Mechanisms of miRNA regulation of early embryonic development

During the development of an embryo, cells transit through a stage where they possess the potential to give rise to all the cell types and tissues that will form the newborn organism. This potential is called pluripotency, and those cells that are pluripotent are stem cells and hold great promise for regenerative medicine. This promise is not only due to their future clinical applications in cellular therapies, but also because now patient specific pluripotent cells can be generated for "disease-in-a-dish" approaches to understand human diseases. Stem cell therapies are being developed for diseases ranging from diabetes to neurodegenerative disorders and a stem cell based mechanism is thought to underlie different forms of cancer.
The cells that are in the pluripotent state need to make choices regarding the cell type that they will give rise to and pass strict quality controls, as only those that are not defective will survive. This implies that pluripotent cells are under tight regulation and understanding this regulation is important not only to gain insight into how an embryo forms but as well to be able to use pluripotent cells for regenerative medicine. This project aims to understand the regulation of the cell survival and differentiation programs in pluripotent cells.
MicroRNAs are small non-coding RNAs that inhibit gene expression and have emerged over the past decade as key regulators involved in many processes including development, cell proliferation, apoptosis and in numerous diseases, including cancer. In the preliminary work leading up to this proposal we have found that microRNAs are required for the survival of pluripotent cells and for these cells to differentiate into neural cell types. These observations therefore have directly implicated microRNAs in the regulation of pluripotent cells. Understanding how microRNAs regulate cell survival and differentiation of the pluripotent state is the main focus of this project.
As a first step we will use mouse genetics as well as pluripotent stem cell lines for "in a dish" approaches to delete Dicer, that is the protein that is essential for microRNA generation. By studying the effects of Dicer deletion, and therefore microRNA depletion, in the embryo and in pluripotent stem cell lines we will be able to uncover precisely what the roles of microRNAs are in the regulation of the pluripotent state.
MicroRNAs fall into families according to the genes that they inhibit, those within a family are thought to target the same genes. Our preliminary work to this project identified microRNAs of the miR-291a-3p, miR-17, miR-19 and miR-92 families as strong candidates for being involved in the regulation of the survival and differentiation of pluripotent cells. For these reasons our work will focus on these microRNAs and test which families can rescue the defects caused by the depletion of microRNAs in pluripotent stem cells.
Finally, we aim to identify what genes are targeted by the microRNAs that regulate pluripotent cell survival and differentiation. This will establish the microRNA-target interactions that regulate pluripotency. Given that microRNAs inhibit gene expression, we will study what genes decrease in expression when cells are treated with a given microRNA. Given that many cell types are saturated with microRNAs, we will use Dicer deficient pluripotent stem cells for these studies, as they represent a microRNA free cell type. Bioinformatic programs to predict microRNA targets will be used to generate a list of likely microRNA target genes from those identified experimentally. The best candidate genes will be validated by mutation of the microRNA target site in it. This will establish which interactions are functionally important.

Understanding the regulatory networks underlying pluripotency and differentiation is essential to effectively use stem cells for therapeutic applications. Two distinct phases of pluripotency have been proposed, a naïve state and a primed state and these states are thought to be regulated by different mechanisms. microRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression and have been shown to be required to for proper proliferation and exit of the naïve pluripotent state. However little is known about their roles in the primed phase of pluripotency. In preliminary work to this proposal we have found that in contrast to the naïve pluripotent state, miRNAs regulate cell survival and neural differentiation of primed pluripotent cells. The main focus of this project is to understand how miRNAs regulate these processes. Dicer is an enzyme that is essential for the production of mature microRNAs. We will first establish the functional requirements of miRNAs in the epiblast by analysing embryos in which Dicer has been specifically deleted in primed pluripotent cells or in derivatives of each of the germ layers. These studies will be complemented with the analysis of the effects that Dicer deletion has on the survival and differentiation of epiblast stem cells (EpiSCs). Next we will identify what microRNAs regulate the primed pluripotent state by determining which miRNAs can rescue the defects of Dicer deleted EpiSCs. Finally we will establish the miRNA-mRNA target interactions that regulate primed epiblast pluripotency. For this we will transfect single miRNAs into Dicer deleted EpiSCs and study what genes become downregulated. These genes will be compared to those obtained by bioinformatic prediction of microRNA targets. The best candidate genes will be validated by mutation of the microRNA target site in them to establish which interactions are functionally important.

The large promise that pluripotent stem cells hold for regenerative medicine, where stem cell are induced to differentiate towards specialised cells that are then transplanted into patients, has carried with it a large degree of interest both from the academic and medical communities Furthermore, with the advances in reprogramming there is an enormous scope for applying in vitro differentiation of patient-derived induced pluripotent stem (iPS) cells to study diseases. However, increased knowledge about how specific cell types are induced in an irreversible way as well as how to differentiate cells down specific lineages is vital to control that the correct cell types are generated before these stem cell therapy becomes meaningful clinically. Our work aims to unravel the roles of microRNAs in the control of pluripotency and neuronal differentiation and therefore is at the core of this interest. Answering the questions of how do the roles of microRNAs differ in the naïve and primed pluripotent states, how do microRNAs control stem cell survival and neuronal differentiation of pluripotent stem cells, what microRNAs have roles in these processes and what their key targets are, will give fundamental insights into the regulatory networks that control pluripotency.
In the academic environment, those working in the UK and abroad, in the fields of developmental biology and stem cell biology as well as those interested in the diseases that are potential targets for stem cell therapy (such as neurodegenerative diseases) as well as those that possibly have a stem cell origin or where the microRNAs families we study have been involved, such as cancer, are potential beneficiaries of our work. The ability of microRNAs to reprogram differentiated cells to the pluripotent state indicates that our work will also be of relevance to those with an interest in reprogramming, as we will identify those microRNAs that are required for pluripotent stem cell survival.
MicroRNAs have a big potential as therapeutic agents/drug targets and given the importance of their role in pluripotency, they will undoubtedly have an impact in regenerative medicine. Therefore the results obtained in this study will be of direct relevance to translational research and have a potential commercial interest. Furthermore, given the role we have identified fore microRNAs in neural differentiation it is likely that our findings will also impact by improving the protocols designed for the differentiation of stem cells towards neuronal subtypes that are of interest to regenerative medicine. We will exploit these potential commercial interests through Imperial College Innovations so as to identify the most suitable partners.
A further beneficiary of our work will be the lay public. Given the medical relevance and ethical implications of stem cells, our work will provide factual input and therefore benefit the public discussion about the advantages and risks of stem cell therapy. We take seriously the responsibility of scientists to engage with the lay public, to raise awareness among them of the results of publically funded research, to openly debate ethical issues relating to our research so that public opinion may be formed in an informed manner and to take the excitement of our research to the children of today, who will be the scientists of tomorrow. For these reasons we engage through the university but as well as individuals in activities aimed at the public diffusion of science.