Materials exploitation of the biointerface to control MSC quality and niche phenotype

We understand that stem cells hold the key to curing many degenerative conditions. Currently lacking, however, are the technologies that will open up use of stem cells for regenerative therapies. In the body, in their niches, adult stem cells are controlled by their environment - a complex mixture of proteins, sugars and other cells. This environmental control allows stem cell growth with maintenance of stem cell phenotype (the cells observable characteristics). However, when we take stem cells out of the body and grow them in the lab they don't have these environmental controls and so quickly loose stem cell characteristics, making it hard to grow large numbers of clinically useful stem cells.
In this project we will refine a material that we use to arrange proteins and cells in a particular way to allow stem cell growth. First, we will investigate mesenchymal stem cells (MSCs) from bone marrow. MSCs are responsible for provision of bone, cartilage, ligament and tendon cells. We will improve on our ability to grow these cells in the lab and will understand how the cells regulate themselves so that we can identify drugs and drug targets that we can exploit to improve growth in larger-scale stem cell cultures. Further we will develop our technology to allow culture without animal products so that the cells are clinically relevant for use in humans. However, in this project, rather than investigating MSC use in skeletal regeneration, we wish to see if we can maintain their ability to modulate the immune system for longer. MSCs have exciting potential to be used almost as a drug along with transplants as they can modulate immune responses to help prevent transplant rejection. The blood transfusion service are investigating this possibility and we will work with them using our approaches to expand high quality MSCs with immune modulatory capacity retained.
Further, we will use our materials systems to investigate haematopoietic stem cell (HSC - stem cells that make blood cells) maintenance. In the bone marrow, HSCs stick to MSCs and this preserves their stem cell characteristics. In the lab HSCs don't proliferate and rapidly loose their stem cell phenotype. HSCs are important as they are central to a widely used stem cell therapy - the bone marrow transplant that remains a successful tool in the fight against conditions such as leukaemia. In leukaemia, HSC progeny cells that go on to make blood cells (red blood cells that carry oxygen around the body and white blood cells that fight infections) become diseased. Thus, bone marrow, that contains stem cells, can be moved from a healthy donor to a recipient who has had their own, diseased, stem cells killed. The donated stem cells have the ability to repopulate the blood of the recipient with disease free cells. This is an amazing example of the ability of a few stem cells to repopulate and regenerate.
There are, however, some major drawbacks. Firstly, this is a one donor to one recipient therapy and the ability to match recipients with tissue-matched donors is very limited. While there have been advances, such as use of mobilised peripheral blood stem cells, supply still falls far short of demand and this severely limits the therapy that can be offered.
While it is very ambitious to say that we believe our technology can be used to grow HSCs in the lab, we believe we can take the first steps towards this. Our aim is to bioengineer niche environments using MSCs and our novel materials. By controlling the characteristics of the MSCs we will increase the number of HSC sticking to them. This achieved, we will investigate HSC phenotype maintenance and look for the tempting possibility of HSC growth.
Understanding stem cells and unlocking their potential is one of the major challenges of this century. This project aims both to improve understanding and unlock potential.

We understand that control of growth factors (GFs) and cell adhesion is critical to maintain MSC phenotype and to drive differentiation. While mechanisms of differentiation are better understood, understanding MSC self-renewal is more challenging.
We have identified a polymer system based on polyethylacrylate (PEA) that facilitates manipulation of cell adhesion and GF presentation to MSCs allowing us to control MSC self-renewal. PEA controls the arrangement of fibronectin adsorbed on its surface, exposing the RGD adhesion tripeptide and the growth factor binding region that we can subsequently decorate with very low, but effective, concentrations of GFs. We have preliminary data that shows that we can both control MSC osteogenesis and expression of MSC and niche marker proteins using this system. Our system is amenable to conversion into 3D matrices and we have developed in-house plasma polymerisation systems to facilitate this. Further, that we use human fibronectin as the interface allows us to move to xeno-free cultures.
In this project we will produce environments that control MSC self-renewal and expression of niche markers to (a) allow us to investigate mechanisms of MSC proliferation and (b) explore HSC homing. Through metabolomic investigation of self-renewal we will identify targets to allow us to improve MSC growth. We will investigate the potential of our expanded cells to support transplantation through improved maintenance of immune modulatory effects.
Further, we will investigate if the improved niche-related MSC phenotypes increase hematopoietic stem cell (HSC) homing and maintenance of HSC phenotype in culture. In the niche, MSCs and HSCs co-exist and HSCs home to MSCs in order to maintain their phenotype. MSCs in the niche are regulated by extracellular matrix interactions and GFs, as with our PEA system. Improvement in HSC maintenance will facilitate gene/drug therapy and is a Scottish National Blood Transfusion Service priority research aim.

Scientists and Healthcare Professionals
We will use high impact, open access publishing to ensure our research is disseminated as broadly as possible (see academic beneficiaries section). This will be of direct interest to the biomaterials, tissue engineering, stem cells and medical communities. Our results will form the basis for new research directions across the world through new collaborations.

Patients
Islet transplant is used to correct type 1 diabetes, particularly in patients with hypoglycaemic unawareness who have limited quality of life due to their inability to know when they may become hypoglycaemic and become unconscious. Widespread us of islet transplant is curtailed by the limited supply of cadaveric organs. Further, the number of islets that are retained in the portal vein after transplantation are limited by immune attack. The immune-modulatory capability of MSCs could thus be central in improving this therapy.
A cGMP compatible culture system that maintains immune-modulatory function would improve the quality of MSC grown in vitro and permit extended proliferation increasing the number of cell that can be harvested for clinical use. Therefore, the investigations proposed in this project could significantly contribute to our aims to produce more and better cells for transplantation.

For our HSC ambitions, a reliable in vitro system permitting HSC maintenance would be invaluable to achieve the transfer of therapeutic genes into HSCs or the use of genome editing tools such as CRISPR/TALENs to correct disease causing mutations in the perspective of novel gene therapies. Such therapies are again of major Pharma interest [1].
In addition to the established use of bone marrow (or peripheral blood stem cells) transplant for the treatment of malignant haematopoietic diseases, there are over 650 clinical trials [2] under way in the world related to HSCs; clinical needs and potential benefits are very significant.
In terms of our future aims of HSC expansion, there are around 25,000 allogeneic bone marrow transplant procedures currently performed worldwide pa [3], and there could be twice as many if matched donors could be found. By working towards 'one donor to multiple recipient' therapies we can revolutionise the capacity to supply improved care.

Health Service Providers
Our work is well aligned to SNBTS future directions and we have discussed this project with them in detail to understand what they want to achieve and what we need to achieve to help them take on our ideas towards translation. For transplant therapies, the side effects of immune suppression can be large and costly (e.g. cancer). For HSC therapies, the health services are restricted in their ability for care due to availability of high-quality HSCs; engineering or expanding HSCs could help overcome this.

Industry
Our work will be of interest to industrialists working in the material science /stem cell space. Companies working in this area include those with interest in stem cell therapies e.g. Admedus, t2cure, Scarless, Lipogems and Roslin Cells and also providers of innovative cell culture platforms e.g. BiogelX, Stem Cell Technologies and Porvair as well as major multinationals in this space e.g. Nunc. These companies would have economic benefit from licensing and exploiting our products, developing and marketing new bone marrow development products.

Public
Our work will be of benefit to the general public, raising interest in science, educating about the challenges faced by researchers, increasing funding for research, increasing awareness of the importance of stem cell growth. We will engage with the next generation of scientists and clinicians to inspire them about opportunities to develop new stem cell technologies.

1. Gersbach. Genome engineering: the next genomic revolution. Nat Meth 11, 1009 (2014)
2. http://clinicaltrials.gov
3. Pasquini. Current use and outcome of HSC transplantation. 2013. www.cibmtr.org