Clonal dynamics of the blood stem cell niche

血液干细胞生态位的克隆动力学

基本信息

批准号：
10569876
负责人：
Chloe Sophie Baron
金额：
$ 9.15万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10569876
关键词：
APLN gene Adult Advisory Committees Affect Algorithms Automobile Driving Award Bar Codes Biochemical Biological Assay Blood Blood Cells Blood Vessels Bone Marrow CRISPR/Cas technology Cell Maintenance Cell Proliferation Cells Clonality Clone Cells Color Computer Analysis Computing Methodologies Confocal Microscopy DNA Data Data Set Disease Disease Progression Disease model Dysmyelopoietic Syndromes Embryonic Development Endothelial Cells Endothelium Equilibrium Feedback Fibroblasts Flow Cytometry Gene Expression Profile Genetic Genetic Transcription Goals Hematological Disease Hematopoiesis Hematopoietic Hematopoietic stem cells Heterogeneity Hormone secretion Human In Vitro Journals Kidney Knowledge Laboratories Ligands Marrow Mediating Mentorship Methods Microscopic Modeling Molecular Mus Mutagenesis Organism Output Pathogenesis Pathway interactions Play Prize Proliferating Proto-Oncogenes Recovery Reporter Research Research Personnel Role Science Signal Pathway Signal Transduction Stromal Cells Technical Expertise Technology Testing Transgenic Organisms Transplantation Vascular Permeabilities Vascular remodeling Visualization Work Zebrafish adrenomedullin angiogenesis c-myc Genes cancer cell candidate identification career career development cell type computational basis confocal imaging hematopoietic stem cell niche improved in silico in vivo in vivo evaluation mosaic mouse model multidisciplinary multiple omics new therapeutic target next generation sequencing novel overexpression prevent progenitor programs promoter receptor reflectance confocal microscopy research and development response single cell mRNA sequencing stem stem cell biology symposium tool transcriptome transcriptomics

项目摘要

PROJECT SUMMARY The mechanisms by which the hematopoietic stem and progenitor (HSPC) niche is affected by clonal hematological disorders such as myelodysplastic syndrome (MDS) remain poorly understood. Furthermore, the heterogeneity and clonal response of endothelial and stromal cells (the main components of the HSPC niche) in MDS in vivo remain unexplored. To tackle these aspects, I developed a new zebrafish model of MDS by driving the protooncogene CMYC overexpression specifically in blood cells. Additionally, I crossed a genetic lineage tracing zebrafish line called GESTALT to a double transgenic zebrafish line carrying two fluorescent reporters allowing to purify specifically niche endothelial and stromal cells. This way, I created a new GESTALT line that permits CRISPR-CAS9 based barcoding during zebrafish embryonic development, purification of adult marrow niche cells and recovery of niche DNA barcodes by sequencing. Combining these novel tools, I induced MDS in barcoded zebrafish and read out the clonality and the transcriptome of endothelial and stromal cells. I discovered that clones of stromal cells selectively expand, and endothelial cells are transcriptionally remodeled in MDS. Given these data, I hypothesize that MDS remodels the clonality and transcriptional profile of the HSPC niche and that mechanisms involved in HSPC-niche interactions promote disease progression. Under the mentorship of Dr. Leonard Zon, I will investigate the mechanisms by which MDS remodels the niche using a combination of in silico computational approaches, genetic (GESTALT) and color based (Zebrabow) lineage tracing, confocal microscopy and in vivo mosaic mutagenesis. Once I establish my laboratory, I will build a multidisciplinary team to deepen my computational analyses and broaden my in vivo genetic and biochemical perturbations of the clonal mechanisms of niche involvement in MDS. My overarching goal is to identify novel targetable mechanisms specific to the HSPC niche that would prevent and/or halt MDS progression. This K99/R00 award will enable me to develop new technical skills, participate in courses that will improve my ability to manage a laboratory, and attend conferences that will broaden my network and my knowledge of hematological disease modeling, Zebrabow lineage tracing paired with confocal microscopy and zebrafish mutagenesis. The scientific advisory committee I have put together includes experts in the fields of hematopoiesis, lineage tracing, and stem cell biology and, along with Dr. Zon, will give me feedback on my research and career progress. These proposed research and career development activities will pave the way for me to become an independent investigator discovering and studying new mechanisms responsible for hematopoietic disorders progression mediated by the blood stem cell niche.

项目概要克隆微环境影响造血干细胞和祖细胞（HSPC）生态位的机制骨髓增生异常综合征 (MDS) 等血液系统疾病仍然知之甚少。此外，内皮细胞和基质细胞（HSPC 生态位的主要成分）的异质性和克隆反应体内 MDS 仍未得到探索。为了解决这些问题，我通过驱动开发了一种新的 MDS 斑马鱼模型原癌基因 CMYC 在血细胞中过度表达。此外，我跨越了遗传谱系追踪称为 GESTALT 的斑马鱼品系到携带两个荧光报告基因的双转基因斑马鱼品系允许特异性纯化利基内皮细胞和基质细胞。这样，我创建了一条新的 GESTALT 线允许在斑马鱼胚胎发育、成体骨髓纯化过程中进行基于 CRISPR-CAS9 的条形码利基细胞和通过测序恢复利基 DNA 条形码。结合这些新工具，我诱导了 MDS 对斑马鱼进行条形码标记并读出内皮细胞和基质细胞的克隆性和转录组。我发现 MDS 中基质细胞克隆选择性扩增，内皮细胞进行转录重塑。鉴于这些数据，我假设 MDS 重塑了 HSPC 的克隆性和转录谱生态位以及参与 HSPC-生态位相互作用的机制促进疾病进展。在下面在 Leonard Zon 博士的指导下，我将研究 MDS 使用以下方法重塑利基的机制：计算机计算方法、遗传 (GESTALT) 和基于颜色 (Zebrabow) 谱系的组合示踪、共焦显微镜和体内嵌合突变。一旦我建立了我的实验室，我将建立一个多学科团队深化我的计算分析并拓宽我的体内遗传和生化 MDS 中涉及生态位的克隆机制的扰动。我的首要目标是识别小说 HSPC 利基特有的可预防和/或阻止 MDS 进展的靶向机制。这 K99/R00 奖项将使我能够发展新的技术技能，参加能够提高我的能力的课程管理实验室并参加会议，这将扩大我的网络和我的知识血液疾病模型、斑马弓谱系追踪与共聚焦显微镜和斑马鱼配对诱变。我组建的科学顾问委员会包括以下领域的专家造血、谱系追踪和干细胞生物学，并将与 Zon 博士一起向我提供有关我的研究的反馈研究和职业进步。这些拟议的研究和职业发展活动将为我成为一名独立调查员，发现和研究负责的新机制由血液干细胞生态位介导的造血障碍进展。