Enhancement of hematopoietic stem cell mobilization and engraftment

增强造血干细胞动员和植入

• 批准号: 8344863
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 140.18万
• 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/8344863
• 关键词: Actins; Adhesions; Allogenic; Animals; Architecture; Autologous; Behavior; Blast Cell; Bone Marrow; CD34 gene; Cell Culture Techniques; Cell Cycle; Cell Separation; Cell surface; Cells; Characteristics; Cholesterol; Clinical Trials; Collaborations; Complex; Data; Engraftment; Environment; Epigenetic Process; Gene Expression Profiling; Goals; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cell Transplantation; Home environment; Homing; Human; Image; Immunodeficient Mouse; Immunohistochemistry; In Vitro; Integrins; KAI1 gene; Knock-out; Laboratories; Lentivirus Vector; Leukemic Cell; Link; Lipids; Macaca mulatta; Maps; Marrow; Mediator of activation protein; Membrane; Methods; Mitosis; Modeling; Mus; One-Step dentin bonding system; Organ; Osteoblasts; Outcome; Oxygen; Phase; Phenotype; Photons; Population; Process; Property; Proteins; Reactive Oxygen Species; Reporting; Research Personnel; Scheme; Series; Signal Transduction; Small Interfering RNA; Stem cells; Stromal Cell-Derived Factor 1; Techniques; Time; Tissues; Translating; Transplantation; Umbilical Cord Blood; Xenograft Model; base; combinatorial; gene therapy; human PHEMX protein; improved; in vitro Model; in vivo; nonhuman primate; novel; peripheral blood; polymerization; prevent; prominin; research study; stem; stem cell population

A series of recent high profile reports suggested that a simple positive selection approach utilizing SLAM cell surface markers results in enrichment of long-term repopulating stem cells to the same degree as much more complex multi-step negative and positive selection schemes. These studies, performed in the murine model, also reported that for the first time immunohistochemistry could be used to identify HSCs in tissue sections, allowing analysis of HSCs in their niches. Investigators hoped that the same markers would translate to human HSCs, but in a series of experiments we have shown that unfortunately SLAM markers are unlikely to be relevant in larger animals and humans. SLAM-purified rhesus macaque and human mobilized peripheral blood, bone marrow and cord blood CD34+ cells were transplanted into immunodeficient mice, and there was no enhancement of engraftment in the SLAM+ versus SLAM- populations. These results are importantsince investigators are already utilizing SLAM purification of human cells without supportive data documenting HSC enrichment. In collaboration with the laboratory of Jennifer Lippincott-Schwartz, we have identified a polarized membrane domain on primitive hematopoietic stem and progenitor cells that is critical for interactions with osteoblasts, central functional components of the hematopoietic niche in vivo. These domains allow attachment to osteoblasts, contain a number of proteins known to be important in organizing membrane domains, including tetraspanins, integrins, and prominin, and have a central lipid component. Cholesterol depletion or disruption of actin polymerization disrupts the domains, and prevents attachment to osteoblasts. Following attachment to osteoblasts, we have documented membrane transfer from the HSCs to the osteoblasts, and initiation of a signaling cascade in osteoblasts, resulting in increased expression of SDF-1, a primary mediator of stem cell mobilization and engraftment. We have now demonstrated these domains to be important for in vivo homing and engraftment of human CD34+ cells, in murine xenograft model. We have found these domains to be disrupted during the S/G2/M phases of the cell cycle, and investigated the link between the loss of these membrane domains and the previous observation from our group and others that HSCs in active cycle do not home or engraft as efficiently as cells in the Go phase of the cell cycle. We are exploring the critical protein components in the domain, studying whether siRNA knockdown or full knockout of tetraspanins, specifically CD82, impacts on domain formation or HSC behavior. Blocking CD82 blocks adhesion and engraftment. Conversely, we are searching for compounds that increase domain formation and tetraspanin expression, in hopes that homing and engraftment of cells exposed to these agents in vitro might be improved. IBD increases expression of the tetraspanin CD9 however this increased expression does not result in higher engraftment. We have begin to compare these domains in primitive normal versus primary human leukemic blasts, and found that the domains are disrupted in leukemic cells. We are now using competitive repopulation studies and multicolor 3D confocal and 2 photon imaging to ask whether leukemic and normal stem cell compete for the same in vivo bone marrow niche. Most recently, we have begun to explore the relationship between oxygen concentration and the localization and behavior of HSCs. Marrow cells sorted for the lowest concentration of reactive oxygen species have characteristics of more primitive long-term engrafting cells, are not in active cell cycle, and purification based on ROS concentration may provide a simple one-step HSC enrichment process. Once cells are mobilized to the peripheral blood, outside of their normal protective niche, ROS concentration no longer correlates with primitive stem cell phenotype and function. We are carrying out gene expression profiling and plan to study epigenetic markers within ROS low and ROS high marrow stem cell populations. We will also ask whether cells cultured under ROS low conditions better maintain HSC properties, and can be genetically transduced and expanded, compared to cells under standard ex vivo culture conditions. Finally we have recently developed a very novel and powerful technique combining combinatorial transduction with 3-5 lentiviral vectors expressing fluorescent proteins with confocal and 2 photon imaging of intact bone marrow and other hematopoietic organ architecture, to be able to localize and map hematopoiesis at a clonal level in the bone marrow environment.

最近一系列备受瞩目的报道表明，利用SLAM细胞表面标志物的简单阳性选择方法导致长期再增殖干细胞的富集达到与复杂得多的多步骤阴性和阳性选择方案相同的程度。这些在小鼠模型中进行的研究还报告称，免疫组织化学首次可用于识别组织切片中的HSC，从而可以分析其小生境中的HSC。 研究人员希望相同的标记物能够转化为人类HSC，但在一系列实验中，我们已经证明，不幸的是，SLAM标记物不太可能与大型动物和人类相关。将SLAM纯化的恒河猴和人动员的外周血、骨髓和脐带血CD 34+细胞移植到免疫缺陷小鼠中，并且在SLAM+与SLAM-群体中没有增强植入。 这些结果很重要，因为研究人员已经在利用SLAM纯化人类细胞，而没有支持性数据记录HSC富集。 在与Jennifer Lippincott-Schwartz实验室的合作中，我们已经确定了原始造血干细胞和祖细胞上的极化膜结构域，该结构域对于与成骨细胞（体内造血生态位的中心功能组分）的相互作用至关重要。这些结构域允许附着到成骨细胞，含有许多已知在组织膜结构域中重要的蛋白质，包括四跨膜蛋白、整合素和膜蛋白，并且具有中心脂质组分。 胆固醇耗尽或肌动蛋白聚合的破坏破坏了结构域，并阻止附着成骨细胞。 附着到成骨细胞后，我们已经记录了从HSC到成骨细胞的膜转移，以及成骨细胞中信号级联的启动，导致SDF-1表达增加，SDF-1是干细胞动员和植入的主要介质。我们现在已经证明这些结构域对于在鼠异种移植模型中人CD 34+细胞的体内归巢和植入是重要的。 我们已经发现这些结构域在细胞周期的S/G2/M期被破坏，并研究了这些膜结构域的丢失与我们小组和其他人先前观察到的HSC在活跃周期中不像细胞周期的Go期中的细胞那样有效地归巢或移植之间的联系。我们正在探索结构域中的关键蛋白质组分，研究siRNA敲除或完全敲除四跨膜蛋白，特别是CD 82，是否会影响结构域形成或HSC行为。阻断CD 82阻断粘附和植入。 相反，我们正在寻找增加结构域形成和四跨膜蛋白表达的化合物，希望体外暴露于这些试剂的细胞的归巢和植入可能得到改善。IBD增加四跨膜蛋白CD 9的表达，然而这种增加的表达不会导致更高的植入。我们开始比较原始正常人白血病原始细胞和原代人白血病原始细胞中的这些结构域，发现这些结构域在白血病细胞中被破坏。我们现在正在使用竞争性再增殖研究和3D共聚焦和双光子成像来询问白血病和正常干细胞是否竞争相同的体内骨髓小生境。 最近，我们已经开始探索氧浓度与HSC的定位和行为之间的关系。 以最低活性氧浓度分选的骨髓细胞具有更原始的长期移植细胞的特征，不处于活跃的细胞周期中，并且基于ROS浓度的纯化可以提供简单的一步HSC富集过程。一旦细胞被动员到外周血中，在其正常保护性小生境之外，ROS浓度不再与原始干细胞表型和功能相关。我们正在进行基因表达谱分析，并计划研究ROS低和ROS高骨髓干细胞群中的表观遗传标记。 我们还将询问与标准离体培养条件下的细胞相比，在ROS低条件下培养的细胞是否更好地保持HSC特性，并且可以进行遗传转导和扩增。 最后，我们最近开发了一种非常新颖和强大的技术，其将组合转导与表达荧光蛋白的3-5种慢病毒载体与完整骨髓和其他造血器官结构的共聚焦和2光子成像相结合，以能够在骨髓环境中以克隆水平定位和映射造血。