Clonal analysis of in vivo hematopoiesis

体内造血克隆分析

• 批准号: 9353127
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 165.36万
• 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/9353127
• 关键词: Allogenic; Animals; Autologous; B-Lymphocytes; Base Pairing; Behavior; Blood Platelets; Blood specimen; Bone Marrow; CD34 gene; Cell Lineage; Cell Ontogeny; Cell Therapy; Cells; Child; Clinical Trials; Collaborations; Common Lymphoid Progenitor; Cues; Cytomegalovirus; Data; Derivation procedure; Development; Engraftment; Environment; Erythroid; FCGR3B gene; Genetic; Goals; Growth; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Human; Imaging Techniques; In Situ; In Vitro; Individual; Infection; Intestines; Investigation; Kinetics; Knowledge; Label; Light; Link; Liver; Lymphoid; Macaca; Maintenance; Malignant Neoplasms; Maps; Marrow; Memory; Methodology; Methods; Modeling; Molecular; Mus; Myelogenous; Myeloid Cells; NCAM1 gene; National Heart, Lung, and Blood Institute; Natural Killer Cells; Outcome; Output; Pattern; Phenotype; Population; Primates; Process; Production; Site; Stem cells; Sweden; T-Lymphocyte; Techniques; Time; Transplantation; Vagina; Virus; Virus Diseases; Waxes; aged; cytotoxic; fighting; gene therapy; improved; in vitro Model; in vivo; insight; juvenile animal; life history; lymph nodes; mouse model; nonhuman primate; novel; precursor cell; response; segregation; stem; therapy development; transcriptome; transcriptome sequencing

We have utilized molecular techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) in vivo. We have continued active development and utilization of lentiviral "barcoding" with high-diversity 31-35 base pair genetic barcodes utilized to label individual hematopoietic stem and progenitor cells to study in vivo 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 15 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to 4.5 years) and in multiple lineages in a quantitative and highly reproducible manner. 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 and life history 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 48 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 in NHLBI, Dr Miti Kaur's group at the Tulane Non-human Primate facility, and Dr Yenan Bryceson's group at the Karolinska Institut in Stockholm Sweden. We have mapped the clonally-unique barcodes to NK cells with an "adaptive" phenotype, linked in murine studies to NK memory, and in humans to putative NK adaptive responses to viruses such as CMV. Within NK cells with an adaptive phenotype, we find oligoclonal and massively expanded individual clones, with no contributions found in other lineages, and with waxing and waning patterns suggesting responses to specific environmental cues such as viral infection or reactivation. Our data provides the first direct demonstration of clonal NK responses, possibly explaining NK memory. We continue to search for the precursor to these cells, tracking dominant clones in phenotypically purified samples from blood, bone marrow, lymph nodes, liver, and vaginal and intestinal lymphoid aggregates. With profound in vivo NK depletion, the same clones arise again, without recruitment from highly polyclonal HSPC. We have extended our analysis of the geographic segregation of individual HSPCs long term in specific marrow sites, confirming our prior findings in mice using LEGO imaging techniques now 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 also followed the output of thousands of individual HSPC clones in several animals for up to 4 years, and demonstrate marked clonal stability of output from long-term repopulating clones producing myeloid, B cell and T cell lineages, along with CD56brightCD16- NK cells. We have shown that myeloid vs lymphoid bias is common, and that this bias remains stable in individual clones over time. We have recently extended the barcoding model in a number of additional 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 now have set up the DropSeq single cell methodology at NHLBI in collaboration with Andre Larochelle, and have evidence that we can retrieve both the barcode and the transcriptome from single cells. This approach offers many investigative opportunities.

我们利用分子生物学技术对造血干细胞和祖细胞（HSPCs）在体内的行为有了新的认识。 我们继续积极开发和利用慢病毒“条形码化”，其具有用于标记个体造血干细胞和祖细胞的高多样性31-35个碱基对遗传条形码，以在非人灵长类动物模型中研究体内造血。 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15只猕猴，并且已经能够以定量和高度可重复的方式跟踪数千个个体HSPC随时间（长达4.5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起源和生活史。即使在移植后48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NHLBI的Rick查尔兹博士小组、Tulane非人灵长类动物机构的Miti Kaur博士小组和瑞典斯德哥尔摩Karolinska研究所的Yenan Bryceson博士小组合作，继续使用这些猕猴的条形码细胞进一步剖析体内NK细胞个体发育和NK细胞的离体扩增过程，这与过继性细胞治疗开发高度相关。 我们已经将克隆独特的条形码映射到具有“适应性”表型的NK细胞，在小鼠研究中与NK记忆相关联，并且在人中与对病毒如CMV的推定NK适应性应答相关联。在具有适应性表型的NK细胞内，我们发现寡克隆和大规模扩增的个体克隆，在其他谱系中没有发现贡献，并且具有暗示对特定环境线索（如病毒感染或再活化）的反应的消长模式。我们的数据提供了克隆NK反应的第一个直接证明，可能解释NK记忆。我们继续寻找这些细胞的前体，追踪来自血液、骨髓、淋巴结、肝脏、阴道和肠道淋巴聚集体的表型纯化样品中的优势克隆。 随着体内NK的大量消耗，相同的克隆再次出现，而没有从高度多克隆的HSPC中募集。 我们已经扩展了我们对特定骨髓部位中单个HSPC长期地理分离的分析，证实了我们先前在小鼠中使用乐高成像技术的发现，现在在猕猴模型中使用条形码。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。 我们还跟踪了几种动物中数千个单个HSPC克隆的输出长达4年，并证明了长期再增殖克隆输出的显著克隆稳定性，这些克隆产生骨髓、B细胞和T细胞谱系，沿着CD 56亮CD 16- NK细胞。我们已经表明，骨髓与淋巴的偏见是常见的，这种偏见保持稳定，随着时间的推移，在个别克隆。 我们最近在许多其他方向扩展了条形码模型，包括：1）年轻与老年HSPC的克隆行为比较，来自两只携带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。我们现在已经在NHLBI与Andre Larochelle合作建立了DropSeq单细胞方法，并且有证据表明我们可以从单细胞中检索条形码和转录组。这种方法提供了许多调查机会。