Decoding the bridges and barriers to cellular reprogramming and lineage identity

项目摘要 人体由大量的细胞类型组成。这些类型通常在 感觉到一种类型细胞一旦建立，通常不会切换到其他类型的细胞。的一个主要目标 再生医学和我们对细胞类型的一般理解是发现我们是否以及如何迫使细胞 从一种类型切换到另一种类型。最近大约在过去十年的结果表明，它是在 使细胞从一种类型转化为一种类型的原理，方法是强迫细胞打开一组小的基因。然而，在 在绝大多数情况下，我们不知道这些基因是什么，在成千上万的潜在基因中 因此，我们的理解在很大程度上取决于根据先验知识挑选候选人。 此外，即使识别了这些基因集，细胞类型的相互转换效率也非常高 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的组合来解决这两个挑战 以及追溯单个细胞的新技术。对于小区标识，最常见的方法是 FIELD是使用任何特定细胞类型中哪些基因处于开启或关闭状态的配置文件来确定特定血统 各种因素。然而，虽然这些基因是特定于家族的，但它们在某种意义上可能并不是家族决定的 它们本身可能不会将细胞驱动到特定类型。我们开发了一种称为摄动ID的方法 它使用一系列系统的扰动来识别特定的基因，这些基因作为回应打开和关闭， 我们已经证明了有能力相互转换细胞。我们建议使用PerturID来识别和验证 在一系列单元格类型之间进行单元格类型转换的候选对象，最终得出一组通用的 细胞重新编程的原则。这将适用于再生医学以及整个 生物学作为一个整体。另一个主要问题，效率，一直是个谜，因为目前没有人 知道为什么有些细胞能够重新编程，而绝大多数细胞不能。挑战在于缺乏 回溯分析细胞的工具：我们如何回溯时间来分析最终将采用 不同的命运？我们已经开发了用于执行这种回溯性分析的工具。我们将应用这一“时间” 确定细胞唯一特征的低效重编程问题的“机器”方法学 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的通路 这个频率。我们的工作将共同改变我们对细胞类型的概念，并将具有巨大的实用价值 对其在再生医学中应用的启示。

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.