Illuminating molecular mechanisms required for efficient reprogramming and transdiffrentiation

阐明有效重编程和转分化所需的分子机制

• 批准号: BB/L023474/1
• 负责人: Keisuke Kaji
• 金额: $ 52.46万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL023474%2F1
• 关键词: Illuminating; molecular; mechanisms; required; efficient

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.

我们的身体由大约300种不同类型的细胞组成，每种细胞都有不同的专门作用。然而，我们并不总是由所有这些不同的细胞组成。在早期的人类胚胎中，它是一个只有100-150个细胞的球，所有这些细胞都是完全非特异性的。在这个阶段，每个细胞都可以分裂产生身体的任何细胞。这种灵活性被称为“多能性”。随着胚胎的发育和身体的形成，细胞不断分裂，逐渐变得更加专业化，失去了灵活性。为了使细胞保持多能性，我们必须将它们从早期胚胎中取出，并为它们提供保持分裂所需的特殊条件，而不是专门化;复制自己。从早期胚胎中提取的细胞在这样的培养皿中繁殖，称为胚胎干细胞（ESC）。只要有正确的线索，它们就可以在体内产生任何类型的特化细胞。直到最近，我们还认为细胞一旦特化，就不可能改变它们的特性--比如说，将它们从皮肤细胞转化为肌肉细胞。然而，在2006年，一种称为重编程的技术被开发出来，通过这种技术，我们可以从体内的任何细胞中制造出类似ESC的柔性细胞。这些人工柔性细胞被称为“诱导多能干细胞（iPSCs）”。iPSC的发现是一项非常令人兴奋的成就，因为理论上，它允许我们从任何个体中产生iPSC，然后用它们来制造研究或治疗疾病所需的新的特化细胞。受到iPSC发现的启发，研究人员开发了将一种类型的特化细胞直接转化为另一种类型的特化细胞的方法，而无需首先经历类似ESC的灵活阶段。例如，现在可以在实验室中将皮肤细胞直接转化为肌肉细胞或血细胞。我们如何实现这些“重新编程”和“细胞转换”过程，这些过程在正常情况下不会发生在体内？关键是要了解细胞内发现的一组重要蛋白质，称为“主转录因子”。每种不同类型的细胞，无论是特化细胞还是胚胎干细胞，都具有独特的主转录因子组合。这些主转录因子通过控制细胞内DNA的使用方式来决定细胞是什么。为了实现重编程和细胞转化，研究人员将他们想要制造的细胞类型的主转录因子放入另一种细胞中。因此，将通常在ESC中发现的主转录因子放入皮肤细胞中，可以使我们将皮肤细胞重新编程为ESC样细胞（iPSCs），从而恢复细胞的原始皮肤特征。将肌肉主转录因子放入皮肤细胞，将它们从皮肤转化为肌肉。原则很简单，但策略并不总是奏效。我们通常根本无法改写细胞的原始特征，即使我们能够实现转换，效率也可能只有0.1%。这些转换方法效率低的可能原因之一是主转录因子不能单独工作。它们通常需要其他蛋白质来支持它们。在我们的初步实验中，我们已经确定了一种蛋白质，我们认为它可以促进主转录因子，从而更有效地从皮肤细胞中产生iPSC。令人兴奋的是，先前发表的研究表明，这种蛋白质可能是其他主转录因子的通用助推器，包括用于皮肤到肌肉或皮肤到血细胞转换的转录因子。在这个项目中，我们的目标是更详细地研究这种助推因子在重编程和细胞转化中的作用。这项研究将帮助我们理解为什么在大多数情况下，简单地将主转录因子放入细胞中不足以实现细胞转化，并将使我们能够找到改进技术的策略。

项目成果

Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming.

• DOI: 10.1016/j.stem.2017.10.013
• 发表时间: 2017-12-07
• 期刊: Cell stem cell
• 影响因子: 23.9
• 作者: Ruetz T;Pfisterer U;Di Stefano B;Ashmore J;Beniazza M;Tian TV;Kaemena DF;Tosti L;Tan W;Manning JR;Chantzoura E;Ottosson DR;Collombet S;Johnsson A;Cohen E;Yusa K;Linnarsson S;Graf T;Parmar M;Kaji K
• 通讯作者: Kaji K