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. A technologically-advanced and unique imaging approach combining confocal microscopy, 2 photon microscopy and advanced analytic approaches was developed, and has been applied to study the process of hematopoietic engraftment in the marrow of mice and monkeys. Early engraftment is endosteal, and large clones consisting of the progeny of single HSPCs remain distinctly and surprisingly localized in the marrow for up to 3-4 months. This result suggests that following cell division, HSPCs spread contiguously in the marrow instead of recirculating to a new location via mobilization into the blood, and that exit of HSPCs from the marrow may be primarily a death pathway. These studies were performed utilizing total body irradiation conditioning, and new studies are ongoing asking whether the geographic patterns of engraftment and hematopoiesis may be different with alternative conditioning regimens, or no conditioning (utilizing immunodeficient/stem cell deficient recipients that can engraft all donor cells without any conditioning). Similar studies are now also ongoing in the non-human primate model. Contributions of HPSC-derived differentiated progeny cells could also be examined in mice transplanted with LEGO-transduced HPSCs, at very high resolution showing clear morphology of all cell types in various tissues of interest. Intercalating HSPC-derived cells could be easily mapped in all tissues, but there was no evidence for direct contribution of HSPC-derived cells to endodermal or ectodermal tissues. We have also applied 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 5 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to one year) 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 until 9-12 months post-transplant, and in vitro and murine models have not previously been able to shed light on NK cell lineage relationships. We have also demonstrated geographic segregation of individual HSPCs long term in specific marrow sites, confirming the findings described above using imaging techniques. The barcoding projects remain highly active, with numerous new projects shedding light on multiple aspects of hematopoiesis that can now be addressed directly in vivo via this powerful technology. We are investigating 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 preliminary data indicating competition for the same niches, with confocal imaging results also backing up these functional findings. We can also now transduce the MLL-AF9 murine leukemic cells with LEGO vectors and follow leukemic engraftment and progression in vivo in the marrow and other tissues. Clinically we have pursued analysis of the impact of eltrombopag on in vivo expansion of HSPCs, specifically in the bone marrow failure setting. We reported that 40-50% of patients with refractory aplastic anemia respond to eltrombopag, becoming transfusion independent and in the majority of cases manifesting a trilineage response. As described in Dr. Young's annual report, he has built on my group's initial observation and has now utilized this thrombopoietin analog to try and improve outcomes and maintain stem cell numbers in patients with severe new onset aplastic anemia. My group has also investigated the use of a new laboratory parameter, the immature platelet fraction, measured in clinical samples in an analogous manner to reticulocytes for the red cell lineage, to measure marrow function post-transplantation, post-transfusion, and in bone marrow failure.
|Wu, Chuanfeng; Li, Brian; Lu, Rong et al. (2014) Clonal tracking of rhesus macaque hematopoiesis highlights a distinct lineage origin for natural killer cells. Cell Stem Cell 14:486-99|
|Malide, Daniela; Metais, Jean-Yves; Dunbar, Cynthia E (2014) In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy. J Vis Exp :|
|Kim, Sanggu; Kim, Namshin; Presson, Angela P et al. (2014) Dynamics of HSPC repopulation in nonhuman primates revealed by a decade-long clonal-tracking study. Cell Stem Cell 14:473-85|
|Bat, Taha; Leitman, Susan F; Calvo, Katherine R et al. (2013) Measurement of the absolute immature platelet number reflects marrow production and is not impacted by platelet transfusion. Transfusion 53:1201-4|
|Townsley, Danielle M; Desmond, Ronan; Dunbar, Cynthia E et al. (2013) Pathophysiology and management of thrombocytopenia in bone marrow failure: possible clinical applications of TPO receptor agonists in aplastic anemia and myelodysplastic syndromes. Int J Hematol 98:48-55|
|Bat, T; Steinberg, S M; Childs, R et al. (2013) Active thrombopoiesis is associated with worse severity and activity of chronic GVHD. Bone Marrow Transplant :|