Defining the mechanisms regulating MLLT3 expression in human hematopoietic stem cells

定义人类造血干细胞中 MLLT3 表达的调节机制

• 批准号: 9894797
• 负责人: Hanna Katri Annikki Mikkola
• 金额: $ 28.08万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-16 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894797
• 关键词: Binding; Bioinformatics; Biology; Blood Cells; CRISPR/Cas technology; Cell Culture Techniques; Cell physiology; ChIP-seq; Chromatin; Clinical; Data; Development; EVI1 gene; Engineering; Engraftment; Enhancers; Epigenetic Process; Experimental Models; Fetal Liver; Future; Gene Expression; Generations; Genes; Genetic Enhancer Element; Goals; Hematological Disease; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; High-Throughput Nucleotide Sequencing; Histones; Human; Immunologics; In Vitro; Individual; KDM1A gene; Lead; Lentivirus Vector; Life; Lysine; MLLT3 gene; Maintenance; Malignant - descriptor; Mediating; Modification; Monitor; Mus; Names; Patients; Protein Isoforms; Proteins; Protocols documentation; Regenerative Medicine; Regulation; Regulator Genes; Regulatory Element; Reporter; Repression; Reverse Transcriptase Polymerase Chain Reaction; Role; Savings; Second Pregnancy Trimester; Testing; Tetanus Helper Peptide; Time; Transcript; Transcription Initiation Site; Transcriptional Elongation Factors; Transplantation; Umbilical Cord Blood; aptamer; base; blood formation; cell type; epigenome editing; gain of function; hematopoietic stem cell expansion; hematopoietic stem cell self-renewal; hematopoietic tissue; hemogenic endothelium; high throughput analysis; human RNA sequencing; human embryonic stem cell; improved; in vivo; insight; novel; novel strategies; overexpression; p300/CBP-Associated Factor; population based; programs; recruit; self-renewal; single-cell RNA sequencing; stem cell therapy; stemness; success

SUMMARY Hematopoietic stem cells (HSCs) sustain life-long blood formation due to their ability to self-renew and differentiate into all mature blood cell types (referred to as “stemness”). Transplantation of HSC-containing grafts is a life-saving therapy for multiple blood disorders; however, shortage of immunologically matched donors limits the number of patients that can be treated. Expansion/generation of human HSCs in culture would greatly improve transplantation therapy but has been unsuccessful due to poor understanding of the underlying biology of HSC stemness. We identified MLLT3 as HSC regulator that is highly enriched in human HSC at all stages once they have emerged from AGM and expand in the fetal liver (FL); however, MLLT3 expression declines in cultured HSC. Loss and gain of function studies on human FL and cord blood (CB) HSCs showed that MLLT3 is critical for their self-renewal, and when its expression is restored, it enables HSC expansion in culture by protecting their “stemness” program. Importantly, FL and CB HSC expanded in culture showed 10-30 fold increase in human engraftment in NSG mice, and ability to sustain the HSPC compartment and multilineage hematopoiesis without malignant transformation or differentiation block. This finding offers an unprecedented opportunity to understand how human HSC stemness is controlled, and harness MLLT3 for clinical use. So far, the regulatory mechanisms that govern MLLT3 expression in HSC are completely unknown. Using a combination of epigenetic studies and bioinformatic approaches, we identified several candidate enhancers in MLLT3 gene that may regulate its two isoforms. Our data suggests that these enhancers are epigenetically remodeled during differentiation and silenced during HSC culture, providing new avenues to understand why cultured HSC lose MLLT3 expression. To understand how MLLT3 expression is regulated in human HSCs, we propose two complementary approaches: 1) CRISPR/Cas9-mediated epigenome editing to dissect the role of MLLT3 enhancers, 2) combination of bioinformatic and experimental approaches using lentiviral overexpression to identify MLLT3 upstream regulators. Success in these approaches could help generate/expand human HSCs in culture and thereby increase the availability of HSCs for transplantation.

摘要 造血干细胞(HSCs)由于具有自我更新和再生能力，可维持终生造血。 分化为所有成熟的血细胞类型(称为“干性”)。含HSC的移植物的移植 是一种治疗多种血液疾病的救命药物；然而，缺乏免疫匹配的捐赠者限制了 可以治疗的病人数量。在培养中扩增/生成人类造血干细胞将大大 改善移植治疗，但由于对潜在生物学缺乏了解而未获成功 HSC的茎。我们确定MLLT3是一种在所有阶段都高度富含人HSC的HSC调节因子 一旦它们从AGM中出现并在胎肝(FL)中扩张；然而，MLLT3的表达在 培养的HSC。人外周血和脐血HSCs功能丧失和获得的研究表明，MLLT3是 对他们的自我更新至关重要，当其表达恢复时，它通过以下方式使HSC在文化中扩张 保护他们的“茎”计划。重要的是，FL和CB HSC在培养中扩增10-30倍 人在NSG小鼠体内的植入增加以及维持HSPC间隔室和多系的能力 无恶性转化或分化障碍的造血细胞。这一发现提供了史无前例的 有机会了解人类干细胞是如何控制的，并利用MLLT3进行临床使用。到目前为止, 在HSC中控制MLLT3表达的调控机制是完全未知的。使用组合 通过表观遗传学研究和生物信息学方法，我们确定了MLLT3基因的几个候选增强子 这可能会调节它的两种异构体。我们的数据表明，这些增强子在表观遗传过程中被重塑 在HSC培养过程中的分化和沉默，为理解培养的HSC失败的原因提供了新的途径 MLLT3的表达。为了了解MLLT3在人HSCs中的表达是如何调节的，我们提出了两个 互补方法：1)CRISPR/Cas9介导的表观基因组编辑以剖析MLLT3的作用 增强剂，2)使用慢病毒过表达的生物信息学和实验方法的组合 确定MLLT3上游调节器。这些方法的成功可能有助于产生/扩大人类造血干细胞 培养，从而增加供移植的造血干细胞的可用性。