Clonal analysis of in vivo hematopoiesis

体内造血克隆分析

• 批准号: 8939842
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 120.54万
• 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/8939842
• 关键词: Address; Allogenic; Animals; Autologous; B-Lymphocytes; Back; Behavior; Blood; Blood Cells; Blood specimen; Bone Marrow; CD34 gene; Cell Lineage; Cell Ontogeny; Cell Therapy; Cell division; Cells; Cessation of life; Child; Clinical Trials; Collaborations; Color; Common Lymphoid Progenitor; Confocal Microscopy; Data; Derivation procedure; Disease model; Dose; Embryo; Engraftment; FCGR3B gene; Freezing; Future; Genes; Genetic; Goals; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Human; Image; Imaging Techniques; In Vitro; Individual; Infusion procedures; Intestines; Knowledge; Lentivirus Vector; Leukemic Cell; Light; Liver; Location; Lymphoid; MLL-AF9; Macaca; Maps; Marrow; Mesenchymal; Methodology; Methods; Microscopy; Modeling; Monkeys; Morphology; Mouse Strains; Mus; Myeloid Cells; NCAM1 gene; Natural Killer Cells; Natural regeneration; Outcome; Output; Pathway interactions; Pattern; Photons; Population; Primates; Process; Proteins; Regimen; Research Personnel; Resolution; Site; Stem cells; Stromal Cells; T-Lymphocyte; Technology; Time; Tissues; Transplantation; United States National Institutes of Health; Vagina; Whole-Body Irradiation; cell type; combinatorial; conditioning; cytotoxic; gene therapy; graft vs host disease; improved; in vitro Model; in vivo; insight; interest; leukemia; lymph nodes; molecular imaging; nonhuman primate; novel; segregation; self-renewal; stem; therapy development; 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, now successfully re-derived from frozen embryos as required to bring new mouse strains into the NIH). 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. In collaboration with NCI investigators we have utilized this methodology to study the in vivo distribution and behavior of mesenchymal stromal cells in the context of murine graft-versus-host disease models. We have developed 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 8 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to two years) 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 even 24 months post-transplant. In vitro and murine models have not previously been able to shed light on NK cell lineage relationships. We have continued to use barcoded cells from these macaques to further dissect in vivo NK cell ontogeny, and the process of ex vivo expansion of NK cells, highly relevant for adoptive cell therapy development, in collaboration with Dr. Rick Childs' group. We have discussed a unique self-renewing population able to regenerate CD56+ NK cells that is present in a "double negative" population of peripheral blood cells. We continue to search for the precursor to the ontologically-unique CD16+ mature NK subpopulation, tracking dominant clones in phenotypically purified samples from blood, bone marrow, lymph nodes, and in the future liver, vaginal and intestinal lymphoid aggregates. We have also demonstrated geographic segregation of individual HSPCs long term in specific marrow sites, confirming the findings described above using LEGO 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 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.

我们利用分子和成像技术对造血干细胞和祖细胞(HSPC)在体内的行为获得了新的见解。利用慢病毒载体携带5种不同荧光蛋白(FP)的基因，称为LEGO载体，我们利用组合颜色方法能够在体内唯一地标记并跟踪单个HSPC的输出。结合共聚焦显微镜、双光子显微镜和先进的分析方法，发展了一种技术先进和独特的成像方法，并已应用于研究小鼠和猴子骨髓中的造血植入过程。早期植入是骨内的，由单个HSPC的后代组成的大克隆在骨髓中保持明显的、令人惊讶的定位长达3-4个月。这一结果表明，在细胞分裂后，HSPC在骨髓中连续扩散，而不是通过动员进入血液再循环到新的位置，HSPC从骨髓中退出可能主要是一条死亡途径。这些研究是利用全身照射条件进行的，新的研究正在进行中，询问替代条件方案或不条件条件下植入和造血的地理模式是否会不同(利用免疫缺陷/干细胞缺陷受体可以在不进行任何条件的情况下移植所有供体细胞，现在根据需要成功地从冷冻胚胎中重新获得新的小鼠品系)。在移植了乐高转导的hPSCs的小鼠身上，也可以检测到HPSC衍生的分化后代细胞的贡献，在非常高的分辨率下，显示了感兴趣的各种组织中所有细胞类型的清晰形态。HSPC来源的细胞可以很容易地在所有组织中定位，但没有证据表明HSPC来源的细胞对内胚层或外胚层组织有直接贡献。在与NCI研究人员的合作下，我们利用这种方法在小鼠移植物抗宿主病模型的背景下研究了间充质基质细胞的体内分布和行为。 我们开发了具有高多样性31-35bp遗传条形码的慢病毒“条形码”，用于研究非人类灵长类动物模型的造血。我们的合作者荣路首先设计了这一非常强大的方法，并将其应用于研究小鼠的造血。我们现在已经移植了8只带有条形码自体CD34+细胞的猕猴，并首次能够以定量和高度重复性的方式跟踪数千个个体HSPC在一段时间内(长达两年)和多个谱系的造血输出。我们已经有了许多重要和新的发现，包括缺乏证据表明灵长类动物中有共同的淋巴祖细胞产生T和B细胞，直到移植后期才有B和T细胞的共同克隆衍生，以及更早的髓系和B细胞的共同克隆衍生。我们还首次发现了自然杀伤(NK)细胞的主要部分的独特谱系来源。即使在移植后24个月，CD16+CD56-细胞毒性NK细胞与B、T或髓系细胞也没有共享条形码。在体外和小鼠模型之前还不能阐明NK细胞的谱系关系。我们继续使用这些猕猴的条形码细胞，与Rick Childs博士的团队合作，进一步剖析体内NK细胞的个体发育和NK细胞的体外扩增过程，这与过继细胞疗法的开发高度相关。我们已经讨论了一种独特的自我更新群体，能够再生CD56+NK细胞，这种细胞存在于外周血细胞的“双阴性”群体中。我们继续寻找本体论上唯一的CD16+成熟NK亚群的前体，追踪从血液、骨髓、淋巴结以及未来的肝脏、阴道和肠道淋巴聚集物中提取的表型纯化样本中的主要克隆。 我们还证明了特定骨髓部位的单个HSPC的长期地理隔离，并使用乐高成像技术证实了上述发现。 条形码项目仍然非常活跃，许多新项目揭示了造血的多个方面，现在可以通过这项强大的技术在体内直接解决这些问题。 在小鼠模型中，我们正在使用竞争性再繁殖的方法来研究正常的HSPC和白血病移植细胞之间的关系，询问共输注增加剂量的hPSCs是否可以直接与白血病细胞竞争骨髓壁龛，从而减缓白血病的进展。我们有数据表明，对相同的利基市场存在竞争，共聚焦成像结果也支持这些功能发现。我们现在还可以用LEGO载体转导MLL-AF9小鼠白血病细胞，并跟踪白血病在体内骨髓和其他组织中的植入和进展。