Nanoparticles and Nanotopography: A Nano-toolbox to Control Stem Cell Self-Renewal via miRNAs

Mesenchymal stem cells (MSCs) are unspecialised cells that live in a localised area in the bone marrow called the 'niche'. MSCs are important as they can change (differentiate) into a variety of different cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells), depending on when the body needs these cells. For several years now, researchers have been trying to identify methods of controlling how and when MSCs differentiate, so we can either keep them as they are (ie. as stem cells, for transplant studies etc) or make them become a certain tissue (eg. bone, for conditions such as osteoporosis). One well-known method available is to culture and grow the MSCs with special chemical additives that make them become a certain cell type. However this is not ideal, as you are using extra chemicals. We have established a way of growing MSCs and controlling their behaviour, simply by growing them on a particular pattern imprinted onto a material, with no chemicals. This means we can grow cultures of MSCs and either keep them self-renewing (as stem cells) or make them turn into osteoblasts to form bone.

There are certain signals inside the MSC niche that control the MSC's fate. Recent studies have indicated that small sections of RNA, called microRNA (miRNA), are important in this control. In the first part of this project, we want to use our patterns to control MSC behaviour, and then look to see which miRNAs as involved in that control.

Once we have identified these miRNAs, we will then see if we can do the reverse, ie. control MSC behaviour by blocking (or silencing) the particular miRNAs. We will do this by designing blockers of the miRNAs (called antagomirs), which we will deliver into the MSCs. They can be difficult to deliver, so we will guide them into cells by attaching the antagomirs onto small nanoparticles, which will function as taxis to ferry antagomirs into the MSCs. This will give us a nanoparticle tool, which can be ultimately used in clinic to induce bone formation.

However, it must be noted that the MSC niche is located the bone marrow, which is a 3D tissue. Therefore, the final part of our project will look to see if we can also taxi the antagomirs into MSCs grown in 3D, and similarly control the fate of the cells therein. This will give a better idea of whether our technology would work in vivo.

Mesenchymal stem cells (MSCs) represent a cell population with self-renewal ability and multipotent differentiation capabilities. Recent studies have shown that microRNAs (miRNAs) act as regulatory signals both in maintaining self-renewal and instigating differentiation. However, their role in MSC fate is currently poorly understood and remains to be exploited. We have developed two novel nanotopgraphies, which allow us to maintain MSC self-renewal or induce osteogenesis in a chemical free controlled environment (i.e. no changes in medium formulation or surface chemistry is required). We aim to use these topographies to culture our MSCs and build on preliminary data to determine which miRNAs are up- and down-regulated during self-renewal and osteogenesis.
Our team also have expertise in using gold nanoparticle (AuNP) intracellular delivery systems with a view to gene silencing. In this project we aim to design antagomirs against the miRNAs shown to be up- and down-regulated during self-renewal and osteogenesis, and decorate AuNPs with said antagomirs with the intention of influencing MSC fate, and driving cells towards osteogenesis.
Our final aim is to develop a simple in vitro niche mimic model system comprised of MSCs seeded onto our self-renewing nanotopography overlaid with a soft collagen gel (akin to the stiffness of bone marrow). Nanoparticles are excellent platforms for cell delivery in 3D tissues. Injection of the AuNPs (either by syringe or microinjection), which will be further functionalised with MSC targeting moieties, will allow us to assess the efficiency of our antagomir functionalised AuNPs at targeting MSCs in a niche-like environment.
In summary, we will thus utilise both nanotopographies and nanoparticles in a synergistic study to identify key miRNAs involved in MSC renewal and differentiation (osteogenesis) and then take a step towards targeting MSCs with our particles to direct cell fate in an in vitro 'niche-mimic' environment.

Beyond academic impact, this research has clear potential impact in stem cell biology (new tools), nanoparticle synthesis (with entirely novel functionalisation) and in clinic (with promise for emerging new therapies) in the UK and rest of the world.

Nanoparticles are potentially powerful tools in biology, allowing targeted delivery of reagents at high efficiency. Small RNAs, particularly miRNAs, appear to be central to stem cell function and control and so it is timely to move our siRNA delivery technology to miRNA silencing using antogomirs. This is facilitated by our paired nanotopographical controls, presently unique in the world. The development of such a nanotoolbox will have large potential impact on basic and applied research. Furthermore, the manipulation of mesenchymal stem cell phenotype by altering integral miRNAs is a highly novel concept, which will provide a exclusive method of studying MSC behaviour. Finally, the development of simple 3D niche mimics will further aid research into the signaling control that maintains MSC phenotype, and will lead to advances in the study of degenerative conditions.

In clinic, the aging population in the developed world is a major research focus and a funding priority area. Degenerative conditions such as osteoporosis and trauma complications, such as non-union fractures could be targeted through ability to tune MSC response. In these and other conditions, the MSC niche fails in its ability to respond to demand. To address these issues, we need to target the MSCs in the bone marrow to increase osteogenesis. Nanoparticles represent an ideal solution to this due to their ability to be loaded with multiple cargoes (therapy, targeting and efficiency enhancing). We thus team with Mr Meek and Prof Ahmed to help us develop the clinical direction of our research from an early stage. This will clearly aid translation and clinical interest (user acceptance).
We will similarly collaborate with Integrated Magnetic Systems Limited (IMSL), who currently market magnetic nanoparticles for biomedical applications. IMSL are very interested in the outcome of this project, and the subsequent possibility of moving the entire concept of targeting using antagomirs from gold to magnetic nanoparticles. Their advice will help us remain focused on the commercial potential of the nanotools developed during the project.