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

在我们的身体里，皮肤细胞只产生皮肤，肌肉细胞只产生肌肉，骨细胞只产生骨骼，等等。然而，我们的整个身体是由子宫里的一个细胞，一个受精卵产生的。这个细胞，生命之源，受精后就开始分裂，变成2个细胞、4个细胞、8个细胞、16个细胞，不断分裂，逐渐形成胚胎的主体。在胚胎发育的初期，直到受精后大约1周，当细胞群看起来仍然像一个球时，我们的细胞可以产生大约300种不同的细胞类型，这些细胞类型协调了我们成人身体的形成。然而，在这个阶段之后，子宫里的细胞开始分化，在未来的身体中产生特定的细胞类型。细胞开始向体内一个特殊细胞做出承诺的方式通常被描述为一个滚下山的球。在山顶上，球可以进入底部的任何山谷。然而，一旦球从山顶落下，它所经过的山谷就会被限制在山脊的同一侧。当球滚动到山的较低位置时，能够向下移动的山谷的选择变得更加有限。同样，当婴儿出生时，体内细胞产生不同细胞类型的能力非常有限。使细胞具有多种潜能并产生所有细胞类型的唯一方法是在受精后一周左右从子宫取出胚胎，并将其放入塑料培养皿中，培养皿中含有营养丰富的液体，其中含有最佳条件，以防止细胞进一步特化。这些可以制造人体所有细胞类型的未固定细胞因此被称为“多能干细胞”，因为它们来源于胚胎，所以被称为胚胎干细胞（ESCs）。干细胞逐渐且不可逆地特化的一般发育原理，在2006年被一个非常简单的实验重写。一组科学家发现，只要同时人工操纵4个基因，任何特化的细胞都可以变成像ESCs这样的多能细胞。这种让细胞生物钟倒转的技巧被称为“重编程”，而由此产生的人工生成的非定向细胞被称为“诱导多能干细胞”。这一发现给干细胞和医学研究界带来了巨大的兴奋。多能干细胞可以从体内的任何细胞（例如你的皮肤细胞）生成，理论上可以用于生产移植、药物筛选、毒理学测试等所需的任何细胞类型。目前，诱导多能干细胞的成功生成并不是100%有效，重编程的过程需要一个多月的时间，而且是一个昂贵的过程。这项技术还远远没有把它的优点传递给社会。此外，令人惊讶的是，我们仍然不知道为什么仅操纵4个基因就能诱导多能性，从而消除特化细胞的特征。因此，在本项目中，我们旨在利用最先进的分子生物学技术阐明重编程的分子机制。在第一个目标中，我们的目标是揭示有效重编程的抑制或必需基因。在第二个目标中，我们将研究被操纵的4个基因如何简洁有效地执行重编程过程。作为该项目的一个成果，我们期望有策略在更短的时间内以更高的成功率产生多能干细胞，并更好地了解我们如何操纵特化细胞和/或多能细胞来产生医疗应用所需的不同特化细胞。

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.