HT chemical manipulation of foetal and adult stem cells - selection transfection and scaffold identification
Lead Research Organisation:
University of Southampton
Department Name: Development Origin of Health and Disease
Abstract
The cells we will study, so-called Mesenchymal stem cells from bone marrow, are very special as they have the potential to form a variety of tissue types such as cartilage, bone, muscle, tendon, ligament and fat. For this reason stem cells are currently one of the most exciting and promising areas for disease treatment and reparative medicine and in the future this will allow stem cell-based therapies to be developed to treat or cure diseases. Given the fact that we have an ageing population, the development of strategies to exploit stem cells to allow bone formation or the replacement or restoration of diseased or worn-out bone is a major clinical and socio-economic need. However crucial issues to allow this approach to be successful are the development of methods to allow the purification of stem cells to provide sufficient cells for tissue regeneration, and the development of tools to allow the cells to be controlled and converted into the specific cells desired. There is also a need to allow 3D scaffolds to be built to allow stem cell growth to form selected tissues. This study sets out to develop tools to allow selection of these stem cells and the identification of scaffolds on which these cells will grow and can be modulated using a chemical biology approach.
Technical Summary
The use of stem cells in the form of cell-based therapies is currently one of the most exciting and promising areas for disease treatment and reparative medicine. However, for this goal to be achieved, it is critical that platform technologies be generated to enable the isolation of stem cells, their transfection with both DNA and RNAi as well as the generation of scaffolds for stem cell growth and differentiation to form selected tissues. In order to address this issue we will harness the potential of High Throughput chemical arrays, screens and transfection strategies with mesenchymal stem cells to i) identifyof polymers, via micro-array screening, capable of enriching and supporting fetal mesenchymal stem cells and adult mesenchymal stem cell growth, ii) the development of HT stem cell RNAi an didenitfication of bio-mimetic chemical scaffolds / sequences to drive stem cell differentiation and to modulate stem cell plasticity. Finally we propose to examine ex vivo and in vivo our identified innovative scaffolds using bone formation form the mesenchymal stem cells as en exemplar. This programme of research offers a unique opportunity to harness the potential of polymer arrays and transfection chip technology to characterise the chemical fingerprint of fetal mesenchymal stem cells in comparison to adult mesenchymal stem cells as well as specific stromal lineages (adipocyte versus osteoblast). This potentially offers a novel approach to identify select populations and population subsets (whether skeletal or related stromal lineages such as adipogenic), modulate differentiation and will provide appropriate scaffolds for 3D skeletal reconstruction.
People |
ORCID iD |
| Richard Oreffo (Principal Investigator) |
Publications
Berry CC
(2007)
Human fibroblast and human bone marrow cell response to lithographically nanopatterned adhesive domains on protein rejecting substrates.
in IEEE transactions on nanobioscience
Dalby MJ
(2007)
The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder.
in Nature materials
Khan F
(2009)
Polymer microarrays - A high-throughput approach to the identification scaffolds and selection of tissue engineering scaffolds
in European Cells and
Khan F
(2009)
Versatile biocompatible polymer hydrogels: scaffolds for cell growth.
in Angewandte Chemie (International ed. in English)
Richard Oreffo (Author)
(2009)
Polymer microarrays - A high-throughout approach to the identification and selection of tissue engineering scaffolds
in European Cells and Materials (ECM)
Tare R
(2008)
Polyurethane microarrays: A novel platform for selection of skeletal osteoprogenitors
in Calcified Tissue
Tare R
(2009)
Application of
in Calcified Tissue International
Tare R
(2007)
Selection of human Mesenchymal osteoprogenitors from bone marrow stromal cell populations using polymer microarrays
in Tissue Engineering
Tare RS
(2012)
Isolation, differentiation, and characterisation of skeletal stem cells from human bone marrow in vitro and in vivo.
in Methods in molecular biology (Clifton, N.J.)
| Description | 1. Development of polyurethane substrates capable of selectively immobilising STRO-1 immunoselected skeletal progenitors from heterogeneous bone marrow mononuclear cell (BMMC) populations (Biomaterials 2009) 2. Identification of polyurethane substrates for surface modification of tissue engineering scaffolds to enhance skeletal cell growth and differentiation (Biomaterials 2009) 3. Synthesis of a synthetic, but biodegradable, binary polymer blend that forms a hydrogel when the two components, namely chitosan and poly(ethylenimine), are mixed. The hydrogel provides a robust 3-D scaffold for the culture and transfection of foetal skeletal progenitors (Angewandte Chemie International Edition 2009) 4. Generation of 3-D biomimetic scaffolds, by blending poly(L-lactide)/ PLLA and poly(caprolactone)/ PCL, exhibiting porosity and interconnectivity reminiscent of 3-D architecture of normal bone - these scaffolds facilitate bone tissue regeneration in vivo and hold tremendous promise in the development of strategies for skeletal tissue repair (Biomaterials 2010) |
| Exploitation Route | Novel scaffolds generated and high throughput chemical library selection strategies for cells and scaffolds for tissue engineering- the approach can be applied to other cell and tissue types. |
| Sectors | Healthcare |
| Description | The application of high throughput polymer library strategies to identify novel cell selective and bone tissue scaffolds can be applied to other tissues; Thus, from a library of 135 polymer blends, 44% of the polymer blends were identified to bind to adult human skeletal stem cells to varying degrees. The potential of these polymeric substrates to support skeletal stem cell growth and osteogenic differentiation was analysed by in vitro studies before undertaking in vivo bone regeneration studies - this minimised the use of animals. The approach can be applied to other tissues. Selected scaffolds could be scaled to allow preclinical and clinical testing and evaluation. |
| First Year Of Impact | 2010 |
| Sector | Healthcare |
| Impact Types | Economic |
| Title | POLYMER BLENDS |
| Description | A biocompatible polymer mixture for use as a matrix for cellular attachment includes a mixture of at least two polymers selected from the group consisting of : chitosan (CS), polyethylenimine (PEI), poly (L-lactic acid) (PLLA), poly (D- lactic acid) (PDLA), poly (2 -hydroxy ethyl methacrylate) (PHEMA), poly (e-caprolactone) (PCL), poly(vinyl acetate) (PVAc), poly (ethylene oxide) (PEO), poly [ (R) -3-hydroxybutyric acid)] (PHB), cellulose acetate (CA), poly (lactide-co-glycolide) (PLGA) and poly (N- isopropylacrylamide) (PNIPAM). Implants making use of the polymer mixtures can support cell attachment, growth and differentiation, and tissue regeneration in vivo. |
| IP Reference | WO2010023463 |
| Protection | Patent application published |
| Year Protection Granted | 2010 |
| Licensed | No |
| Impact | Allowed additional research in the field via grant support |
| Title | Polymer blends |
| Description | A biocompatible polymer mixture for use as a matrix for cellular attachment includes a mixture of at least two polymers selected from the group consisting of: chitosan (CS), polyethylenimine (PEI), poly(L-lactic acid) (PLLA), poly(D-lactic acid)(PDLA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(µ-caprolactone) (PCL), poly(vinyl acetate) (PVAc), poly (ethylene oxide) (PEO), poly[(R)-3-hydroxybutyric acid)] (PHB), cellulose acetate (CA), poly(lactide-co-glycolide) (PLGA) and poly(N-isopropylacrylamide)(PNIPAM). Implants making use of the polymer mixtures can support cell attachment, growth and differentiation, and tissue regeneration in vivo. |
| IP Reference | EP2385105 |
| Protection | Patent application published |
| Year Protection Granted | 2011 |
| Licensed | No |
| Impact | None to date- only filed in Julyy 2011 - |
| Title | Selective cellular adhesion on polymer microarrays |
| Description | Agarose is used to provide a coating having a cytophobic effect on a support used for screening an array of polymer samples for cell-binding properties. Polymers for cell-binding properties are screened by forming a polymer library, such as a library of polyurethanes, by parallel synthesis, at least partially characterising the library members, forming an array of the library members on a support such as a glass slide, incubating the microarray with a cell suspension and observing the microarray to determine the binding activity of library members with specific cells. The method is used to identify polymers that bind to ostoprogenitor cells, so that the polymers can be used as a substrate for contact with unselected human bone marrow mononuclear cell preparations for selective immobilisation of ostoprogenitor cells. Suitable polyurethanes are made from polyols such as poly(propylene glycol), poly(tetramethylene glycol, poly [1, 6- hexanediol/neopentyl glycol/diethylene glycol-alt-(adipic acid)] diol, poly[1, 6- hexanediol/neopentyl glycol-alt- (adipic acid)] diol, isocyanate such as 1 ,6-diisocyanohexane, 4, 4' methylenebis(phenylisocyanate), 4-methyl- 1,3, phenylene diisocyanate, 1, 4 diisocyanobenzene, 4, 4' methylenebis(cyclohexylisocyanate) and 1, 3 bis(isocyanatomethyl)cyclohexane and chain extenders such as 1, 4 butanediol, ethylene glycol, propylene glycol, ethylene diamine, 3-dimethylamino-1,2-propanediol, 2-nitro-2- methyl-1, 3 propanediol, 2,2,3, 3, 4, 4, 5, 5 -octofluoro-1,6, hexanediol, and diethyl bis(hydroxymethyl)malonate. |
| IP Reference | GB2425074 |
| Protection | Patent application published |
| Year Protection Granted | 2006 |
| Licensed | No |
| Impact | Additional support for further research as a consequence of the patent file. Follow- on Funds achieved |
| Description | Scaffolds and cells - making replacement body parts in the lab - Exhibit at the BBSRC Great British Bioscience Festival |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | Engaged over 6500 visitors from across the UK as part of the BBSRC run GBBF. EPSRC grant generated the centre piece of our exhibit - the Stem cell mountain - a large interactive pinball machine built in collaboration with Winchester Science Centre to communicate the concept of Stem Cell Potential. In response to the feedback question 'Tell us something from your visit that you found particularly interesting' the answer 'Stem Cells' was one of the most frequently given. Through a conversation at this exhibition a talented international MSc student from Queen Mary's was inspired to commence a PhD project (self-funded) in my group. |
| Year(s) Of Engagement Activity | 2014 |
| Description | Science Day at University of Southampton |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | Science Day ('Stem Cell Mountain' & Bone regenerative medicine display') at University of Southampton - 14/03/15. (Open to the general public - 1000+ visitors) |
| Year(s) Of Engagement Activity | 2008,2011,2013,2015,2016 |
| Description | Speaker at UKRMP Conference UK |
| Form Of Engagement Activity | A magazine, newsletter or online publication |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Invited Speaker - Inaugural UK Regenerative Medicine Conference, London. "Size matters - Harnessing biomimetic materials for skeletal tissue engineering." The audience were peer scientists, clinicians and industrialist to inform, educate and debate developments in translational bone research. |
| Year(s) Of Engagement Activity | 2016 |
| Description | Stem Cell Mountain at the Glastonbury Music Festival Science Tent |
| 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 | Stem Cell Mountain exhibit in shared Science Tent, mixed Festival audience (academics, post-docs, science specialists, general public, families and young people) Measure 1: visitors passing stand; 1100 per day = 5500 Measure 2: visitors interacting with researchers on stand; 500 per day = 2500 Engaged classically hard to reached audience. Quote: "I was considering doing a PhD at one point - I didn't think I'd reconsider it here" |
| Year(s) Of Engagement Activity | 2015 |