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

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.

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.

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.