Mesenchymal stem cells (MSCs) offer huge potential in regenerative medicine and we propose their use in treatment of intervertebral disc degeneration (IVDD), a cause of back pain. IVDD is driven by increased expression of pro-inflammatory factors, primarily IL-1. Our preliminary data suggests that MSCs secrete anti-inflammatory factors (including IL-1 receptor antagonist and TSG-6), suggesting that, in addition to their differentiation potential in regenerative strategies, MSCs may modulate the cytokine milieu of the degenerate IVD.
Implantation of MSCs into degenerate discs may aid regeneration via secretion of anti-inflammatory factors which can reduce catabolic cytokine levels thereby restoring a healthy, anabolic phenotype in resident NP cells. This will be tested utilizing established in vitro co-culture models.
The objectives are to:
1. To investigate the effect of co-culture on the phenotype of degenerate human IVD cells and human MSCs;
2. To identify the key mediators (specifically the anti-inflammatory and anti-catabolic molecules) produced during co-culture;
3. To identify the mechanisms by which bi-directional communication occurs between IVD cells and MSCs.
The study will employ primary human cell culture, including 2D and 3D mono-cultures and co-cultures of MSCs and IVD cells. Phenotype will be assessed using quantitative real-time PCR and RNASeq next-generation transcriptomic profiling. Western blotting, flow cytometry/cell sorting and multiplex (Luminex) ELISA methods will be employed to determine expression levels of known anti-inflammatory/anti-catabolic proteins. Mass spectrometry proteomics will be employed to identify novel factors. Confocal live-cell imaging and SEM will be utilised to investigate mechanisms of cellular interaction, including tunneling nanotubes and microvesicle release.
This project will enhance our understanding of the anti-inflammatory potential of MSCs and the mechanisms which control the bi-directional interactions between cells. Findings will inform the development of clinically-viable MSC-based therapies for IVDD and will have implications for use of MSCs in other regenerative therapies.
This multidisciplinary project brings together expertise in a range of cell types and analytical methodology. The student will gain extensive experience in primary human cell culture, including complex co-culture model systems. A wide range of imaging techniques will be employed to visualise cell-cell interactions, including state-of-the-art long-term live cell confocal imaging. Flow cytometry and FACS will be used to analyse and sort cell sub-populations/cell types. Gene and protein analyses will be extensively performed and the student will be exposed to new –omics technologies (RNASeq transcriptomics and MS proteomics). Thus this project will provide the student with a skill set that enables them to succeed in a laboratory-based career.
Applicants are expected to hold, or about to obtain, a minimum upper second class undergraduate degree (or equivalent) in a subject related to biological sciences. A masters degree, with research experience, in stem cells and regenerative medicine, or biomaterials or would beneficial.
This 3-year full-time PhD is open to candidates able to provide evidence of self-arranged funding/ sponsorship and is due to commence from January 2017 onwards.
Any enquiries relating to the project and/or suitability should be directed to Dr Richardson ([email protected]
). Applications are invited on an on-going basis but early expression of interest is encouraged.
1) Richardson SM, Walker RV, Parker S, Rhodes NP, Hunt JA, Freemont AJ, Hoyland JA. Intervertebral disc cell mediated mesenchymal stem cell differentiation. Stem Cells 2006; 24(3):707-16.
2) Strassburg S, Richardson SM, Freemont AJ, Hoyland JA. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regenerative Medicine. 2010;5(5):701-11.
3) Strassburg S, Hodson NW, Hill PI, Richardson SM, Hoyland JA. Bi-directional Exchange of Membrane Components Occurs during Co-culture of Mesenchymal Stem Cells and Nucleus Pulposus Cells. PLoS ONE. 2012;7(3):e33739.
4) Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA. Characterization of the human nucleus pulposus cell phenotype and evaluation of novel marker gene expression to define adult stem cell differentiation. Arthritis and Rheumatism. 2010;62(12):3695-705.
5) Hoyland JA, Le Maitre C, Freemont AJ. Investigation of the role of IL-1 and TNF in matrix degradation in the intervertebral disc. Rheumatology (Oxford). 2008;47(6):809-14.