The use of microRNAs and nanotopography to modulate skeletal stem cell fate and function

Lead Research Organisation: University of Southampton
Department Name: Development Origin of Health and Disease

Abstract

Medical advances have led to a welcome increase in life expectancy. However, increased aging populations pose new challenges and emphasize the need for novel approaches to aid and repair tissue lost through damage or disease. By 2020 approximately 20% of the UK population will be over 65 and the numbers of hip fractures worldwide will increase from 1.7 million in 1990 to 6.3 million in 2050. Thus, there is now an urgent need to understand how to maintain stem cells and then how to direct them into cell types we want such as bone and cartilage to enhance bone repair and improve quality of life for the patient.

However, despite intensive research interest there is limited information on how to reproducibly maintain the bone stem cells (known as skeletal or mesenchymal stem cells); or indeed how to tell a bone stem cell to make bone or cartilage. Fortunately, human bone marrow contains these special skeletal stem cells. These stem cells can easily be obtained from these tissues and have the potential to form a variety of tissue types such as cartilage, bone, muscle, tendon, ligament and fat (for this reason, skeletal stem cells are currently one of the most exciting and promising areas for tissue engineering and reparative medicine and in the future this will allow stem cell-based therapies to be developed to treat or cure diseases).

We are particularly interested in understanding how to maintain stem cells and to switch (differentiate) these bone stem cells to new bone and cartilage fat for regenerative medicine. For this approach to be successful, it is crucial to understand the way in which these skeletal stem cells change to become mature bone or retain their stem characteristics. Unlocking the molecular signals is the key to developing understanding and being able to undertake these studies in the absence of chemical cues is critical (to avoid confusing signals due to the chemicals used); we have powerful early data showing small nucleotides called microRNAs are key.

MicroRNAs (miRNAs), are very small (only 18-25 nucleotides) non-protein-coding single-stranded RNAs that have the ability to regulate gene transcription. They have important and varied roles in many biological and disease related processes. There is new and exciting data to suggest i) a number of miRNAs are specifically expressed in stem cells, ii) they can control stem cell self-renewal, and, iii) they can control the ability of stem cells to form different tissue types. In addition we have data on nanotopographical surfaces (surfaces that can change or maintain our stem cells without any chemical cues) indicating the key role of microRNAs in keeping or changing bone stem cells. We will look at how these microRNAs affect cell behaviour and cytoskeleton (proteins involved in cell adhesion, spreading, metabolism and signalling), cell growth and differentiation.

The results of this proposed project will open the way to modulate bone stem cells and thus drive the stems cells towards the desired cell type and provides exciting healthcare opportunities that will benefit many.

Technical Summary

With an increasing ageing population the clinical requirement to replace degenerated tissues, such as musculoskeletal tissue, is a major socio-economic requirement.

Skeletal (SSC) or Mesenchymal Stem Cells (MSCs) have the potential to form a variety of stromal lineages including bone, cartilage, fat and muscle and display plasticity between lineages. Lineage modulation offers significant therapeutic potential for using these cells in regenerative medicine. A key issue is an understanding of skeletal stem cell differentiation and maintenance of skeletal stem cell properties.

MicroRNAs (miRNAs) are a class of endogenous non-protein-coding single-stranded RNAs, ranging from 18-25 nucleotides in length that regulate gene transcription post-transcriptionally with fundamental and diverse roles in a variety of biological and pathological processes. A number of miRNAs are specifically expressed in stem cells, control stem cell self-renewal and differentiation, while other miRNAs have been linked with disease or prognosis. We have emergent proof of concept data that miRNAs regulate skeletal stem cell fate and function. We propose that miRNAs play a critical role in the regulation of skeletal stem cell fate and function.

We will determine the role of miRNAs in the maintenance of stem phenotype or enhancement of bone stem cell differentiation and mechanisms involved including cytoskeletal or epigenetic changes. We have recently shown that defined nanotopographies are capable of robustly regulating SSC fate in the absence of chemical cues. We will therefore use custom designed nanotopographies to control SSC phenotype (for instance, to induce osteogenesis or maintain SSC numbers) and examine how miRNAs affect stem cell responses through modulation of epigenetic, biochemical and biomechanical processes.

This study will facilitate our understanding of the mechanisms involved in the regulation of skeletal stem cell fate and function.

Planned Impact

Our research project aims to bring together a multidisciplinary team to elucidate the role of microRNAs in the regulation of skeletal stem cell fate and function, and will have a number of academic, industrial, societal, economic and awareness impacts.

ACADEMIA
Many optional training opportunities will be available to the Research Fellow, Dr Carmen de Andres - RO was, until August 2013, Associate Dean International and Enterprise at the University of Southampton and thus has seven years' experience in being able to tie in with the aims of the RC UK, to develop entrepreneurship within the academic culture and the development of enterprise skills for researchers that will further enhance Dr de Andres and critically, the impact of the research from this project grant. We will engage on an international basis to promote the UK as a centre of excellence in all aspects of stem cells, microRNA/epigenetic and regenerative medicine.

Our track records show evidence of impact in Japan (joint publications, student research exchange with Kyoto University (Oreffo-Tabata); Europe (Leverhulme visiting fellowship - Oreffo to Dr de Andres Santiago de Compostela University), Middle East (Oreffo adjunct chair and joint research programmes at Stem Cell Unit, King Saud University, China /Hong Kong (Joint Stem Cell lab between Centre for Human Development, Stem cells and regeneration and Chinese University of Hong Kong) and many other linkages.

BUSINESS AND INDUSTRY
We have a number of industry links (see track record for ROCO, NG and MD) and will communicate openly with appropriate industry partners (IP discussions will be undertaken under CDAs) especially towards the final phase of our programme as we develop robust strategies for growth factor delivery from our various scaffolds for tissue regeneration. See 'Pathways to Impact' for more details. ROCO established the University of Southampton Health and Pharma Industry Sector Theme (Director 2011-2013) and will utilise established links in this programme.

GENERAL PUBLIC - PUBLIC AWARENESS AND POLICY
We will engage with the general public to explain sensitively (management of expectation is key in this area of research) the clinical and commercial potential of our work. We believe that stem cell biology and the life sciences - encapsulated by our project have a special responsibility to explain stem cell science to schoolchildren. There is significant potential in this area including vast potential from a reparative standpoint for bone stem cells in orthopaedics and skeletal stem cell biology across a range of clinical areas and the importance of multidisciplinary science - our outreach programme will provide impact over the next 3 years through active schools outreach by ROCO and all the applicants.

GENERAL PUBLIC - HEALTH AND WELL-BEING
TSB Cell Therapy Catapult Centre: We will engage with the Cell Therapy Catapult as appropriate especially from a stem cell clinical translation perspective in the end phase of our programme again as appropriate (preclinical and early clinical development).
Improving Public Services: Oreffo, Dalby and Gadegaard work closely with the clinical community (see Pathways to Impact) including specifically the orthopaedic community (RO - 7 clinical MD/PhD trained in the last eight years and over 30 papers and 20 awards to clinical fellows) - we will look to translate our stem cell science to through to the clinic, if appropriate, in regenerative projects, programmes as appropriate including the UK Regenerative Medicine and Medical Technologies Innovation and Knowledge Centre programmes.

Publications

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Black CR (2015) Bone Tissue Engineering. in Current molecular biology reports

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Cidonio G (2021) Nanocomposite Clay-Based Bioinks for Skeletal Tissue Engineering. in Methods in molecular biology (Clifton, N.J.)

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Costa Moura C (2018) Quantitative temporal interrogation in 3D of bioengineered human cartilage using multimodal label-free imaging. in Integrative biology : quantitative biosciences from nano to macro

 
Title Stem Cell Mountain 
Description Large 2m diameter stem cell mountain exhibit with interactive pinball - marble run that aids the participant to understand stem cell transition form pluripotent state ( top of mountain) and progression to specialised developed tissues. The participant can determine stem cell fate and tissue orientation and development using the pinball levers 
Type Of Art Artwork 
Year Produced 2014 
Impact 1) Impact on public: (all numbers are robust estimates unless specified) Southampton Science and Engineering Festival (1 day) - regional general public, predominantly families Measure 1: visitors passing stand; 1300 Measure 2: visitors interacting with researchers on your stand; 400 Thomas Hardye School science day, Dorchester - Dorset secondary school students Measure 1: visitors passing stand; 300 Measure 2: visitors interacting with researchers on your stand; 300 [i.e. managed throughput so all students stayed at stand for length of time] Cheltenham Science Festival (6 days) - central exhibit in Discover Zone, mixed public audience (science specialists, general public, schools): Measure 1: visitors passing stand; 1250 per day = 7500 Measure 2: visitors interacting with researchers on your stand; 300 per day = 1800 Glastonbury science tent (5 Days) - 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 your stand; 500 per day = 2500 Winchester Science Festival (3 days - some bad weather!) - exhibition stand, family audience, SciFest talk attendees Measure 1: visitors passing stand; 300 per day = 900 Measure 2: visitors interacting with researchers on your stand; 150 per day = 450 Bestival Science Tent (4 days) - exhibit in shared Science Tent, mixed Festival audience (mainly general public and especially young people 17-25) Measure 1: visitors passing stand; 1400 per day = 5600 Measure 2: visitors interacting with researchers on your stand; 500 per day = 2000 Qualitative feedback from visitors: "This model illustrates stem cell science brilliantly, and it's fun too" "The stem cell machine was so brilliant - it really helped to explain how the body works in such an interactive and easy way" "Helpful, informative team of staff on hand" "Interesting to learn why stem cell research is so controversial" "Great explanation from volunteers about the stem cell model. My Year 6 pupils could then explain the purpose of the model - great" "That's how you teach science!" "It's great to see this activity is in the busiest tent in the field" "I'm glad we saw this - it makes a difference" "That's amazing - a really great way to show stem cell analysis" "I was considering doing a PhD at one point - I didn't think I'd reconsider it here" "The stem cell mountain - thank you for having this idea. It's such a clever way to explain the concept" "Such a simple way to represent a very complicated thing" "We need science like this in school. It's so much more interesting" The exhibit has substantially enhanced our engagement activities both in terms of quality and quantity of impact. As well as touring with the Bringing Research to Life Roadshow - which recognised it as the 'Best Engagement Activity - and through which over 7000 people have interacted with it to learn about stem cell biology, the exhibit is on permanent display at the Winchester Science Centre (>100,000 visitors annually). So popular is the exhibit there that we have now produced a replica to 
URL https://issuu.com/university_of_southampton/docs/3297_uos_ris_e-mag_aw/c/spysdbe
 
Description The work has shown the importance of nanotopography in the modulation of bone stem cell fate and function. Specifically, we have now shown how different nanotopographies created in titanium can affect bone stem cell function.
A new area we have researched is how the surface profile of a cell (imagine a plaster cast) can affect stem cell and bone cell function.
We have taken forward with manufacturing colleagues new topographical approaches and shown in new publications, building, on the original platform the importance of topography in skeletal cell function
Exploitation Route We will be examining the potential to generate 3D scaffolds with distinct patterns in future studies to see if this can generate new devices that could affect cell fate and function.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.southampton.ac.uk/medicine/about/staff/roco.page?roco#research
 
Description Development of student exhibits and art work to explain the science and importance of nanotopography on stem cell fate and function.
First Year Of Impact 2015
Sector Education
Impact Types Societal

 
Title Computer tomopgraphy (uCT) 
Description ? New methodology (uCT) developed to analyse changes in bone structure after a short period of time such as the one experienced after the CAM assay 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact ? New methodology (uCT) developed to analyse changes in bone structure - improved data analysis across research spectrum 
 
Description BBSRC GB Bioscience 2014 Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Large science festival over four days - reaching politicians and policy makers on day 1, school children on day 2 and public on days 3 and 4

Event held November 13-16th - over 6000 attended our stand over four days form school children to aged individuals, research councils and various education stakeholders
Year(s) Of Engagement Activity 2014
URL http://www.bbsrc.ac.uk/society/exhibitions/gb-bioscience-festival/scaffolds-cells-making-replacement...
 
Description Cheltenham Science Festival - Central Exhibit in Discover Zone 
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 The Stem Cell Mountain featured in 'Doc Brown's Lab' (in keeping with the 2015 Back to the Future Theme) at the famous Cheltenham Science Festival Discover Zone. The team interacted with an estimated 1800 visitors over 6 days. Received feedback such as the following: "Great explanation from volunteers about the stem cell model. My Year 6 pupils could then explain the purpose of the model - great" and "That's how you teach science!"
Year(s) Of Engagement Activity 2015
URL http://www.cheltenhamfestivals.com/science/
 
Description Invited Speaker - DASCS 2017 Conference, Odense 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact 2nd November 2017: Invited Speaker - DASCS (Danish Stem Cell Society) 2017 Conference, Odense - Skeletal Cell Based Strategies for Bone Repair - Opportunities and Challenges
Year(s) Of Engagement Activity 2017
 
Description Invited Speaker at my own institution for the Institute of Developmental Sciences 10 Year Celebration Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact 8th November 2017: IDS 10th Anniversary Event
Celebrating 10 Years of Research Excellence
The theme of the day was 'A Life Course of Health' and this conference was designed to be a discovery of medical research across the life course, bringing together academics from the Insititute of Developmental Sciences (IDS) at the Faculty of Medicine, University of Southampton to share their insight. The day consisted of six engaging hot science talks and I presented in the Regenerative Medicine section.
Year(s) Of Engagement Activity 2017
 
Description Invited lecture at the Medical Research Council Lifecourse Epidemiology Unit - Musculoskeletal Lecture Series 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Third sector organisations
Results and Impact Online Seminar: "Regenerative Musculoskeletal Medicine: Skeletal cell based strategies for bone regeneration from bench to clinic" on Tuesday 26th January 2021
Year(s) Of Engagement Activity 2021
 
Description NHS OPEN DAY 
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 A full day presentation on Scaffolds and cells - making replacement body parts in the lab at Southampton General Hospital- attended by over 1000 people

Strong interest from children as a result of a new "Stem Cell Pinball" display built specifically for the event
Year(s) Of Engagement Activity 2014
URL http://www.stemcells.org.uk/
 
Description National Science Week 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Several thousand visitied University of Southampton Science event- the Bone and Joint Musculoskeletal Activity drew approx 200-300 individuals who activelty participated (made models, discussed bone repair, handeled samples) in the last science week exhibition.

Candidates asked specifically about Medicine Bachelor of Medicine programmes and how to apply. Patients asked if they could jopin clinical trials in the future as availalble for impaction bone grafting - so extremely positive feedback for the researchers on this project.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2015,2016
 
Description Open Conference "Stem cells for bone regeneration: state-of-the-art research and solutions" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited Speaker - Bonebank Conference, Odense, Denmark. "Skeletal stem cells for bone tissue regeneration.".
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 Postgraduate Day 
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 Postgraduate students
Results and Impact Scaffolds and cells - making replacement body parts in the lab - an exhibition showcasing the ability to harness cells and scaffolds - including 3D print technology for orthopaedic regenerative medicine applications

Interest for future research projects and collaborations
Year(s) Of Engagement Activity 2014
URL http://www.stemcells.org.uk/
 
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 Southampton Science and Engineering Festival 
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 Stem Cell Mountain at the Southampton Science and Engineering Festival (1 day) - regional general public, predominantly families Measure 1: visitors passing stand; 1300 Measure 2: visitors interacting with researchers on the stand; 400
Year(s) Of Engagement Activity 2015
 
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 Outreach Programme (New Forest Country Show) 
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 Stem Cell Mountain outreach programme at the New Forest Country Show 28/07/15 (general public, farmers, agricultural community. 105,000 people visited over 3 days).
Year(s) Of Engagement Activity 2015
 
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
 
Description Stem Cell Mountain on Tour - Overview 
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 The EPSRC funded Stem Cell Mountain Roadshow Exhibit combines the fun of a pin ball machine with a profound metaphor for a key biological concept. The stem cell mountain was designed in collaboration with Winchester Science Centre to bring to life the complex idea of stem cell potential. The exhibit has engaged festival goers at Glastonbury and Bestival, science aficionados at the UK's top Science Festivals, as well as the 100000 visitors per year that visit the Winchester Science Centre where a replica is full time resident (see individual entries for numbers at each event) Qualitative feedback on Stem Cell Mountain from the public: "This model illustrates stem cell science brilliantly, and it's fun too" "The stem cell machine was so brilliant - it really helped to explain how the body works in such an interactive and easy way" "Helpful, informative team of staff on hand" "Interesting to learn why stem cell research is so controversial" "Great explanation from volunteers about the stem cell model. My Year 6 pupils could then explain the purpose of the model - great" "That's how you teach science!" "It's great to see this activity is in the busiest tent in the field" "I'm glad we saw this - it makes a difference" "That's amazing - a really great way to show stem cell analysis" "I was considering doing a PhD at one point - I didn't think I'd reconsider it here" "The stem cell mountain - thank you for having this idea. It's such a clever way to explain the concept" "Such a simple way to represent a very complicated thing" "We need science like this in school. It's so much more interesting" Qualitative feedback from the Stem Cell Mountain team of researchers: "One of the amazing perks that come with researching! Great day, talked to many interesting people from various backgrounds and ages (from 5yr olds to 70 year old ex-specialists in the area). Very good and helpful for both the people that we talk to and for us as researchers" "It made me think my research matters" "It helps you to step back and see the amazing things you get to study in university with fresh eyes" "People were really interested in what are doing in our research" "It is a great opportunity to leave the academic bubble and a useful and interesting attempt to explain science research to a wide audience. The best thing is seeing people's reactions when everything clicks and makes sense to them!" "The stem cell mountain is very impressive. Proud to represent" "This festival was great to see how amazed people are about the potential of stem cells" " A brilliant way to inform the public about the work we do" " A pleasure to meet and collaborate with the range and depth of skills and knowledge in our roving science tent" "I was blown away by the strength, diversity and great nature of the team" "I've really enjoyed manning the stem cell volcano - my first experience of the exhibit. I'd quite like one in my living room!" "It's good to see so many people interested in stem cell research"
Year(s) Of Engagement Activity 2014,2015,2016
 
Description Thomas Hardye School Science Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A Dorset secondary school open day engaging 300 students with translational stem cell medicine. First experience of public engagement for several colleagues who reported great satisfaction with the experience.
Year(s) Of Engagement Activity 2015
URL http://www.southampton.ac.uk/per/university/roadshow.page
 
Description Winchester Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Stem Cell Mountain at a three day up and coming science festival. Engaged largely family audience and science festival talk attendees Measure 1: visitors passing stand; 300 per day = 900 Measure 2: visitors interacting with researchers on stand; 150 per day = 450
Year(s) Of Engagement Activity 2015