Summary Analysis of hematopoietic stem cell function in non-human primates provides insights that are relevant for human biology and therapeutic strategies. In this study, we applied quantitative genetic barcoding to track the clonal output of transplanted autologous rhesus macaque hematopoietic stem and progenitor cells over a time period of up to 9.5 months. We found that uni-lineage short-term progenitors reconstituted myeloid and lymphoid lineages at one month, but were supplanted over time by multi-lineage clones, initially myeloid-restricted, then myeloid-B clones, and then stable myeloid-B-T multi-lineage long-term repopulating clones. Surprisingly, reconstitution of the natural killer cell lineage, and particularly the major CD16+/CD56− peripheral blood NK compartment, showed limited clonal overlap with T, B or myeloid lineages, and therefore appears to be ontologically distinct. Thus, in addition to providing insights into clonal behavior over time, our analysis suggests an unexpected paradigm for the relationship between NK cells and other hematopoietic lineages in primates.

Summary Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable, however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2-treatment using human and non-human primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB following xenotransplantation. In non-human primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over one year post-infusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and non-human primates, and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.

SUMMARY In mice, clonal tracking of hematopoietic stem cells has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3–10 years) and likely represent self-renewing hematopoietic stem cells (HSCs), while the remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid or both lineages. Over time, the 4–10% of clones with robust dual lineage contribution predominated in repopulation capacity. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically-relevant model over a long time frame, and provides a substantial system-level dataset that is a reference point for future work.

IntroductionThe ability to transfer genes into repopulating hematopoietic stem cells ex vivo and to achieve regulated expression in specific lineages following hematopoietic reconstitution would create many therapeutic opportunities. 1 Although the initial use of murine oncoretroviral vectors to transfer genes into primitive murine hematopoietic cells was reported 20 years ago, 2 translation of this approach to clinical application has been slow and has required considerable effort. Despite progress being achieved in the murine system with correction of single gene defects in murine models of human immunodeficiencies [3][4][5][6] and chronic granulomatous disease, 7,8 the much lower efficiency of gene transfer into human stem cells 1 has hampered success. The necessity for high-level oncoretroviral vector gene transfer to achieve therapeutic benefit, however, was circumvented in 2 recent clinical trials designed to cure severe combined immunodeficiency due to a deficiency of the common ␥-chain of the lymphoid cytokine receptor 9 or adenosine deaminase. 10 In these trials, a potent selective repopulating advantage of the gene-corrected lymphoid cells resulted in therapeutically relevant numbers of functional lymphocytes.Despite this success, 2 barriers appear to limit the ability of murine oncoretroviral vectors to achieve adequate transduction of primitive hematopoietic stem cells for treatment of other disorders in which the gene-corrected cells do not have a selective advantage. Because the human homolog of the receptor for murine ecotropic vector particles does not interact with the ecotropic envelope protein, amphotropic particles have been used in both human studies and in large animal models. The amphotropic receptor, however, is expressed at low levels on human stem cells. 11 Alternative envelopes have been tested, such as those derived from the gibbon ape leukemia virus, 12 feline endogenous virus (RD114), 13 or feline leukemia virus type C, 14 the receptors for which are expressed at higher levels on primitive hematopoietic cell populations. However, large animal studies have failed to clearly identify a superior pseudotype that consistently yields high-level stem cell gene transfer. These data suggest that a second barrier-namely, the requirement for mitosis to allow integration of the oncoretroviral vector genome 15 -along with the relative instability of the preintegration complex 16 may be the main limitations of oncoretroviral-mediated stem cell gene transfer. In an effort to induce stem cell cycling, cytokines such as stem cell factor (SCF), Flt-3 ligand (Flt3-L), and megakaryocyte growth and development factor (MGDF) 17,18 are added to culture medium and a fragment of fibronectin is used to colocalize vector particles and target cells, 19 leading to improved stem cell transduction efficiency in large animal models. 12,20,21 Nonetheless, there remains significant variability among animals, with many animals having low marking and only occasional animals having proportions of genetically modifi...