Clonal analysis of in vivo hematopoiesis

体内造血克隆分析

• 批准号: 9157391
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 130.35万
• 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/9157391
• 关键词: Allogenic; Animals; Autologous; B-Lymphocytes; Back; Behavior; Blood Cells; Blood Platelets; Blood specimen; Bone Marrow; CD34 gene; Cell Lineage; Cell Ontogeny; Cell Therapy; Cells; Characteristics; Child; Clinical Trials; Collaborations; Color; Common Lymphoid Progenitor; Data; Derivation procedure; Development; Dose; Engraftment; Erythroid; FCGR3B gene; Future; Genes; Genetic; Goals; Growth; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Human; Image; Imaging Techniques; In Situ; In Vitro; Individual; Infusion procedures; Intestines; Investigation; Kinetics; Knowledge; Lentivirus Vector; Leukemic Cell; Light; Liver; Lymphoid; Macaca; Marrow; Memory; Methodology; Methods; Modeling; Mus; Myeloid Cells; NCAM1 gene; Natural Killer Cells; Natural regeneration; Outcome; Output; Pattern; Phenotype; Population; Primates; Process; Production; Proteins; Publishing; Site; Stem cells; T-Lymphocyte; Time; Transplantation; Vagina; aged; combinatorial; cytotoxic; gene therapy; improved; in vitro Model; in vivo; insight; interest; journal article; juvenile animal; leukemia; lymph nodes; molecular imaging; nonhuman primate; novel; precursor cell; segregation; self-renewal; stem; therapy development; transcriptome sequencing; 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. 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.

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。我们对这种方法产生了浓厚的兴趣，并被邀请发表一篇视频期刊文章，从技术上演示这种方法。 我们继续积极开发和利用具有高多样性31- 35 bp遗传条形码的慢病毒“条形码”来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12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3.5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。 我们有证据表明，克隆不同的NK细胞具有“适应性”表型，并且可能具有“记忆NK”细胞的特征，这在NK领域中是当前非常感兴趣的，然而，由于缺乏克隆标记物，它们的克隆特征和来源以前无法在人类中进行研究。 我们已经扩展了我们对特定骨髓部位中个体HSPC长期地理分离的分析，证实了上述使用乐高成像技术在猕猴模型中使用条形码的发现。我们可以直接证明在局部骨髓龛中原位产生B细胞、CD 56 + NK细胞和髓样细胞，令人惊讶的是，我们现在有了原位骨髓产生T细胞的有力证据。 我们最近在许多新的方向上扩展了条形码模型，包括：1）年轻与老年HSPC的克隆行为的比较，来自两个带有条形码细胞的老年猕猴移植的初步数据表明，与年轻动物相比，动力学和克隆模式非常不同。2)分析干细胞扩增的新方法，对体内扩增的细胞与未扩增的细胞进行定量和谱系分析。3)红细胞系和血小板系克隆个体发生的研究。4)与纽约大学的Rahul Sajita合作，将单细胞RNAseq应用于条形码群体，以进一步定义个体发育并识别罕见的前体细胞群体。 我们正在完成一项在小鼠模型中使用竞争性再增殖的正常HSPC和白血病移植细胞之间关系的研究，询问共输注增加剂量的HPSC是否可以直接与白血病细胞竞争骨髓小生境，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。