Abstract
BackgroundCurrent 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.MethodsHumanized 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.ResultsA 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.ConclusionsHuman 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.
Highlights
Current understanding of hematopoiesis is largely derived from mouse models that are physiologi‐ cally distant from humans
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
Our study revealed the in vivo dynamics of clonal expansion, emergence of stable stem cell clones, and consequences of vector integration bias on repopulation of human hematopoietic stem and progenitor cells (HSPC) in mouse environment
Summary
Current understanding of hematopoiesis is largely derived from mouse models that are physiologi‐ cally distant from humans. To mimic anti-HIV gene therapy in HIV patients, a recent study used an HIV-1 pre-infected hu-BLT mouse model and demonstrated that HIV-1 infection induces selective expansion of antiHIV-1 dual shRNA gene-modified (protected) CD4 + T cells over control vector-modified unprotected CD4 + T cells [9]. Another recent study utilized HIV-infected huBLT mouse models to iteratively test CD28 and 4-1BB costimulation for optimizing anti-HIV CAR T cell therapy [10] These studies reaffirm that hu-BLT mouse model provides an invaluable in vivo system for cancer, virus, and stem cell research and is indispensable for preclinical gene therapy studies.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
