Illuminating molecular mechanisms required for efficient reprogramming and transdiffrentiation

Our bodies are made up of around 300 different types of cells, each with a different, specialized role. However, we were not always composed of all these different cells. In an early human embryo, it is a ball of just 100-150 cells, all of which are completely unspecialized. At this stage, each cell can divide to produce any of the body's cells. This flexibility is called 'pluripotency'. As the embryo develops and the body takes shape, the cells divide repeatedly, gradually becoming more specialized and losing their flexibility. To make the cells stay pluripotent, we have to take them out of the early embryo and provide them with the particular conditions they need to keep dividing without specialization; making copies of themselves. Cells extracted from an early embryo and multiplied in a dish like this are called embryonic stem cells (ESCs). Given the right cues, they can generate any type of specialized cell in the body.

Until recently, we thought that once cells were specialized it was not possible to change their character - to convert them from, say, skin cells into muscle cells. However, in 2006 a technology called reprogramming was developed, with which we can make ESC-like flexible cells from any cells in the body. These artificial flexible cells are called 'induced pluripotent stem cells (iPSCs)'. The discovery of iPSCs is a very exciting achievement because it allows us, in theory, to generate iPSCs from any individual and then use them to make new specialized cells that might be needed for studying or treating disease. Inspired by the discovery of iPSCs, researchers have since developed methods for converting one type of specialized cell directly into another, without first going through the ESC-like flexible stage. For example, it is now possible to convert skin cells directly into muscle cells or blood cells in the lab.

How do we achieve these 'reprogramming' and 'cell conversion' processes, which do not happen under normal circumstances inside the body? The trick is to understand a set of important proteins found inside cells called 'master transcription factors'. Each different type of cell, whether it is a specialized cell or an embryonic stem cell, has a unique combination of master transcription factors. These master transcription factors determine what a cell is by controlling how the DNA inside the cell is used. To achieve reprogramming and cell conversion, researchers take the master transcription factors of the kind of cell they want to make, and put them into another type of cell. So, putting the master transcription factors normally found in ESCs into skin cells allows us to reprogram the skin cells into ESC-like cells (iPSCs), overwriting the original skin characteristics of the cells. Putting muscle master transcription factors into skin cells converts them from skin to muscle. The principal is simple but the strategy does not always work well. We often cannot overwrite the cells' original character at all, and when we can achieve conversion it may be with as little as 0.1% efficiency.

One of the possible reasons for the low efficiency of these conversion methods is that the master transcription factors cannot work alone. They often need other proteins to support them. In our preliminary experiments, we have identified a protein that we think boosts the master transcription factors to generate iPSCs more efficiently from skin cells. Excitingly, previously published studies suggest this protein could be a general booster for other master transcription factors, including those used for skin-to-muscle or skin-to-blood-cell conversion. In this project we aim to work out in more detail how this booster factor acts in reprogramming and cell conversion. This study will help us to understand why simply putting the master transcription factors into the cells is not sufficient to achieve cell conversion in most cases, and will enable us to find strategies to improve the technology.

We have previously developed an efficient reprogramming system, in which an iPSC line generated with a single piggyback transposon carrying 4 doxycycline (dox)-inducible reprogramming factors (c-Myc, Klf4, Oct4, Sox2 = MKOS) was used to generate mouse embryonic fibroblasts (secondary MEFs), and subsequently the secondary MEFs were reprogrammed by administration of dox. Using this so-called secondary reprogramming system, as well as the newly identified cell surface markers CD44 and ICAM1, we succeeded in tracking the paths taken by cells becoming iPSCs and performed gene expression profiling of the intermediate subpopulations (O'Malley, et al., Nature, 2013). This work revealed that reprogramming of MEFs is not simply the loss of fibroblastic genes and gain of pluripotency genes, but also includes transient up- and down-regulation of unexpected genes including multiple epidermis genes.

Using this secondary reprogramming system and cell surface markers, we found a signaling molecule which potentially plays a key role for the progression of reprogramming. An inhibitor specific for this molecule retains cells undergoing reprogramming in an intermediate stage. Importantly this inhibitor does not affect proliferation or survival of MEFs and iPSCs, indicating this molecule is important in an intermediate stage specific manner. Literatures suggest that this molecule has potential to act as a co-activator of not only reprogramming factors, but also other master transcription factors in different cell types. In this project we aim to 1) investigate function of this molecule in reprogramming, and 2) understand its function in other master transcription factor-mediated transdifferentiation processes, using knockout MEFs, constitutively active and dominant negative forms of this molecule. We will also address co-occupancy of this molecule and master transcription factors on the target genes during reprogramming and transdifferentiation.

The outcome of the project will have an impact on the stem cell and regenerative medicine research community. While generation of induced pluripotent stem cells (iPSCs), brought large excitement in the stem cell field, mechanisms of the process are not well understood. Although several improved approaches have been developed to generate non-genetically modified iPSCs, the successful rate of iPSC line generation is not yet 100%, quality of iPSCs could vary, and kinetics of iPSC generation has not advanced. A factor we are focusing on in this project has potential to solve some of those issues improving reprogramming efficiency. In addition to the practical benefits, understanding molecular mechanisms underlying successful reprogramming will provide ideas how to generate desired cell types faithfully and efficiently. Following generation of iPSCs, several similar approaches - overexpression of cell type specific master transcription factors - have been taken to achieve transdifferentation. This strategy has a large potential to be applied in order to generate various different cell types, with fully functional outputs. This project addresses a hypothesis that there is a common mechanism which can enhance both reprogramming and transcription factor-mediated transdifferentiation, and has the potential to make an impact on these newly arising technologies.

Scientific achievements from the proposed research will be shared with world-wide scientific communities through national and international conferences as well as by being published in high profile scientific journals. In the long term, impacts on the stem cell research community can be transferred to other research areas such as medicine, developmental biology, and also generate a broader impact on society such as deeper understanding of human biology, medicine, influence on policy, and increasing enthusiasm for science,. There is also a high probability that this research will give rise to intellectual property (IP) associated with reprogramming and transdifferentiation technologies. The Edinburgh BioQuarter adjacent to the SCRM building is established as the commercialization arm of Europe's fastest-growing academic medical centre. IP from the proposed research will be developed by the business development department of the BioQuarter for rapid commercialization.
Scientific outreach is also very active in MRC CRM. Five specialist science communicators in the MRC CRM organize and arrange outreach events involving researchers to explain the hope from stem cell research whilst dispelling hype. My publications in 2009 and 2013 in Nature had world-wide media coverage (BBC, Times, Independent, Guardian, etc), and a press release that was prepared by the science communicators and the university press officer. Outreach events in the past include Science festivals, School visits, Open days, Teacher workshops, Health care meetings, Film events, which involved over 35,000 people in total between 2008 and 2012. My lab is actively involved in those activities and findings from this project will be presented to the general public.