Decoding the bridges and barriers to cellular reprogramming and lineage identity

项目摘要 人体是由大量的细胞组成的。这些类型通常在 感测到一种类型的细胞一旦建立，通常不会切换到其他细胞类型。的主要目标 再生医学和我们对细胞类型的理解是为了发现我们是否以及如何能够迫使细胞 从一种类型转换到另一种类型。在过去十年中，最近的结果表明， 这是一种通过迫使细胞开启一小部分基因而将细胞从一种类型转化为另一种类型的原理。但在 在绝大多数情况下，我们不知道这些基因组是从成千上万的潜在基因中分离出来的。 因此，我们的理解在很大程度上取决于根据先前的知识挑选候选人。 此外，即使当这些基因组被鉴定时，细胞类型的相互转化的效率也是非常低的。 低，只有一小部分源单元格转换为目标类型。 我们提出的研究使用细胞身份的新概念组合来解决这两个挑战 以及追踪单个细胞的新技术。对于小区标识， 一个领域是使用在任何特定细胞类型中哪些基因开启或关闭的概况来确定谱系特异性 因素然而，虽然这些基因是谱系特异性的，但它们在某种意义上可能不是谱系决定基因。 它们本身不会将细胞驱动为特定类型。我们开发了一种方法，我们称之为PerturbID 它使用一系列系统扰动来识别特定的基因，这些基因在反应中打开和关闭， 我们已经证明了它们具有相互转换细胞的能力。我们建议使用PerturbID来识别和验证 在一系列细胞类型的细胞类型转换的候选人，最终达到一组通用的 细胞重编程的原理这将应用于再生医学以及跨 生物学作为一个整体另一个主要问题，效率，仍然是个谜，因为目前没有人 他知道为什么有些细胞能够重新编程，而绝大多数细胞不能。挑战在于 回顾分析细胞的工具：我们如何让时间倒流，分析最终将采用 不同的命运我们已经开发了用于执行这种回顾性分析的工具。我们将应用这个“时间 机器”的方法来解决低效的重编程问题，以确定细胞的独特签名 为细胞类型转换做好准备，并将进行遗传筛选，以分离能够操纵 这个频率。总之，我们的工作将改变我们的细胞类型的概念，并将有巨大的实际意义。 其在再生医学中的应用。

Project Summary The human body is composed of a large number of cell types. These types are generally quite stable in the sense that cells of one type, once established, typically do not switch to other cell types. A major goal for regenerative medicine and our understanding of cell type in general is to discover if and how we can force cells to switch from one type to another. Recent results over roughly the last decade have shown that it is in principle possible to convert cells from one type by forcing them to turn on small sets of genes. However, in the vast majority of cases, we have no idea what these sets of genes are out of the many thousands of potential ones, and so our understanding has largely been dictated by picking candidates based on prior knowledge. Furthermore, even when these sets of genes are identified, the efficiency of interconversion of cell type is very low, with only a small percentage of source cells converting to the target type. Our proposed research tackles both of these challenges using a combination of new concepts of cell identity and new technology for tracing individual cells back in time. For cell identity, the approach most common in the field is to use profiles of which genes are on or off in any particular cell type to determine lineage-specific factors. However, while these genes are lineage-specific, they may not be lineage-determining in the sense that they may not drive a cell to a particular type per se. We have developed a methodology we call PerturbID that uses a series of systematic perturbations to identify specific genes that turn on and off in response, which we have shown have the capacity to interconvert cells. We propose to use PerturbID to identify and validate candidates for cell type transformation across a range of cell types, ultimately arriving at a set of general principles for cellular reprogramming. This will have applications for regenerative medicine as well as across biology as a whole. The other major problem, of efficiency, has remained mysterious because nobody currently knows why some cells are capable of reprogramming while the vast majority are not. The challenge is the lack of tools for retrospective profiling of cells: how do we rewind time to profile the cells that will ultimately adopt a different fate? We have developed tools for performing this retrospective profiling. We will apply this “time machine” methodology to the problem of inefficient reprogramming to determine the unique signature of cells primed for cell type conversion, and will perform genetic screens to isolate pathways capable of manipulating this frequency. Together, our work will transform our concepts of cell type and will have enormous practical implications for its application in regenerative medicine.