Stem cells and regenerative medicine hold great promise for human health and healthy ageing in the future. A number of exciting technologies have recently been developed by which the derivation of embryonic stem cells from adult cell types is now possible, albeit inefficiently. This includes the transfer of reprogramming factors into adult cells, resulting in the generation of induced pluripotent stem cells (iPS cells). This technology, however, is inefficient and a particular bottleneck appears to be the reprogramming of the epigenome. Epigenetics or the epigenome refers to all the modifications to DNA and the chromatin that are important for the function of the genome in the context of development and in the adult organism in different organs and tissues. Importantly, in germ cells and early embryos, the epigenome is reprogrammed on a genome-wide scale, so that development of a new organism and of new stem cells is possible. Work in our laboratory and that of others has revealed several features of this genome-wide epigenetic reprogramming. In collaboration with the company CellCentric, we have begun to set up a number of systematic screens for the isolation of epigenetic reprogramming factors, including those that can erase DNA methylation from the genome. The current programme of work, again with CellCentric, aims to assess the function of these new reprogramming factors in germ cells and early embryos, embryonic stem cells, and in the generation of iPS cells. CellCentric will, in particular, work on the isolation of small molecules that can alter the function of our reprogramming factors, which will then be tested in embryonic stem cells and iPS cells. The combined programme of work will shed new light on the fundamental process of epigenetic reprogramming, and provide new tools for the manipulation of stem cells and the translation into approaches to regenerative medicine and healthy ageing.

Definition: A rapidly developing area at the interfaces of engineering/physical sciences, life sciences and medicine. Includes:- cell therapies (including stem cells), three dimensional cell/ matrix constructs, bioactive scaffolds, regenerative devices, in vitro tissue models for drug discovery and pre-clinical research.Social and economic needs include:Increased longevity of the ageing population with expectations of an active lifestyle and government requirements for a longer working life.Need to reduce healthcare costs, shorten hospital stays and achieve more rapid rehabilitationAn emergent disruptive industrial sector at the interface between pharmaceutical and medical devicesRequirement for relevant laboratory biological systems for screening and selection of drugs at theearly development stage, coupled with Reduction, Refinement, Replacement of in vivo testing. Translational barriers and industry needs: The tissue engineering/ regenerative medicine industry needs an increase in the number of trained multidisciplinary personnel to translate basic research, deliver new product developments, enhance manufacturing and processing capacity, to develop preclinical test methodologies and to develop standards and work within a dynamic regulatory environment. Evidence from N8 industry workshop on regenerative medicine.Academic needs: A rapidly emerging internationally competitive interdisciplinary area requiring new blood ---------------------

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The bone marrow is a site of health and disease. In health, it produces all of the blood cells that we rely on to carry oxygen and protect us from infection. However, the stem cells that produce the blood and that reside in the marrow, the haematopoietic stem cells (HSCs), age and can tip over into disease states, such as developing leukaemia. Factors such as smoking and treatment of cancers elsewhere in the body (toxic effects of chemotherapy/radiotherapy) can accelerate ageing, and therefore, drive the transition to disease. Further, it forms a home to other cancer cells, that leave their original tumour and move, or metastasise, to the bone marrow. Once in the marrow, they can become dormant, hiding from chemotherapies and activating sometime later to form devastating bone cancers. The cues that wake cancer cells from dormancy are largely unknown. If models of the bone marrow that contain human cells and that can mimic key facets of the niche in the lab, such as blood regeneration, cancer evolution and dormancy, can be developed it would be a big help in the search for better cancer therapies. We are developing the materials and technologies required to meet this challenge. In this programme of research, we will tackle three biomedical challenges: 1) HSC regeneration. Bone marrow transplantation (more correctly HSC transplantation) is a one-donor, one-recipient therapy that can be curative for blood diseases such as leukaemia. It is limited as HSCs cannot be looked after well out of the body. Approaches to properly look after these precious cells in the lab could allow this key therapy to become a one-donor, multiple recipient treatment. Further, the ability to look after the cells in the lab would open up the potential for genetically modifying the cells to allow us to cure the cells and put them back into the patient, losing the need for patient immunosuppression. 2) Cancer evolution. As we get older, our cells collect mutations in their DNA and these mutations can be drivers of cancer. Lifestyle choices such as smoking, and side effects of treatments of other diseases can also add mutations to the cells. As blood cancers develop, the bone marrow changes its architecture to protect these diseased HSCs. Our 3D environments will allow us to better understand this marrow remodelling process and how drugs can target cancers in this more protective environment. The models will also allow us to study the potential toxicity of gene-edited HSCs to make sure they don't produce unwanted side effects or are not cancerous in themselves. 3) Dormancy. What triggers dormancy and activation from dormancy are poorly understood. By placing our 3D environments in a miniaturised format where we can connect other models that include infection and immune response, we can start to understand the factors involved in the activation of cancer cells from dormancy. Our vision is driven by materials and engineering, as the bone marrow niche is rich in structural and signalling biological materials (proteins). Therefore, we will establish three engineering challenges: (1) Cells can be controlled by the stiffness and viscous nature of materials (viscoelasticity). We will therefore develop synthetic-biological hybrid materials that can be manufactured to have reproducible physical properties and that have biological functionality. (2) We will develop these materials to interact with growth factors and bioactive metabolites, both of which are powerful controllers of cell behaviours. These materials will be used to assemble the HSC microenvironments in lab-on-chip (miniaturised) format to allow high-content drug and toxicity screening. (3) We will develop real-time systems to detect changes in cell behaviour, such as the transition from health to cancer using Raman and Brillouin microscopies. The use of animals in research provides poor predictivity. We will offer better than animal model alternatives.

We are carrying out genome-wide epigenomics screens using state of the art array and Next Generation Sequencing (NGS) methods (MeDIP Seq, BS Seq, ChIP Seq and others). In collaboration with CellCentric and funding by TSB we have completed one such screen in which we identified 69 genes which are hypomethylated and expressed in ES cells while being hypermethylated and repressed in primary fibroblasts (pMEF) (Farthing et al 2008 PLoS Genet, Ng et al 2008 Nature Cell Biol). Likewise we identified 67 genes hypomethylated in TS cells and hypermethylated in pMEFs. Some of the genes that are expressed in ES and repressed in pMEFs are known regulators of pluripotency (eg Nanog) while others are of unknown function. Many of the gene candidates are suspected to have roles in transcriptional or epigenetic regulation. Our hypothesis is therefore that this gene set is enriched for new regulators of pluripotency and epigenetic reprogramming. Pluripotency in ES cells is maintained by a core circuitry of transcription factors which includes Oct4, Nanog, and Sox2. These transcription factors bind to a network of other genes which they either activate or repress. This must also include epigenetic reprogramming factors since ES cells are able to reprogramme somatic cells when they are fused to them, but the nature of these epigenetic modifiers is not known. Excitingly, a core set of four transcription factors (Oct4, Sox2, Klf4, c-myc) have been shown to be able to reprogramme pMEFs and other differentiated cell types to a pluripotent state (so called induced pluripotent stem, or iPS cells). Further work has shown that currently only two factors (either Oct4 and Klf4, or Oct4 and Sox2) in combination with drugs that affect epigenetic pathways (VPA affecting histone acetylation, BIX affecting histone methylation) are needed for induction of iPS cells, highlighting again the need for the induction of the pluripotency gene circuitry and that for epigenetic reprogramming of the genome. However, generation of iPS cells is a very inefficient process and many iPS lines are not completely epigenetically reprogrammed to a pluripotent state. The isolation of new pluripotency factors and epigenetic modifiers is therefore urgently needed in order to improve the prospects for the effective use of stem cells in regenerative medicine.