Clonal dynamics of the blood stem cell niche

血液干细胞生态位的克隆动力学

• 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
• 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

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.

项目摘要 造血茎和祖细胞（HSPC）小裂的机制受克隆的影响 血液学疾病（例如骨髓增生性综合征（MDS））仍然了解不足。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 MDS IN VIVO仍未开发。为了解决这些方面，我通过开车开发了一种新的斑马鱼模型 原子能中的CMYC过表达专门在血细胞中。此外，我越过了遗传谱系 追踪斑马鱼线，称为格式塔（Gestalt 允许纯化特定的小众内皮细胞和基质细胞。这样，我创建了一条新的格式塔线 允许基于CRISPR-CAS9的条形码在斑马鱼胚胎开发过程中，成人骨髓的纯化 利基细胞和通过测序恢复小裂DNA条形码。结合了这些新颖的工具，我诱导了MD 条形码斑马鱼并读取内皮细胞和基质细胞的克隆性和转录组。我发现 基质细胞的克隆有选择地扩展，内皮细胞在MDS中转录重塑。 鉴于这些数据，我假设MD重塑了HSPC的克隆性和转录曲线 利基和HSPC-Niche相互作用涉及的机制促进了疾病的进展。在下面 Leonard Zon博士的指导，我将研究MDS使用A进行改造的机制 在计算机计算方法中的组合，遗传（格式塔）和颜色（Zebrabobow）谱系 追踪，共聚焦显微镜和体内镶嵌诱变。建立实验室后，我将建立一个 多学科团队加深我的计算分析并扩大我的体内遗传和生化 利基参与MD的克隆机制的扰动。我的总体目标是确定小说 可预防和/或停止MDS进展的HSPC利基特异性机制。这 K99/R00奖将使我能够发展新的技术技能，参加可以提高我能力的课程 管理实验室，参加会议，以扩大我的网络和我的了解 血液学疾病建模，斑马谱系跟踪与共焦显微镜和斑马鱼配对 诱变。我组成的科学咨询委员会包括 造血，谱系跟踪和干细胞生物学以及Zon博士将为我提供有关我的反馈 研究和职业进步。这些拟议的研究和职业发展活动将为 我成为一名独立研究人员，发现和研究负责的新机制 血液干细胞生态位介导的造血疾病进展。