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.

项目总结 难以在培养中扩增自我更新的人类造血干细胞(HSC)或从 人类多能干细胞(PSC)阻碍了体外工程HSC用于治疗的应用 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的自我更新会影响全功能细胞的生成/扩增 体外培养的人肝星状细胞。高纯度人胎肝(FL)转录图谱的指导 HSC，我们试图确定管理人类HSC自我更新的关键转录调控因子，与长期- 学期目标是开发新的策略来改善体外来源的造血细胞的功能。我们 超长复合体(SEC)成分MLLT3/AF9被鉴定为人HSC的新调节因子 茎干。MLLT3在出生前的人类发育(FL)中高度富含自我更新的HSC (脐带血)和成人(骨髓，BM)，但在HSPC分化过程中表达下调 和体外培养。慢病毒敲除人FL和CB HSC中MLLT3导致HSC功能丧失 在体外和体内，MLLT3的过表达显著促进了细胞的体外扩增和植入 HL和CB的HSPC，部分挽救了hESC来源的HSPC的增殖潜能。一个重要的 MLLT3的特点是它不重新编程或转化造血细胞，而只是增强自身的 适当特定的造血干细胞的更新和增殖潜力。我们现在将研究MLLT3是如何合作的 具有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茎干计划(目标1)。然后我们将研究通过以下方式挽救培养中的MLLT3水平 慢病毒过表达或瞬时RNA电穿孔增加体内可植入细胞的扩张 人类造血干细胞(目标2)。最后，我们将确定一个迄今未被描述的短片的独特功能 MLLT3的异构体，在人类HSCs中也高度丰富，可能具有完全相反的功能 长度MLLT3(目标3)。这项提议将有助于理解MLLT3如何作为一种上游监管机构发挥作用 人类HSC中的“茎”，以及它在调节HSC命运决定中的功能可能如何被 不同的异构体。这些研究不仅将增加我们对基本监管的知识 管理人类HSC命运决定的机制，但也为开发新的方法铺平了道路 肝星状细胞的体外扩增和操作以用于治疗。