Clonal analysis of in vivo hematopoiesis

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。我们对这种方法产生了浓厚的兴趣，并应邀发表了一篇视频期刊文章，从技术上演示了这种方法。 我们继续积极开发和利用具有高多样性31-35bp遗传条形码的慢病毒条形码，以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12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3.5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。我们有证据表明，克隆性不同的NK细胞具有“适应性”表型，并可能具有当前NK领域非常感兴趣的“记忆性NK”细胞的特征，但由于缺乏克隆标记，其克隆特征和起源在人类中尚不能被研究。 我们对特定骨髓部位的单个HSPC的地理隔离进行了长期的分析，在使用条形码的猕猴模型中使用乐高成像技术证实了上述发现。我们可以直接证明B细胞、CD56+NK细胞和髓系细胞在局部的骨髓壁龛中原位产生，令人惊讶的是，我们现在有强有力的证据表明T细胞在骨髓中原位产生。 我们最近在许多新的方向上扩展了条形码模型，包括：1)比较年轻和老年HSPC的克隆行为，通过移植两只带有条形码细胞的老年猕猴的初步数据，显示出与年轻动物非常不同的动力学和克隆模式。2)分析干细胞扩增的新方法，对体内扩增的细胞和未扩增的细胞进行定量和谱系分析。3)红系和血小板系克隆个体发育的研究。4)与纽约大学的Rahul Sajita合作，将单细胞RNAseq应用于条形码种群，以进一步定义个体发育和识别罕见的前体细胞种群。 我们在小鼠模型中通过竞争性再繁殖完成了正常HSPC与白血病移植细胞之间的关系的研究，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。

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. We have generated significant interest in this approach and were invited to publish a video journal article demonstrating the approach technically. We have continued active development and utilization of 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 12 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to 3.5 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 evidence that the clonally-distinct NK cells have an "adaptive" phenotype, and may have characteristics of "memory NK" cells that are of great current interest in the NK field, however their clonal characteristics and derivation have not previously been able to be investigated in humans, given the lack of clonal markers. We have extended our analysis of the geographic segregation of individual HSPCs long term in specific marrow sites, confirming the findings described above using LEGO imaging techniques in the macaque model utilizing barcoding. We can directly demonstrate in situ production of B cells, CD56+ NK cells and myeloid cells in localized marrow niches, and surprisingly, we now have strong evidence for in situ marrow production of T cells. We have recently extended the barcoding model in a number of new directions, including: 1) Comparison of the clonal behavior of young versus aged HSPC, with preliminary data from transplants of two aged macaques with barcoded cells demonstrating a very different kinetic and clonal pattern compared to young animals. 2) Analysis of novel methodologies for stem cell expansion, with quantitative and lineage analytics performed on expanded versus non-expanded cells in vivo. 3) Investigation of the clonal ontogeny of erythroid and platelet lineages. 4) Collaboration with Rahul Sajita at NYU to apply single cell RNAseq to barcoded populations in order to further define ontogeny as well as identify rare precursor cell populations. We are completing an investigation of 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.