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microRNA regulation of apoptosis and differentiation during early mammalian development

Final Report Summary - MEMD (microRNA regulation of apoptosis and differentiation during early mammalian development)

Executive Summary:

Soon after fertilization, the mammalian embryo is composed of cells that have the ability to turn into all the cell types that are found in the body. These are called embryonic stem cells and as the embryo develops they gradually form more specialized cell types that are no longer able to contribute to all body tissues. This process is called cell differentiation. In the mouse embryo we can find stem cells with the ability to form any cell type until approximately the seventh day after fertilization, however the characteristics of these stem cells change depending on how close they are to differentiating into more specialized cell types. For example stem cells found in the mouse embryo three days after fertilization have different properties from the ones found six days after fertilization. Embryonic stem cells can be obtained from embryos and grown in a dish in conditions that allow them to retain their identity and by changing these conditions we can make them form particular cell types –such as neurons, skin or muscle. For this reason they have the potential to be powerful tools for therapies like tissue regeneration. Most of the work performed using mouse embryonic stem cells as a model have focused on the study of cells obtained from the three-day embryo, however recent studies show that human stem cells are actually more similar to the ones found in the six-day mouse embryo. Studying the events that occur in the mouse embryo at around this time of development is therefore of special relevance to gain insight into the biology of human stem cells. Understanding the biology of stem cells in the context of embryo development also has important implications in diseases like cancer, where healthy fully differentiated/specialised adult cells undergo the reverse process to become a cell type that resembles the stem cells found in the embryo.

The identity and fate of any cell are given by the specific combination of proteins present in it. Every cell type contain the same set of instructions, encoded in the genes, for making all the different proteins found in an organism. Which parts of these instructions are used in a cell determines which proteins are made. Therefore the process of instruction selection and usage is tightly controlled by numerous factors. Among these factors are microRNAs, small molecules that block the formation of specific proteins when these are not required. MicroRNAs have been found to be essential for the maintenance of healthy cells and their miss-function has been associated with the appearance of numerous illnesses l such as heart disease and cancer.

In this project we aimed to study the role microRNAs play in the development of the mammalian embryo and specifically how these molecules affect the identity and survival of the stem cells found in the mouse embryo six days after fertilization, which would be equivalent to human stem cells. Each cell type contains a specific combination of microRNAs and each microRNA controls the formation of a particular group of proteins. Therefore as a first objective we identified the specific combination of microRNAs present in the stem cells that form the six-day embryo. We found that this set of microRNAs not only is similar to the one found in human stem cells, but also contains microRNAs that are very abundant in cancer cells and that have been related to the formation or progress of tumours. This confirmed the relevance of the model used to study the importance of microRNAs in stem cell biology.

Once the set of microRNAs present in the stem cells of the embryo was identified, the second objective of the project involved determining how important these molecules are to the correct function of stem cells. We first asked what would happen to embryonic stem cells if they did not have these regulators. We found that if we eliminate all the microRNAs from the stem cells of the six-day old embryo the cells die. However if we eliminate them from stem cells of the three-day old embryo (which are further away from beginning to become specialized), the cells survive. This allowed us to conclude that microRNAs are essential for the survival of stem cells but only when they are about to start differentiating.

As the role of microRNAs is to stop protein formation, the elimination of microRNAs from stem cells leads to the formation of proteins that should not be there, and these are likely to be the ones that are killing the cells. We compared the levels of a number of proteins in stem cells with and without microRNAs in order to determine which ones are more abundant when microRNAs are not present. This allowed us to identify a number of proteins whose production is blocked by microRNAs during early embryo development. Among these proteins we found one, called Bim, that promotes cell death and which is normally produced by unhealthy cells that need to be eliminated. Because Bim promotes cell death its production needs to be blocked in healthy cells and we found that microRNAs are in charge of performing this function in the stem cells of the six-day old embryo. Surprisingly, high levels of Bim do not kill the stem cells of the three-day old embryo; this probably explains why microRNAs are not required for the survival of these cells.

Further studies confirmed that as embryonic stem cells get closer to changing into more specialized cell types (i.e. as the mouse embryo develops from three to six day old) they become sensitive to cell death inducing agents and develop a requirement for microRNAs to stop the formation of proteins that would otherwise kill them. This finding is of special interest because when healthy adult cells become cancerous they develop some of the features normally seen in embryonic cells, including becoming more difficult to kill using cell death inducing agents. Indeed, the development of drugs capable of killing tumour cells is one of the main approaches to cancer treatment. Therefore knowing what occurs when stem cells start the differentiation process that makes them susceptible to cell death-inducing agents will open up new research lines that could contribute to our understanding of cancer cell biology.

Overall, the results obtained in this project represent an important advance to out knowledge of the processes that occur during early mammalian development and that allow the maintenance of stem cells survival. However the findings of this study could have a much wider importance for the understanding of the roles that microRNAs play in disease. Unraveling the control of cell death has important implications for diseases such as cancer, were some developmental features are reactivated and cells become resistant to cell death-inducing agents. Indeed, the microRNAs that we have found to be very abundant in embryos have been related to oncogenic processes. In addition, this study contributes to the understanding of stem cell biology which is of great importance to regenerative medicine, where the main challenges are to efficiently and safely convert fully differentiated or specialized adult cells into ‘embryonic-like’ cells with the ability to form all cell types, and the use of those cells for the generation of specific tissues and organs.