Genome-wide exploration of reprogramming mechanisms using CRISPR/Cas9 and DamID technologies

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In our body, skin cells generate only skin, muscle cells make only muscle, bone cells make only bone and so forth. However, our whole body was generated from a single cell, a fertilized egg, in the womb. This cell, the source of life, starts to divide just after fertilization and become 2 cells, 4 cells, 8 cells, 16 cells, continuing to divide and gradually form the body of an embryo. In the beginning of embryo development, until about 1 week after fertilization when the mass of cells still look like a ball, we have cells that can generate all ~300 different cell types which orchestrate the formation of our adult body. However, after this stage, cells in the womb start being specialized to generate specific cell types in the future body. The way cells start making commitments to a specialized cell in a body is often described as a ball rolling down a mountain. On the top of the mountain, the ball can go in to any valley at the bottom. However, once the ball drops from the top of the mountain, the valleys it can go to become restricted to the same side of the ridge. When the ball rolls down to a lower position of the mountain, choices of valleys to be able to travel down become more limited. Similarly, when a baby is born, cells in the body have very limited capacity to generate different cell types. The only way to keep cells with mulit-potential to generate all cell types is taking an embryo from the womb about 1 week after fertilization and put it in a plastic dish with nutrient rich liquid containing optimal conditions to prevent further specialization of the cells. These uncommitted cells that can make all cell types of the body are therefore called 'pluripotent stem cells', and because they arederived from an embryo they are called embryonic stem cells (ESCs). The general principles of development, whereby stem cells gradually and irreversibly become specialized, were re-written by a very simple experiment in 2006. A group of scientists found that any specialized cells can become uncommitted pluripotent cells like ESCs by artificially manipulating only 4 genes simultaneously. This trick that rewound the cells' biological time clock is called 'reprogramming' and the resulting artificially generated uncommitted cells are called 'induced pluripotent stem cells (iPSCs)'. The findings brought tremendous excitement to the stem cell and medical research community. iPSCs can be generated from any cell in the body (e.g. your skin cells) and in theory can be used for the production of any desired cell types for transplantation, drug screening, toxicology tests and so forth. At the moment, successful generation of iPSCs is not 100% efficient, the process of reprogramming takes over 1 month and is an expensive procedure. The technology is still far from delivering its merits to the society. In addition, surprisingly, we still do not know why the manipulation of only 4 genes can induce pluripotency, erasing the character of specialized cells. Therefore in this project we aim to illuminate molecular mechanism of reprogramming using state-of-the-art molecular biology techniques. In the first objective, we aim to reveal genes that are inhibitory or essential for efficient reprogramming. In the second objective, we will investigate how the manipulated 4 genes concisely and effectively execute the process of reprogramming. As an outcome of the project we expect to have strategies to generate iPSCs with higher success and in a shorter time period, and also better understanding how we can manipulate specialized cells and/or pluripotent cells to generate different specialized cells required for medical applications.