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

以单细胞分辨率剖析和重建 iPSC 细胞重编程的分子路线图

• 批准号: 10320077
• 负责人: Jian Shu
• 金额: $ 24.14万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320077
• 关键词: ATAC-seq; Advisory Committees; Award; Bar Codes; Biological Assay; Biology; CRISPR-mediated transcriptional activation; Cell Therapy; Cell physiology; Cells; ChIP-seq; Clinical; Collecting Cell; Communication; Communities; Complex; Computational Biology; Computer Analysis; Computer Models; Coupled; Data; Data Set; Development; Development Plans; Developmental Process; Embryo; Environment; Exposure to; Fibroblasts; Foundations; Generations; Genes; Genetic; Genome engineering; Genomics; Goals; Grant; Heterogeneity; Homeobox; Homeobox Genes; Individual; Institutes; Lead; Lentivirus Vector; Longitudinal Studies; Mathematics; Measures; Mentors; Mentorship; Methods; Modeling; Modernization; Molecular; Oocytes; Pathway interactions; Population; Predictive Factor; Process; Research; Research Support; Resolution; Resources; Role; Route; Stem Cell Research; Techniques; Testing; Therapeutic; Time; Training; United States National Institutes of Health; Writing; base; c-myc Genes; career development; cell type; collaborative environment; combat; combinatorial; computerized tools; embryonic stem cell; engineered stem cells; experimental study; gene regulatory network; induced pluripotent stem cell; insight; loss of function; novel; novel strategies; overexpression; pluripotency; programs; single-cell RNA sequencing; skills; tool; tool development; zygote

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.

项目摘要 了解指导细胞命运转换的分子程序将为细胞的进化提供基础。 开发工具以转换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研究大量的 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，单细胞的大规模分析为系统地解剖细胞开辟了新的前景。 细胞命运转换的基础过程。然而，对大规模单细胞数据的适当分析仍然是一个难题。 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 在成纤维细胞重编程过程中，在16天内的10个时间点收集了65，781个细胞的数据， 通过Oct 4、Sox 2、Klf 4和cMyc的iPSC。在我的K99/R 00提案中，我假设解剖复合体 单细胞分辨率的重编程过程将有助于我们理解重编程的机制 对于iPSCs，识别新的重编程因子，可以提高重编程效率并产生高水平的 可用于临床环境的优质iPSC。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- 通过谱系追踪（K99）的seq数据; 3）通过比较 不同的鸡尾酒（K99/R 00）; 4）开发新的方法来增强细胞命运转换（R 00）。我们一起努力， 拟议的目标将对理解发展进程的旅程产生广泛影响， 为科学界提供了丰富的资源。从长远来看，这些研究可能会揭示新的策略， 以产生治疗相关的细胞。为了实现这些目标，我需要接受额外的培训， 计算分析和干细胞研究，由我的共同导师埃里克兰德博士（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Aviv Regev博士（计算生物学和单细胞技术）、Feng Zhang博士（基因组 工程）。我的职业发展计划整合了计算和实验工具的实践培训 以及沟通、管理、导师、资助撰写等方面的培训。布罗德研究所是一个 理想的环境，提供所有的设施所需的拟议的研究和丰富的跨学科 环境通过NIH K99/R 00 Pathway的支持获得了这些额外的技能， 获得独立奖后，我将有资格执行这些目标，在STEM接口方面取得长足进步 细胞研究和单细胞技术。