Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias.
Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias.
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人源化小鼠中的纵向克隆追踪揭示了基因修饰的人HSPC的持续多克隆再增殖，尽管存在载体整合偏倚。
DOI：
10.1186/s13287-021-02601-5
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发表时间：
2021-10-07
影响因子：
7.5
通讯作者：
Chen ISY
中科院分区：
文献类型：
--
作者：
Suryawanshi GW;Arokium H;Kim S;Khamaikawin W;Lin S;Shimizu S;Chupradit K;Lee Y;Xie Y;Guan X;Suryawanshi V;Presson AP;An DS;Chen ISY
目前对造血的了解主要来自小鼠模型，这些模型在生理上与人类相距甚远。人源化小鼠为研究人类疾病提供了最具生理学相关性的小动物模型，尤其是临床前基因治疗研究。然而，人类造血干细胞和祖细胞（HSPC）在这些动物模型中的克隆再生动力学仅部分了解。使用一种新的克隆跟踪方法设计的小样本量，我们的目标是揭示潜在的克隆动力学的人类细胞在小鼠环境中的再生。采用慢病毒载体转导的人胎肝HSPC （FL-HSPC）在NOD中移植产生人源化骨髓-肝-胸腺（hu-BLT）小鼠。Cg-PrkdcscidIl2rgtm1Wjl/SzJ （NSG）小鼠植入人胎胸腺片。我们开发了一种方法，在仅25µl的小鼠血液中跟踪载体整合位点（VIS），用于小鼠环境中人类HSPC再群集的纵向和定量克隆分析。我们探索了人类HSPC的转录和表观遗传特征，以寻找可能存在的VIS偏见。在hu-BLT小鼠中，共追踪了897个HSPC克隆，首次证明了治疗性和对照性载体修饰的人类细胞群同时在同一人源化小鼠中再生的克隆动力学和协调扩展。移植后19周多克隆再群体稳定，最大克隆的贡献在4周内翻了一番。此外，550个（约60%）克隆持续6周以上，并在不同器官之间高度共享。正常克隆谱证实了基因治疗载体的安全性。人类FL-HSPC的多组学分析显示，54%的载体整合发生在h3k36me3富集区域的±1 kb内。人类在小鼠中的繁殖是多克隆的，并且比以前在人类中观察到的稳定得更快。对H3K36me3的VIS偏好对HSPC的再种群没有明显的负面影响。我们的研究提供了一种方法来纵向跟踪小动物模型中的克隆繁殖，该模型广泛用于干细胞和基因治疗研究，并为临床应用设计了慢病毒载体。本研究的结果为理解人类HPSC在小鼠环境中繁殖的克隆行为提供了一个框架，这对于将人源化小鼠模型的结果转化为人类环境至关重要。在线版本包含补充材料，可在10.1186/s13287-021-02601-5获得。
Current understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment. Humanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias. A total of 897 HSPC clones were longitudinally tracked in hu-BLT mice—providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions. Human repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings. The online version contains supplementary material available at 10.1186/s13287-021-02601-5.
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