicles, knee joint swelling significantly reduced at both 24 and 48 hours after treatment in our pre-clinical model (Figure 2A). Reductions were highly significant in comparison to control animals where knee joints continued to swell in the absence of extracellular vesicles. Histological analysis reviled improved scores for cartilage and arthritis index (Figure 2B). 

The significant reduction in joint swelling following EVs treatment demonstrates their effectiveness as a new therapeutic intervention. Further investigation will identify the mechanisms involved. Cell free treatments such as EVs have advantages over living cells and conditioned medium, including having a membrane that protects constituents from degradation. These benefits might aid more rapid translation from research in the laboratory to clinical use.

Figure 2. The effects of intra-articular injections of EVs in AIA model. (A) Change in knee diameter (swelling) of AIA mice following EVs treatment (or control) shows significantly greater alleviation of swelling at days 2 and 3 post arthritis induction; (B) Significant reductions in the histopathological symptoms of AIA, including cartilage depletion and arthritis index.

The in vitro effect of syndecan-3 gene knockout on bone marrow derived mesenchymal stem cells’ properties

Andrei Stefan, Alasdair Kay, Nick Forsyth, Fiona Jones*, Dada Pisconti*, Oksana Kehoe*University of Liverpool

Funded by Orthopaedic Institute Ltd, Keele University School of Medicine, Oswestry Rheumatology Association

Andrei previously demonstrated that the absence of syndecan-3(Sdc-3) molecule has a significant on stem cell morphology (Figure 3A)

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Figure 3. (A) Morphometric results – the spread surface area of cells lacking syndecan-3 is significantly lower than the one of wild type cells; (B) Changes in signalling pathways in Sdc-3 -/- cells.

 

Recently, Andrei used biochemical assays to investigate which signalling pathways are affected by syndecan-3 loss in mMSCs. The morphological changes were confirmed by the significant reduced cell surface spread of cells lacking syndecan-3 and appeared to be driven by changes in cell signalling pathways (Figure 3B).  

 

Uncovering the role of proteoglycans in exosome biogenesis for improved rheumatoid arthritis therapies

Rebecca Morgan, Cathy Merry*, Oksana Kehoe*University of Nottingham

Funded by EPSRC Centre for Doctoral Training in Regenerative Medicine

Rebecca’s PhD work focused on a particular component of cells conditioned medium known as extracellular vesicles (exosomes). These are membrane-bound vesicles released from cells including MSCs. They contain many proteins, lipids and RNA collectively known as cargo.  It may be possible to increase exosome production and alter their cargo (which defines the activity of the exosomes), to provide more effective rheumatoid arthritis therapies. This could be done by altering a proteoglycan called syndecan which is found on the surface of MSCs and their exosomes.

Work carried out to date, involves isolation of exosomes from conditioned medium of MCF-7 cell line. The size of isolated exosomes was determined by transmission electron microscopy (TEM) (Figure 4).

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Figure 4. Characterisation of exosomes isolated from MCF-7s.Histogram was generated from a total of 568 particle diameters. Representative electron microscopy image demonstrates the range of exosome sizes (arrows). Scale bar= 200 nm.

MCF-7s were also shown to have positive staining for heparan sulphate on their surfaces as confirmed by flow assay using specific antibody (Figure 5).

Figure 5. Expression of heparan sulphate on MCF-7 surfaces using 10E4 staining.

 

Mesenchymal Stem Cell-Derived Extracellular Vesicles as Therapeutic Agents for Juvenile Idiopathic Arthritis

Mairead Hyland, Claire Mennan, Karina Wright, Karen Davies*, Emma Wilson#, Aled Clayton*#, Oksana Kehoe *Royal Wolverhampton NHS Trust and the RJAH Orthopaedic Hospital NHS Foundation Trust; #University of Chester; *#Cardiff University

Funded by Funded by the Orthopaedic Institute Ltd, RJAH Orthopaedic Hospital Charity Keele University School of Medicine

Chronic arthritis of children, known as juvenile idiopathic arthritis (JIA), affects 1 in 1000 children, and one of the leading causes of childhood-acquired disability.  JIA is a complex inflammatory disease with a multifactorial immune pathogenesis. The standard first line of management is to use disease modifying anti-rheumatic drugs, however, a third of patients do not respond, and during this time, these children suffer pain, fatigue and loss of quality of life and may develop growth abnormality and disability. 

Mairead Hyland started her PhD project last October with aim to determine if extracellular vesicles derived from conditioned media of human bone marrow mesenchymal stem cells (MSCs) and umbilical cords stem cells (UC-MSCs) can supress proliferation of activated immune cells taken from the blood of patients with JIA.

Work carried out to date, involves isolating human MSCs from bone marrow aspirates (commercial source, Lonza). Cells were characterised by the ability to produce bone, cartilage and fat (typical trait of MSCs).

Work has also been carried out to isolate extracellular vesicles from human MSCs and UC-MSCs conditioned media. This was done using differential centrifugation where the conditioned media was subjected to multiple centrifugation steps with increasing speeds and time. Successful isolation of exosomes was determined using flow cytometry where markers including typical vesicles surface proteins (CD9, CD61 and CD83) were assessed.