Defining the Self-Renewal Program in Human Hematopoietic Stem Cells

定义人类造血干细胞的自我更新程序

• 批准号: 10210386
• 负责人: Hanna Katri Annikki Mikkola
• 金额: $ 41.51万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-20 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10210386
• 关键词: Adult; Affect; Binding; Biological Assay; Bone Marrow; Cells; ChIP-seq; Complex; DNA Polymerase II; DNA-Binding Proteins; Data; ERG gene; Electroporation; Engineering; Engraftment; Enzymes; Epigenetic Process; Equilibrium; Failure; Fetal Liver; Future; Gene Expression Profile; Generations; Genes; Genetic Transcription; Goals; Hematological Disease; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Histones; Human; Human Development; In Vitro; Knowledge; Length; Lentivirus Vector; Link; MLLT3 gene; Metabolic; Methods; Molecular; Phosphorylation; Polymerase; Process; Property; Protein Isoforms; RNA; Reader; Regulation; Stimulus; System; Testing; Tetanus Helper Peptide; Therapeutic Uses; Transcription Elongation; Transcriptional Regulation; Translations; Umbilical Cord Blood; Western Blotting; Work; base; cell type; global run on sequencing; hematopoietic stem cell expansion; hematopoietic stem cell fate; hematopoietic stem cell self-renewal; human embryonic stem cell; human fetal hematopoietic stem cells; human pluripotent stem cell; improved; improved functioning; in vivo; knock-down; novel; novel strategies; overexpression; prenatal; programs; promoter; recruit; ribosome profiling; self-renewal; small hairpin RNA; stem cell engraftment; stem cell function; stem cell genes; stem cell therapy; stemness; transcriptome sequencing

PROJECT SUMMARY Difficulty to expand self-renewing human hematopoietic stem cells (HSC) in culture or generate them from human pluripotent stem cells (PSC) has hampered the use of in vitro engineered HSCs for therapeutic purposes. Our data suggest that the failure to induce and maintain the correct transcriptional networks governing HSC self-renewal during in vitro culture compromises the generation/expansion of fully functional human HSC in an in vitro setting. Guided by the transcriptional profile of highly purified human fetal liver (FL) HSC, we sought to identify key transcriptional regulators that govern self-renewal in human HSC, with the long- term goal to develop new strategies to improve the function of in vitro derived hematopoietic cells. We identified MLLT3/AF9, a component of superelongation complex (SEC) as a novel regulator of human HSC stemness. MLLT3 is highly enriched in the self-renewing HSC during human development (FL), pre-natally (cord blood, CB) and in the adult (bone marrow, BM), but becomes downregulated during HSPC differentiation and in vitro culture. Lentiviral knockdown of MLLT3 in human FL and CB HSC resulted in loss of HSC function in vitro and in vivo, while overexpression of MLLT3 greatly improved the ex vivo expansion and engraftment of FL and CB HSPCs, and partially rescued the proliferative potential of hESC-derived HSPCs. An important feature of MLLT3 is that it does not reprogram or transform hematopoietic cells, but only enhances the self- renewal and proliferative potential of properly specific HSCs. We will now examine how MLLT3 co-operates with transcription elongation machinery and epigenetic mechanisms to regulates its target genes and maintain proper HSC stemness program (Aim 1). We will then examine whether rescuing MLLT3 levels in culture by lentiviral overexpression or transient RNA electroporation increases the expansion of in vivo engraftable human HSCs (Aim 2). Finally, we will determine the unique function of a hitherto uncharacterized shorter isoform of MLLT3 that is also highly enriched in human HSCs, and may have an opposing function to the full length MLLT3 (Aim 3). This proposal will help understand how MLLT3 functions as an upstream regulator of “stemness” in human HSCs, and how its function in regulating HSC fate decisions may be modulated by the different isoforms. These studies will not only increase our knowledge of the fundamental regulatory mechanisms governing human HSC fate decisions, but also pave the way for developing novel approaches for the ex vivo expansion and manipulation of HSC for therapeutic use.

项目摘要 难以扩大培养物中的自我更新人造血干细胞（HSC）或从 人多能干细胞（PSC）阻碍了体外工程HSC的使用 目的。我们的数据表明，未能诱导和维护正确的转录网络 在体外培养过程中管理HSC自我更新会损害功能齐全的产生/扩展 人类HSC在体外环境中。在高度纯化的人胎儿肝（FL）的转录谱的指导下 HSC，我们感觉到要确定控制人类HSC自我更新的关键转录调节剂，长期 术语目标是制定新策略以改善体外衍生的造血细胞的功能。我们 确定的MLLT3/AF9，是超级复合物（SEC）的组成部分，是人类HSC的新型调节剂 茎。 MLLT3在人类发展期间（FL）中高度丰富了自我更新HSC （脐带血，CB）和成人（骨髓，BM），但在HSPC分化过程中被下调 和体外培养。人类FL和CB HSC中MLLT3的慢病毒敲低导致HSC功能的损失 体外和体内，MLLT3的过表达极大地改善了体内扩张和植入 FL和CB HSPC，并部分反应了hESC衍生的HSPC的增殖潜力。一个重要的 MLLT3的特征是它不重编程或转化造血细胞，但仅增强了自我 适当特定的HSC的更新和增殖潜力。现在，我们将研究MLLT3如何合作 具有转录伸长机制和表观遗传机制，以调节其靶基因并保持 适当的HSC STEMNENS计划（AIM 1）。然后，我们将检查是否通过 慢病毒过表达或瞬态RNA电穿孔增加体内植入的膨胀 人类HSC（目标2）。最后，我们将确定隐藏的未表征短的独特功能 MLLT3的同工型也高度富集了人类HSC，并且可能具有相反的功能 长度mllt3（目标3）。该建议将有助于了解MLLT3如何作为作为上游调节器的功能 人类HSC中的“茎”及其在调节中的功能HSC命运决定如何受到调节 不同的同工型。这些研究不仅会增加我们对基本监管的了解 管理人类HSC脂肪决策的机制，但也为开发新方法的方式铺平了道路 用于治疗的HSC的实体扩展和操纵。