Dissecting and reconstructing the molecular roadmaps of cellular reprogramming to iPSCs in single-cell resolution

项目摘要 了解引导细胞命运转换的分子程序将为 开发工具来改变细胞命运并最终促进治疗相关细胞的产生 类型。理解这种机制的实验方法通常涉及到对大量 人口。这些实验受到严重限制，因为它们只能测量平均效应。 最近，大规模的单细胞图谱为系统解剖细胞开辟了新前景 处理潜在的细胞命运转换。然而，对大规模单元格数据的适当分析仍然是一个 挑战。为了解决这个问题，我们开发了实验和计算方法来研究scrna-seq。 在成纤维细胞重新编程期间，16天内在10个时间点收集的65,781个细胞的数据 IPSCs by Oct4、Sox2、Klf4和cMyc。在我的K99/R00提案中，我假设解剖复合体 单细胞分辨率的重新编程过程将帮助我们理解重新编程的机制 对于IPSC，确定可以提高重新编程效率并产生高- 可在临床环境中使用的优质IPSCs。我建议1)描述候选人的角色 重编程因子(K99)；2)验证从单细胞RNA预测的重编程轨迹- 通过血统追踪(K99)获得SEQ数据；3)通过比较 不同的鸡尾酒(K99/R00)；4)开发新的方法来提高细胞命运转换(R00)。一起， 拟议的目标将对了解发展过程和 为科学界提供丰富的资源。从长远来看，这些研究可能会揭示新的战略。 以产生治疗相关的细胞。为了实现这些拟议的目标，我需要在以下方面进行额外的培训 计算分析和干细胞研究，由我的合作导师Eric Lander博士(遗传学和 )和Rudolf Jaenisch博士(干细胞和基因组工程)以及一个咨询委员会 包括阿维夫·雷格夫博士(计算生物学和单细胞技术)、张峰博士(基因组 工程学)。我的职业发展计划包括计算工具和实验工具方面的实践培训 以及沟通、管理、指导、拨款撰写等方面的培训。博德学院是一个 理想的环境，提供拟议研究所需的所有设施和丰富的跨学科 环境。通过NIH K99/R00途径的支持获得的这些额外技能 独立奖，我将有资格执行这些目标，在干的界面上大踏步前进 细胞研究和单细胞技术。

Project Summary Understanding the molecular programs that guide cell fate conversion will provide a foundation for the development of tools to convert cell fate and eventually facilitate the generation of therapeutically relevant cell types. Experimental approaches to understand such mechanisms have typically involved studying bulk populations. These experiments are severely limited because they can only measure averaged effects. Recently, large-scale profiling of single cells has opened new prospects for systematically dissecting the processes underlying cell fate conversion. However, proper analysis of large-scale single cell data remains a challenge. To combat this, we developed experimental and computational approaches to study scRNA-seq data from 65,781 cells collected at 10 time points over 16 days during the reprogramming of fibroblasts to iPSCs by Oct4, Sox2, Klf4, and cMyc. In my K99/R00 proposal, I hypothesize that dissecting complex reprogramming processes in single-cell resolution will help us understand the mechanisms of reprogramming for iPSCs, identify novel reprogramming factors that can enhance reprogramming efficiency and generate high- quality iPSCs that can be used in clinical settings. I propose to 1) characterize the role of candidate reprograming factors (K99); 2) validate the reprogramming trajectory predicted from single-cell RNA- seq data by lineage tracing (K99); 3) investigate the reprogramming process through comparison of different cocktails (K99/R00); 4) develop new methods to enhance cell fate conversion (R00). Together, the proposed aims will have a broad impact on the journey to understand developmental processes and provide rich resources for the scientific community. In the long term, these studies may reveal novel strategies to generate therapeutically relevant cells. To succeed in these proposed aims, I will need additional training in computational analysis and stem cell research, supported by my co-mentors Dr. Eric Lander (genetics and genomics) and Dr. Rudolf Jaenisch (stem cells and genome engineering) as well as an Advisory Committee including Dr. Aviv Regev (computational biology and single-cell techniques), Dr. Feng Zhang (genome engineering). My career development plan integrates practical training in computational and experimental tools as well as trainings in communication, management, mentorship, grant writing, etc. The Broad Institute is an ideal environment, providing all of the facilities needed for the proposed research and a rich interdisciplinary environment. With these additional skills gained through support by the NIH K99/R00 Pathway to Independence Award, I will be qualified to execute these goals to make great strides at the interface of stem cell research and single-cell techniques.