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 难以在培养物中扩增自我更新的人造血干细胞（HSC）或从 人多能干细胞（PSC）阻碍了体外工程化HSC用于治疗性疾病的应用。 目的我们的数据表明，未能诱导和维持正确的转录网络， 在体外培养过程中控制HSC自我更新损害了全功能造血干细胞的产生/扩增， 体外环境中的人HSC。以高纯度人胎肝（FL）的转录谱为指导 HSC，我们试图确定关键的转录调控，管理自我更新的人HSC，与长期的， 长期目标是开发新的策略来改善体外衍生造血细胞的功能。我们 鉴定了MLLT 3/AF 9，其是作为人HSC的新调节剂的超伸长复合物（SEC）的组分 干性MLLT 3在人类发育（FL）期间（出生前）在自我更新HSC中高度富集 (cord血液，CB）和成人（骨髓，BM）中，但在HSPC分化期间下调 和体外培养。人FL和CB HSC中MLLT 3的慢病毒敲低导致HSC功能丧失 在体外和体内，虽然MLLT 3的过表达大大改善了细胞的离体扩增和植入， FL和CB HSPC，并部分挽救hESC衍生的HSPC的增殖潜力。一个重要 MLLT 3的特征是它不重新编程或转化造血细胞，而只是增强自身免疫。 适当特异性HSC的更新和增殖潜力。现在我们将研究MLLT 3如何合作 通过转录延伸机制和表观遗传机制来调节其靶基因并维持 适当的HSC干性程序（目标1）。然后，我们将研究是否通过以下方法来挽救培养物中的MLLT 3水平： 慢病毒过表达或瞬时RNA电穿孔增加了体内可植入的细胞的扩增， 人HSC（Aim 2）。最后，我们将确定一个迄今为止没有特点的短的独特功能， MLLT 3的同种型，其也在人HSC中高度富集，并且可能具有完全相反的功能 长度MLLT 3（目标3）。该提案将有助于了解MLLT 3如何作为上游调节器发挥作用， 人类HSC中的“干性”，以及它在调节HSC命运决定中的功能如何受到细胞因子的调节 不同的同种型这些研究不仅会增加我们对基本调控机制的认识， 控制人类HSC命运决定的机制，但也为开发新的方法铺平了道路， 用于治疗用途的HSC的离体扩增和操作。