Hematopoietic stem cell (HSC) development, self-renewal and differentiation

造血干细胞 (HSC) 发育、自我更新和分化

• 批准号: 8746716
• 负责人: Andre LaRochelle
• 金额: $ 82.68万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746716
• 关键词: Adult; Agonist; Aplastic Anemia; Autologous; Autologous Transplantation; Biological Assay; Birds; Blood; Blood Platelets; CD34 gene; Categories; Cell Count; Cell Maintenance; Cells; ChIP-seq; Clinic; Clinical; Coculture Techniques; Culture Media; Cultured Cells; Data; Development; Disease; Dose; Embryo; Embryonic Development; Employee Strikes; Endothelial Cells; Endothelium; Engraftment; Evaluation; Family; Future; Gene Expression; Generations; Genes; Genetic; Goals; Gold; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Homeostasis; Human; Hypoxia; Hypoxia Inducible Factor; Hypoxia Pathway; Immune; Immunoprecipitation; In Vitro; Individual; Interleukin-3; Lead; Life; Ligands; Link; Maintenance; Mediator of activation protein; Methylation; Modification; Molecular; Mus; Mutation; Myelogenous; Natural regeneration; Notch Signaling Pathway; Oxygen; PTPRC gene; Pancytopenia; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Population; Protocols documentation; Regulation; Risk; Role; Sickle Cell Anemia; Signal Transduction; Stem Cell Development; Stem Cell Factor; Stem cells; Stress; Suspension substance; Suspensions; Syndrome; System; Technology; Thrombopoietin; Time; Transplantation; Umbilical Cord Blood; Undifferentiated; Work; analog; bHLH-PAS factor HLF; base; clinical application; cytokine; experience; gene therapy; hypoxia inducible factor 1; improved; in vivo; induced pluripotent stem cell; metabolomics; mouse model; notch protein; novel; novel strategies; progenitor; reconstitution; response; self-renewal; stem; stem cell differentiation; stem cell technology; stemness; therapeutic transgene; transcription factor; transcriptome sequencing; transduction efficiency; tumorigenesis

Ojective 1: Develop approaches for expansion of hematopoietic stem cells (HSCs) Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivoexpanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines (e.g. thrombopoietin TPO, stem cell factor SCF) have failed to support reliable amplification of immature stem cells in culture, suggesting that alternative cytokines or additional factors are required and highly desirable. ELTROMBOPAG. Eltrombopag is a novel TPO agonist with an intrinsic ability to expand HSPC in vivo given observed clinical benefits in patients with aplastic anemia. We have cultured human CD34+ cells for up to 21 days in our standard culture medium supplemented with Eltrombopag or with the combination Eltrombopag + TPO. We found that Eltrombopag was not superior to TPO for HSPC expansion in vitro. However, the combination TPO + Eltrombopag favored expansion of total CD34+ cells, platelets and myeloid progenitors better than TPO or Eltrombopag alone. Transplantation of these expanded cells in immune-deficient mice, the gold standard for evaluation of the in vivo repopulating potential of these cells, will determine the clinical utility of this drug for in vitro expansion of HSCs. NOTCH PATHWAY AND HYPOXIA. The primary mediators of hypoxic adaptation are hypoxia-inducible factors (HIF), a family of transcription factors composed of two subunits, an oxygen-labile subunit that rapidly stabilizes in response to low O2 tensions (HIF-1 and HIF-2), and a subunit (HIF-1) that is constitutively expressed. In immunoprecipitation assays, HIF-1 physically interacted with the intracellular domain of Notch, a critical component for the maintenance of undifferentiated stem and progenitor cell populations, providing a striking molecular link between hypoxia and stemness. Given this recent evidence of a convergence of pathways involved in hypoxia sensing and stem cell maintenance, we are investigating the possibility of stem cell expansion under hypoxic conditions by activation of the canonical Notch signaling pathway using Delta-1 ligand. We showed that culture of human CD34+ cells for 21 days in the presence of Delta-1 ligand under hypoxic conditions maintains the CD34+ phenotype in 25% of cultured cells compared to <1% in the absence of Delta-1 ligand. Transplantation of these expanded cells in immune-deficient mice will determine the clinical utility of this approach for in vitro expansion of HSCs. NOVEL PATHWAYS FOR HSC EXPANSION. During homeostasis the HSC pool is maintained at a relatively constant level. In contrast, several murine studies have shown that during hematopoietic stress, such as transplantations, HSCs can and will self-renew extensively, suggesting that HSCs are exposed in vivo to specific factors/signals that promote their self-renewal and amplification. We have transplanted human CD34+ cells into immune-deficient mice and showed evidence of extensive self-renewal in vivo. To identify novel factors/pathways involved in HSC expansion, we are comparing gene expression/methylation patterns between HSC at steady state (before transplant) and after transplant, using RNA-Seq, CHIP-Seq and metabolomics approaches. These studies will have a significant impact on the global understanding of human HSC self-renewal and could lead to the development of novel approaches for HSC expansion in vitro. Objective 2: Develop approaches for differentiation of iPSCs into HSCs With the development of induced pluripotent stem cell (iPSC) technologies emerged the concept of generating iPSCs from an individual patient, correcting the genetic defect using gene specific targeting for safe integration of the therapeutic transgenes, and differentiating the disease-free iPSCs into transplantable HSCs. The hematopoietic system serves as a perfect opportunity to cure diseases using these types of approaches. Decades of HSCT performed in the clinic provide a roadmap for clinical use and the fact that the hematopoietic system constantly regenerates itself provides an opportunity to replenish a patients blood system with new or corrected cells. The proof-of-principle of using iPSC technologies to cure hematopoietic disorders attributed to a genetic defect has been performed in a humanized sickle-cell anemia mouse model. Proof-of-concept was also demonstrated in human cells by generating iPSCs from patients with various hematologic disorders and correcting them to generate disease-free hematopoietic cells. These studies indicate that iPSC technologies could provide a novel long-awaited treatment option for patients with life-threatening bone marrow failure syndromes. Currently used protocols for iPSC differentiation into HSCs can generally be divided into two main categories: those that co-culture stem cells with stromal layers (e.g. OP9), and those that culture stem cells in suspension to form embryoid bodies (EBs). However, these protocols remain inefficient at producing the quantity and quality of HSCs required for clinical applications. On the basis of recent data demonstrating that definitive HSCs are generated from a unique population of endothelial cells known as hemogenic endothelium (HE), we have established a novel system for de novo generation of transplantable HSCs from iPSCs. Human iPSCs derived from normal individuals are cultured in the presence of a cytokine combination that favors development of an adherent layer of HE in vitro. Over a period of 12-14 days, these cells further differentiate to produce and release cells in suspension. Up to 70% of these cells have a CD45+CD34+ phenotype compared to 10-15% CD45+CD34+ using current co-culture or EB-based protocols. We have characterized these cells further and demonstrated a subpopulation with the most defined HSC phenotype described (CD34+CD38-CD45RA-CD90+CD49f+Rholo). The ability of these cells for establish hematopoiesis in vivo in underway. Future work will focus on the application of this technology to patients with life-threatening bone marrow failure syndromes.

目标 1：开发造血干细胞 (HSC) 扩增方法 由于无法产生足够的干细胞数量以及起始细胞群的过度分化，改善离体扩增的造血干细胞和祖细胞（HSPC）的造血重建和植入潜力的尝试基本上不成功。体外研究的经验表明，在培养物中控制 HSPC 的自我更新和分化仍然很困难。基于造血细胞因子（例如血小板生成素 TPO、干细胞因子 SCF）的方案未能支持培养中未成熟干细胞的可靠扩增，这表明需要并且非常需要替代细胞因子或其他因子。 艾尔腾博帕格。艾曲波帕是一种新型 TPO 激动剂，具有在再生障碍性贫血患者中观察到的临床益处，具有体内扩增 HSPC 的内在能力。我们已在补充有艾曲波帕或艾曲波帕 + TPO 组合的标准培养基中培养人类 CD34+ 细胞长达 21 天。我们发现，对于 HSPC 体外扩增，艾曲波帕并不优于 TPO。然而，TPO + 艾曲波帕组合比单独使用 TPO 或艾曲波帕更有利于总 CD34+ 细胞、血小板和骨髓祖细胞的扩增。将这些扩增的细胞移植到免疫缺陷小鼠体内，这是评估这些细胞体内增殖潜力的金标准，将决定该药物体外扩增 HSC 的临床效用。 缺口通路和缺氧。缺氧适应的主要介质是缺氧诱导因子 (HIF)，这是一个由两个亚基组成的转录因子家族，一个氧不稳定亚基，可响应低 O2 张力而快速稳定（HIF-1 和 HIF-2），以及组成型表达的亚基 (HIF-1)。在免疫沉淀试验中，HIF-1 与 Notch 的细胞内结构域发生物理相互作用，Notch 是维持未分化干细胞和祖细胞群的关键成分，在缺氧和干性之间提供了惊人的分子联系。鉴于最近证据表明缺氧传感和干细胞维持涉及的途径趋同，我们正在研究通过使用 Delta-1 配体激活经典 Notch 信号通路来在缺氧条件下进行干细胞扩增的可能性。我们发现，在缺氧条件下，在存在 Delta-1 配体的情况下培养人 CD34+ 细胞 21 天，25% 的培养细胞维持 CD34+ 表型，而在不存在 Delta-1 配体的情况下，维持 CD34+ 表型的比例为 <1%。将这些扩增的细胞移植到免疫缺陷小鼠中将决定这种体外扩增 HSC 的方法的临床实用性。 HSC 扩展的新途径。在体内平衡期间，HSC 池保持在相对恒定的水平。相比之下，一些小鼠研究表明，在造血应激期间，例如移植，HSC 能够并且将会广泛地自我更新，这表明 HSC 在体内暴露于促进其自我更新和扩增的特定因素/信号。我们将人类 CD34+ 细胞移植到免疫缺陷小鼠体内，并显示出体内广泛自我更新的证据。为了确定参与 HSC 扩增的新因子/途径，我们使用 RNA-Seq、CHIP-Seq 和代谢组学方法比较稳态（移植前）和移植后 HSC 之间的基因表达/甲基化模式。这些研究将对人类 HSC 自我更新的全球理解产生重大影响，并可能导致 HSC 体外扩增新方法的开发。 目标 2：开发 iPSC 分化为 HSC 的方法 随着诱导多能干细胞 (iPSC) 技术的发展，出现了从个体患者生成 iPSC、使用基因特异性靶向纠正遗传缺陷以安全整合治疗性转基因、并将无病 iPSC 分化为可移植 HSC 的概念。造血系统是使用这些类型的方法治疗疾病的绝佳机会。几十年来在临床上进行的 HSCT 为临床使用提供了路线图，造血系统不断自我再生的事实提供了用新的或修正的细胞补充患者血液系统的机会。使用 iPSC 技术治疗遗传缺陷导致的造血系统疾病的原理验证已在人源化镰状细胞性贫血小鼠模型中进行。通过从患有各种血液系统疾病的患者身上生成 iPSC，并对其进行纠正以生成无病的造血细胞，也在人类细胞中进行了概念验证。这些研究表明，iPSC 技术可以为患有危及生命的骨髓衰竭综合征的患者提供期待已久的新型治疗选择。目前使用的 iPSC 分化为 HSC 的方案通常可分为两大类：干细胞与基质层（例如 OP9）共培养的方案，以及悬浮培养干细胞形成胚状体（EB）的方案。然而，这些方案在生产临床应用所需的 HSC 数量和质量方面仍然效率低下。 最近的数据表明，最终的 HSC 是由称为造血内皮 (HE) 的独特内皮细胞群产生的，我们建立了一个新的系统，用于从 iPSC 中从头产生可移植的 HSC。源自正常个体的人 iPSC 在细胞因子组合的存在下进行培养，该细胞因子组合有利于体外 HE 粘附层的发育。在 12-14 天的时间内，这些细胞进一步分化，产生并释放悬浮细胞。使用当前共培养或基于 EB 的方案时，这些细胞中高达 70% 具有 CD45+CD34+ 表型，而 10-15% CD45+CD34+ 表型。我们进一步表征了这些细胞，并证明了具有最明确的 HSC 表型的亚群 (CD34+CD38-CD45RA-CD90+CD49f+Rholo)。这些细胞在体内建立造血的能力正在进行中。未来的工作将集中于将该技术应用于患有危及生命的骨髓衰竭综合征的患者。