Identifying the skeletal stem cell for regeneration: harnessing smart nanoparticles and single cell DropSeq molecular profiling platforms

We are now living much longer than we used to. These are exciting times in medical research and indeed, medical advances have led to a welcome increase in life expectancy. However, an unfortunate consequence of this is that as we reach later life the chances that we can become ill or injured increase dramatically. One big problem that elderly people face is illness and injury associated with the bones and joints.

Diseases like osteoporosis and arthritis cause pain, cause bone fractures and lead to immobility and distress to tens of thousands of people each year, costing the taxpayer tens of millions of pounds (in the UK there are some 150,000 osteoporosis-associated wrist, spine and hip fractures, with an estimated healthcare cost of £1.7 billion per annum). So new treatments that enable the skeleton to heal better are urgently required.

Cell-based therapies are currently some of the most exciting and promising areas for bone disease treatment and reparative medicine. However, despite intensive research interest there are currently no reliable methods to isolate (or enrich sufficiently) the rare bone stem cells (known as skeletal stem cells) needed for these strategies. Thus, it is critical that new technologies be generated to enable the robust isolation of bone stem cells from patients (from their bone marrow) for clinical use.

We propose that new approaches, which combine stem cell biology with the latest approaches to identify and cells together with innovative statistical methods will allow us to enrich and isolate the bone stem cell.
In this unique programme of work we will be able, for the first time, to use very small (nanoscale) probes to further refine our cell selection and identification strategy. In particular, we will combine a novel single cell identification procedure (which produces a "barcode" for each cell) with unique cell sorting devices to allow us to isolate bone stem cells. We will combine the unique bar codes (derived from the molecular (RNA) machinery) in the individual cells in combination with the smart nano-probes to allow us to tag cells that will separate and examine if they are stem cells and their ability to make bone and cartilage. Through this unique approach we will enrich for bone stem cells that we will characterise and examine if they are stem cells and test their ability to make bone and cartilage.

This exciting programme of research offers the ability to isolate the bone stem cell and provide substantially enriched, and potentially pure populations of bone stem cells. This will advance considerably our understanding of bone stem cell basic biology and offer significant therapeutic impact for bone repair and the treatment of bone disorders in our ageing population.

To date, it is not possible to isolate a homogenous skeletal stem cell population for research characterisation, functional analysis or clinical application. This multidisciplinary project grant combines smart nanoparticles with the ability to sense unique mRNA signatures in live cells in real time, to identify stem and progenitor skeletal populations and state of the art single cell RNA sequencing platform (DropSeq) to identify the SSC and subsequently validate their functionality in vitro and in vivo.

This programme of research will, for the first time, generate skeletal cell fractions with distinct molecular and functional identity. We hypothesise that "Smart nano-probes able to detect specific skeletal mRNA signatures and single cell RNA-sequencing can be used together to identify human skeletal stem and progenitor cell populations from heterogeneous marrow samples".

We have identified the following research objectives:

1a) To synthesise nano-probes conjugated with skeletal cell and stem cell specific mRNA targets and demonstrate delivery and subsequent analysis of target populations

1b) Single cell RNA sequencing of unsorted and sorted (Stro-1/CD146/Stro-1 bright) skeletal populations to identify new targets for RNA sequence generation

2) To undertake nano-probe target analysis of sequences identified from DropSeq studies and subsequent separation of cell populations and analysis of colony formation

3) To separate and isolate, nano-probe conjugated skeletal populations using FACS and their analysis using single cell RNA sequencing (DropSeq) as well as RT-qPCR of populations and analysis of colony (CFU-F) formation

4) To synthesise multiplexed nano-probes using sequences and targets from objectives 1-3 and subsequent FACS separation and analysis and colony formation analysis.

5) To test the functional capacity of the isolated SSC fractions ex vivo and in vivo in relevant models of bone injury and formation.

Academia

Developing Entrepreneurship: A number of optional training opportunities will be available to the Research Fellows including business skills that complement our various entrepreneurship programmes (Oreffo was Associate Dean International and Enterprise 2008-2013 and developed the Business Fellow model used throughout the University of Southampton) to develop entrepreneurship within the academic culture and the development of enterprise skills for researchers that will further not only further enhance the careers of Drs Stumpf and Jungemann but also will aid the impact of the research from this project.

International Community: We will engage on an international basis to promote the UK as a centre of excellence in all aspects of regenerative medicine and specifically in the field of stem cells, microfluidics and statistical learning. We (RO) have established a Joint Stem Cell laboratory with the Chinese University of Hong Kong (Biomedical Sciences) and BDM is a visiting professor at Kumamoto University. Through these connections our work will impact Chinese and Japanese scientists. 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 Catalonia University); Middle East (Oreffo adjunct chair and joint research programmes at Stem Cell Unit, King Saud University).

General Public and Schools: We will use Science week, Lifelab, speaker4schools (Oreffo) and other mechanisms that we have developed over the last decade to engage with the general public to explain, sensitively, the clinical and commercial potential of our work (see Impact statement) using our Award winning Stem Cell Mountain as a vehicle. We believe that stem cell biology and the life sciences - encapsulated by our project - have a special responsibility to explain science and engineering to schoolchildren. There is significant potential 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 look to ensure that the impact of this research to schools through active schools outreach by all the applicants.

Media and Public Activities: All applicants have enjoyed significant media coverage for their work with presentations on Sky News, BBC Local TV stations, Internet, Radio Stations and the national press and will use similar strategies for this project (see Impact statement).

Business and Industry

UK Industry: We have a number of industry links (e.g. enhanced through our FortisNet (largest Musculoskeletal network in UK - RO co-I) and will communicate openly with appropriate industry partners especially towards the final phase of our programme as we develop robust isolation strategies that have the potential to transform the skeletal regenerative medicine landscape. Furthermore, RO established the University of Southampton Health and Pharma Industry Sector Theme (Director 2011-2013) and will utilise established links in this programme.

TSB Cell Therapy Catapult Centre: We will engage with the Cell Therapy Catapult as appropriate especially from a stem cell clinical translation perspective (preclinical and early development).

NHS and Clinicians: Oreffo has worked closely with clinicians and as appropriate will develop clinical translation of our cell isolation at the close of the project. ROCO works closely with the orthopaedic community (RO has trained 9 clinical MD/PhD in the last eight years with over 35 papers and 20 awards to the clinical fellows).