Clonal analysis of in vivo hematopoiesis

我们已经利用分子和成像技术，以获得新的见解造血干细胞和祖细胞（HSPCs）在体内的行为。 利用携带5种不同荧光蛋白（FP）基因的慢病毒载体（称为LEGO载体），我们利用组合颜色方法能够在体内时间和空间上独特地标记并跟踪来自个体HSPC的输出。 本研究将共聚焦显微镜、双光子显微镜和先进的分析方法相结合，建立了一种技术先进、独特的成像方法，并应用于小鼠和猴骨髓造血移植过程的研究。 早期植入是骨内的，并且由单个HSPC的后代组成的大克隆在骨髓中保持明显且令人惊讶的定位长达3-4个月。 这一结果表明，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种死亡途径。 这些研究是利用全身照射预处理进行的，新的研究正在进行中，询问植入和造血的地理模式是否可能与替代预处理方案不同，或者没有预处理（利用免疫缺陷/干细胞缺陷受体，可以在没有任何预处理的情况下移植所有供体细胞，现在成功地从冷冻胚胎中重新衍生，以将新的小鼠品系带入NIH）。 还可以在移植有LEGO转导的HPSC的小鼠中检查HPSC衍生的分化后代细胞的贡献，以非常高的分辨率显示各种感兴趣组织中所有细胞类型的清晰形态。嵌入HSPC衍生的细胞可以很容易地映射在所有组织中，但没有证据表明HSPC衍生的细胞对内胚层或外胚层组织的直接贡献。 在与NCI研究人员的合作中，我们利用这种方法来研究小鼠移植物抗宿主病模型中间充质基质细胞的体内分布和行为。 我们已经开发了具有高多样性31- 35 bp遗传条形码的慢病毒“条形码化”，以研究非人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强大的方法，并将其应用于研究小鼠造血。我们现在已经用条形码自体CD 34+细胞移植了8只猕猴，并且首次能够以定量和高度可重复的方式跟踪数千个HSPC随时间（长达两年）和多个谱系的造血输出。 我们已经取得了许多重要的新发现，包括缺乏证据表明灵长类动物中有一个共同的淋巴祖细胞产生T和B细胞，直到移植后晚期才有B和T细胞的共享克隆衍生，以及更早的骨髓和B细胞的共享克隆衍生。我们还首次发现了自然杀伤（NK）细胞主要部分的独特谱系来源。即使在移植后24个月，CD 16 + CD 56-细胞毒性NK细胞也不与B、T或骨髓细胞共享条形码。体外和鼠模型以前不能阐明NK细胞谱系关系。 我们与Rick查尔兹博士的小组合作，继续使用来自这些猕猴的条形码化细胞来进一步剖析体内NK细胞个体发育和NK细胞的离体扩增过程，这与过继性细胞疗法开发高度相关。 我们已经讨论了一个独特的自我更新的人口能够再生的CD 56 + NK细胞是目前在一个“双阴性”的外周血细胞群体。 我们继续寻找本体上独特的CD 16+成熟NK亚群的前体，追踪来自血液、骨髓、淋巴结的表型纯化样品中的显性克隆，以及未来的肝脏、阴道和肠道淋巴聚集体。 我们还证明了在特定的骨髓部位长期存在个体HSPC的地理分离，证实了上述使用乐高成像技术的发现。 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，现在可以通过这种强大的技术直接在体内解决。 我们正在研究正常HSPC和白血病移植细胞之间的关系，在小鼠模型中使用竞争性再增殖，询问是否增加剂量的HPSC的共输注可以直接与白血病细胞竞争骨髓龛，从而减缓白血病进展。我们有数据表明竞争相同的壁龛，共聚焦成像结果也支持这些功能性发现。我们现在还可以用LEGO载体来接种MLL-AF 9小鼠白血病细胞，并在骨髓和其他组织中体内跟踪白血病移植和进展。

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.