In vivo hematopoiesis

体内造血

• 批准号: 8746638
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 100.95万
• 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/8746638
• 关键词: Address; Allogenic; Animals; Annual Reports; Aplastic Anemia; Autologous; B-Lymphocytes; Back; Behavior; Blood; CD34 gene; Cell Count; Cell Lineage; Cell division; Cells; Cessation of life; Clinical; Clinical Trials; Color; Common Lymphoid Progenitor; Confocal Microscopy; Data; Derivation procedure; Dose; Engraftment; Erythrocytes; FCGR3B gene; Genes; Genetic; Goals; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Human; Image; Imaging Techniques; Immature Platelet; In Vitro; Individual; Infusion procedures; Knowledge; Laboratories; Lentivirus Vector; Leukemic Cell; Light; Location; MLL-AF9; Macaca; Maps; Marrow; Measures; Methods; Microscopy; Modeling; Monkeys; Morphology; Mus; Myeloid Cells; NCAM1 gene; Natural Killer Cells; Outcome; Output; Pancytopenia; Pathway interactions; Patients; Pattern; Photons; Primates; Process; Proteins; Refractory; Regimen; Reporting; Resolution; Reticulocytes; Sampling; Site; Stem cells; T-Lymphocyte; Technology; Thrombopoietin; Time; Tissues; Transfusion; Transplantation; Whole-Body Irradiation; analog; cell type; combinatorial; conditioning; cytotoxic; gene therapy; improved; in vitro Model; in vivo; insight; interest; leukemia; molecular imaging; nonhuman primate; novel; response; segregation; stem; vector

We have utilized molecular and imaging techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) in vivo. Utilizing lentiviral vectors carrying genes for 5 distinct fluorescent proteins (FPs) termed LEGO vectors, we have utilized a combinatorial color approach to be able to uniquely mark and then track output from individual HSPCs in time and space in vivo. A technologically-advanced and unique imaging approach combining confocal microscopy, 2 photon microscopy and advanced analytic approaches was developed, and has been applied to study the process of hematopoietic engraftment in the marrow of mice and monkeys. Early engraftment is endosteal, and large clones consisting of the progeny of single HSPCs remain distinctly and surprisingly localized in the marrow for up to 3-4 months. This result suggests that following cell division, HSPCs spread contiguously in the marrow instead of recirculating to a new location via mobilization into the blood, and that exit of HSPCs from the marrow may be primarily a death pathway. These studies were performed utilizing total body irradiation conditioning, and new studies are ongoing asking whether the geographic patterns of engraftment and hematopoiesis may be different with alternative conditioning regimens, or no conditioning (utilizing immunodeficient/stem cell deficient recipients that can engraft all donor cells without any conditioning). Similar studies are now also ongoing in the non-human primate model. Contributions of HPSC-derived differentiated progeny cells could also be examined in mice transplanted with LEGO-transduced HPSCs, at very high resolution showing clear morphology of all cell types in various tissues of interest. Intercalating HSPC-derived cells could be easily mapped in all tissues, but there was no evidence for direct contribution of HSPC-derived cells to endodermal or ectodermal tissues. We have also applied lentiviral "barcoding" with high-diversity 31-35bp genetic barcodes to study hematopoiesis in the non-human primate model. Our collaborator Rong Lu first devised this very powerful approach and applied it to study murine hematopoiesis. We have now transplanted 5 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to one year) and in multiple lineages in a quantitative and highly reproducible manner, for the first time. We have already made a number of important and novel discoveries, including the lack of evidence for a common lymphoid progenitor producing T and B cells in primates, with no shared clonal derivation of B and T cells until late after transplant, and much earlier shared clonal derivation of myeloid and B cells. We have also for the first time discovered the unique lineage derivation of the major fraction of natural killer (NK) cells. CD16+CD56- cytotoxic NK cells did not share barcodes with B, T or myeloid cells until 9-12 months post-transplant, and in vitro and murine models have not previously been able to shed light on NK cell lineage relationships. We have also demonstrated geographic segregation of individual HSPCs long term in specific marrow sites, confirming the findings described above using imaging techniques. The barcoding projects remain highly active, with numerous new projects shedding light on multiple aspects of hematopoiesis that can now be addressed directly in vivo via this powerful technology. We are investigating the relationship between normal HSPCs and leukemia engrafting cells using competitive repopulation in the murine model, asking whether co-infusion of increasing doses of HPSCs can compete directly with leukemic cells for marrow niches, thus slowing leukemic progression. We have preliminary data indicating competition for the same niches, with confocal imaging results also backing up these functional findings. We can also now transduce the MLL-AF9 murine leukemic cells with LEGO vectors and follow leukemic engraftment and progression in vivo in the marrow and other tissues. Clinically we have pursued analysis of the impact of eltrombopag on in vivo expansion of HSPCs, specifically in the bone marrow failure setting. We reported that 40-50% of patients with refractory aplastic anemia respond to eltrombopag, becoming transfusion independent and in the majority of cases manifesting a trilineage response. As described in Dr. Young's annual report, he has built on my group's initial observation and has now utilized this thrombopoietin analog to try and improve outcomes and maintain stem cell numbers in patients with severe new onset aplastic anemia. My group has also investigated the use of a new laboratory parameter, the immature platelet fraction, measured in clinical samples in an analogous manner to reticulocytes for the red cell lineage, to measure marrow function post-transplantation, post-transfusion, and in bone marrow failure.

我们利用分子和成像技术来获得对体内造血茎和祖细胞（HSPC）行为的新见解。 利用携带5种不同荧光蛋白（FPS）的慢病毒载体称为LEGO载体，我们采用了一种组合颜色方法来唯一标记，然后在体内时间和空间中跟踪单个HSPC的输出。 开发了一种技术增强和独特的成像方法，结合了共聚焦显微镜，2个光子显微镜和先进的分析方法，并已应用于研究小鼠和猴子骨髓中造血植入的过程。 早期的植入是内传的，由单个HSPC的后代组成的大型克隆在骨髓中保持了明显且令人惊讶的位置，持续3-4个月。 该结果表明，在细胞分裂之后，HSPC在骨髓中连续扩散，而不是通过动员到血液中再循环到新位置，而从骨髓中的HSPC退出可能主要是死亡途径。 利用总体辐照调节进行了这些研究，并正在进行新的研究，询问植入和造血的地理模式是否会因替代条件方案而有所不同，还是没有条件（使用免疫缺陷/干细胞缺陷的受体，可以植入所有供体细胞的情况下所有没有任何条件的供体细胞）。 现在，在非人类灵长类动物模型中也正在进行类似的研究。 还可以在用乐高转移的HPSC移植的小鼠中检查HPSC衍生的分化后代细胞的贡献，并以非常高的分辨率显示出各种感兴趣的组织中所有细胞类型的明显形态。插入HSPC衍生的细胞可以很容易地映射在所有组织中，但是没有证据表明HSPC衍生细胞对内胚层或外皮组织的直接贡献。 我们还使用高多样性31-35bp遗传条形码应用慢病毒“条形码”来研究非人类灵长类动物模型中的造血。 我们的合作者Rong Lu首先设计了这种非常有力的方法，并将其应用于研究鼠造血。现在，我们已经使用条形码自体CD34+细胞移植了5个猕猴，并且能够在第一次以定量和高度再现的方式中跟踪数千个单独的HSPC的造血输出。 我们已经做出了许多重要的和新颖的发现，包括缺乏证据表明，在移植后末直到后期才能在灵长类动物中产生T和B细胞的常见淋巴样祖细胞和B细胞，并且早些时候共享了髓样和B细胞的克隆衍生物。我们也第一次发现了自然杀手（NK）细胞主要部分的独特谱系推导。 CD16+CD56-细胞毒性NK细胞直到移植后9-12个月才与B，T或髓样细胞共享条形码，并且在体外和鼠模型之前尚未能够阐明NK细胞谱系关系。 我们还在特定的骨髓位点长期证明了单个HSPC的地理隔离，并使用成像技术证实了上述发现。 条形码项目仍然高度活跃，许多新项目阐明了造血的多个方面，现在可以通过这项强大的技术直接在体内解决。 我们正在使用鼠模型中使用竞争性重新培养的竞争性重培养的正常HSPC和白血病之间的关系，询问增加剂量的HPSC是否可以直接与白血病细胞竞争骨髓壁ni，从而减慢白血病进展。我们拥有初步数据，表明竞争相同的壁ni，共聚焦成像结果也支持这些功能性发现。现在，我们还可以用乐高载体将MLL-AF9鼠白血病细胞转导，并跟随骨髓和其他组织中体内的白血病植入和进展。 在临床上，我们对Eltrombopag对HSPC体内扩张的影响进行了分析，特别是在骨髓衰竭设置中。我们报道说，40-50％的难治性性性贫血患者对Eltrombopag有反应，独立于输血，并且在大多数情况下表现出了三琳反应。 如Young博士的年度报告中所述，他建立在我小组的最初观察基础上，现在利用这种血小板素类似物来尝试改善预后，并维持严重的新发作性性贫血患者的干细胞数量。 我的小组还研究了新的实验室参数的使用，即未成熟的血小板馏分，以类似方式在临床样品中测量红细胞谱系的网状细胞，以测量移植后移植，转移后输血后和骨髓衰竭。