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Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo . However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34 + cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1 –/– -IL2Rγc –/– (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD- Prkdc scid/scid - IL2Rγc –/– (NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W 41 /W 41 derivatives showed superior chimerism in the W 41 -deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice.

Plerixafor (P) together with granulocyte colony–stimulating factor (G) is now recognized as an important strategy for mobilizing hematopoietic cells for use in patients given myelosuppressive therapies. However, quantitative comparisons of their ability to mobilize human cells with different hematopoietic activities in vitro or in vivo (in immunodeficient mice) and their interrelationships have not been investigated. To address these questions, we collected samples from 5 normal adult volunteers before and after administering P alone and from another 5 before and after a 4-day course of G and again after a subsequent injection of P. Measurements of their blood content of CD34+ cells, in vitro myeloid colony–forming cells, 3- and 6-week long-term culture (LTC) cell outputs, and levels of circulating human platelets, as well as myeloid and lymphoid cells obtained in immunodeficient mice that received transplants, showed all activities were maximal 4 hours after P preceded by G, and 3-week LTC outputs showed the highest concordance with the 3-week circulating human neutrophil levels obtained in mice that received transplants. Thus, human cells capable of producing neutrophils rapidly in vivo were optimally mobilized by the G + P protocol, and the 3-week LTC assay appears to offer a more specific predictor of their levels than conventional CD34+ cell or colony-forming cell counts.

Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.

Better methods to characterize normal human hematopoietic cells with short-term repopulating activity cells (STRCs) are needed to facilitate improving recovery rates in transplanted patients.We now show that 5-fold more human myeloid cells are produced in sublethally irradiated NOD/SCID-IL-2Receptor-γchain-null (NSG) mice engineered to constitutively produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel factor (NSG-3GS mice) than in regular NSG mice 3 weeks after an intravenous injection of CD34 human cord blood cells. Importantly, the NSG-3GS mice also show a concomitant and matched increase in circulating mature human neutrophils. Imaging NSG-3GS recipients of lenti-luciferase-transduced cells showed that human cells being produced 3 weeks posttransplant were heterogeneously distributed, validating the blood as a more representative measure of transplanted STRC activity. Limiting dilution transplants further demonstrated that the early increase in human granulopoiesis in NSG-3GS mice reflects an expanded output of differentiated cells per STRC rather than an increase in STRC detection.NSG-3GS mice support enhanced clonal outputs from human short-term repopulating cells (STRCs) without affecting their engrafting efficiency. Increased human STRC clone sizes enable their more precise and efficient measurement by peripheral blood monitoring.

Background: Assessment of the growth and differentiation of human hematopoietic cells in immunodeficient mice has become vital to much basic and translational research. Historically, the primary focus has been to quantify different types of repopulating cells, based on the diversity and longevity of their clonal outputs in transplanted mice, assuming the results would be relevant to clinical transplants in humans. With the development of mice that lack B, T and NK cells on a variety of backgrounds and that have normal life expectancies but differences in cellular DNA repair ability, an increasing application is to use mice repopulated with human cells to interrogate and perturb mechanisms controlling normal, genetically modified and malignant cell behavior. We have previously shown that C57Bl6 mice homozygous for the W41 mutation of c-kit are fertile with a normal lifespan but have a functionally compromised hematopoietic stem cell (HSC) population. This enables single transplanted syngeneic HSCs to be detected at high frequency in these mice when they have been given a sublethal irradiation dose. Importantly, the HSC-derived clones produced in these mice display the same growth, self-renewal and differentiation abilities as in myeloablated recipients that require a co-transplant of normal mouse bone marrow (BM) cells to support their survival. We now report the development and improved repopulation by human cord blood (CB) CD34+ cells of mice that have the same genetically determined B, T, NK immunodeficiency as NOD/Rag1-/--IL2Rγc-/- (NRG) into which a homozygous W41gene has been introduced. This was achieved by crossing and backcrossing the progeny of NRG x C57Bl6-W41/W41 matings and selecting mice that were homozygous for the Sirpα allele of the NOD mouse, the null Rag1 and null IL2Rgamma chain, genes of the NRG mouse and the W41 gene to obtain all of these on an otherwise mixed NODxC57Bl/6 background (NRG-W41 mice). The NRG mouse was chosen because the Rag1 KO has no effect on the radiosensitivity of other tissues as is the case with the scid (S) gene in the NSG mouse. As a result, use of the NRG mouse allows exploitation of the radioprotective effect of a reduced irradiation dose rate and hence delivery of a selectively higher dose to the HSCs of the host. Results: Initial studies showed that parental NRG mice given 900 cGy split or spread continuously over 3 hrs show similar repopulation by human CD34+ CB cells as NSG mice given 315 cGy, but are more robust with consistent longterm survival. We then performed a pilot experiment using the same transplant design (2x104 CD34+ CB cells/mouse) to compare chimerism obtained in NRG-W41 mice given an estimated “equivalent” radiosensitizing regimen of 150 cGy. The levels of multiple lineages of human cells measured in the BM and spleen 20 weeks post-transplant revealed these were greatly increased in the NRG-W41 mice (>95% human CD45+ cells in the BM vs 40% in NRG mice). Kinetic analysis of human cells in the blood also showed an enhanced output of human myeloid and B-lymphoid cells over time (5-fold higher in the NRG-W41 mice after >3 weeks). Particularly notable was the selectively increased (20-fold) and sustained output of human glycophorin A+ (GPA+) erythroid cells in the NRG-W41 mice (5% human GPA+ cells in the BM of 20-week NRG-W41 mice given 150 cGy and 5x104 CD34+ CB cells/mouse vs 0.25% in the BM of the matched NRG mice given 900 cGy). A similar marked increase (20-fold) was seen on the level of circulating human platelets (SSClowCD41+ CD61+ cells) in comparable groups of transplanted NRG-W41 and NRG mice. We then investigated the extent of repopulation achievable in untreated NRG-W41 recipients. We therefore transplanted mice of both strains with 5x104 CD34+CB cells each and have now followed the levels of human cells in their circulation and BM for up to 20 weeks. Human cells were barely detectable at 3 weeks post-transplant in either strain, but then in the unirradiated NRG-W41 mice only, their levels (all lineages) increased to close to those attained in NRG mice given 900 cGy. Conclusion: NRG-W41 mice support robustly enhanced and long term generation in vivo of a wide range of human hematopoietic cell types including erythrocytes and platelets, with high levels of chimerism achieved even in unirradiated primary recipients transplanted with relatively low numbers of human CD34+ CB cells. Disclosures No relevant conflicts of interest to declare.

Background: Allografting of adult patients remains a widely utilized strategy but rates of treatment-related morbidity and mortality are still high. Granulocyte colony stimulating factor (G-CSF)-stimulated peripheral blood (G-PB) is now the most common source of clinically used hematopoietic allografts. G-PB grafts provide faster rates of neutrophil and platelet recovery compared to unstimulated bone marrow (BM), but G-PB is also associated with an increased risk of graft versus host disease (GVHD). More recently plerixafor has been demonstrated to be an effective hematopoietic cell mobilizing agent, and plerixafor is also now widely used alongside G-CSF to procure PB cells for clinical allografting. However, little is known as to whether plerixafor treatment alone or in combination with G-CSF might have an effect (positive or negative) on the repopulating activity of a BM harvest. Methods: In this study we have evaluated the affect over time of plerixafor ± G-CSF on the hematopoietic activity of the BM and PB of 10 normal adult donors. Donors received one treatment of plerixafor alone (240 μg/kg) vs plerixafor after 4 days of prior G-CSF (5 μg/kg/day). BM and PB samples were taken pre-treatment, post-G-CSF/pre-plerixafor, 4 hrs post-plerixafor, and 24 hours post plerixafor/48 hours post G-CSF. These samples were then analyzed for total nucleated cells (TNCs), CD34+ cells, in vitro colony-forming cells (CFCs), and cells capable of regenerating mature (TNCs) and CD34+ cells in 3-week stroma-containing cultures (LTC-ICs-3 wks) and in short-term (3-week) repopulating cell (STRC) assays in immunodeficient mice. The STRC assessment took advantage of a modified assay with improved sensitivity recently developed by our group, which utilizes NOD/Rag1-/--IL2Rγc-/- mice that constitutively produce human IL-3, GM-CSF and Steel factor (NRG-3GS mice) as the transplant recipients. Results: The G-CSF+plerixafor treatment protocol produced the largest increase in hematopoietic cells in the PB of the donors, as compared to either G-CSF or plerixafor alone. All counts reached peak values 4 hours after administration of plerixafor and 24 hrs after the last administration of G-CSF resulting in maximum increases at that time of 6-fold above baseline for TNCs, 15 for CD34+ cells and 25 for LTC-ICs-3 wks based on absolute measurements of each cell type per µL of PB. In the BM, where corresponding absolute values are not possible, there was a relative decrease in each of these parameters compared to BM TNC values. To assess in vivo STRC activity, collection time-points and sample types were pooled, depleted of CD3+ cells (using diptheria-conjugated OKT3), and 3x106 total cells transplanted IV into each of 2-3 replicate NRG-3GS mice. Four days of G-CSF reduced the STRC activity in donor BM asprates below baseline levels which was rescued by 4 hours after administration of plerixafor to the same donors. This restored human STRC activity included progenitor (CD34+) cells as well as maturing erythroid (glycophorin A+), granulopoietic (CD33+) cells and circulating SSClow human CD41+/CD61+ platelets. Conclusions: A time course study of plerixafor ± G-CSF administration in normal volunteers aligns with previous findings showing that G-CSF+plerixafor treatment induces a transiently higher output of multiple primitive hematopoietic cell types compared to G-CSF or plerixafor alone. These findings suggest an optimal time for harvesting PB and BM for a future clinical trial to determine which may serve as the superior allograft in patients. Disclosures No relevant conflicts of interest to declare.

Accelerated phase (AP) CML is marked by a rising leucocyte count in the face of maintenance treatment, exaggerated basophilia and accumulating blasts. However, AP is typically short-lived and rapidly succeeded by blast crisis. Genetic alterations resulting in loss of IKAROS activity are associated with the development of both BCR-ABL1+ myeloid and lymphoid leukemias, as well as the related BCR-ABL1-negative MPNs. However, how loss of IKAROS functionality contributes to the malignant features of these leukemias is not clear. Here we demonstrate that direct suppression of IKAROS activity in CD34+ cells from chronic phase (CP) CML patients leads to their rapid acquisition of features of AP without causing progression to acute leukemia. To suppress IKAROS activity in CD34+ CP CML cells, we transduced samples from 4 patients with a lentiviral GFP vector encoding a dominant-negative isoform of IKAROS (dnIK6), or an empty control YFP vector, and compared their progeny generated in cultures containing mouse stromal cells engineered to produce human SCF, IL-3, FLT3L and G-CSF. In all cases, we found dnIK6 transduction caused a rapid (evident within 2 weeks) and sustained (for at least 6 weeks) increase in total cell output (4-20x higher than YFP+ controls at 4-6 weeks, p To interrogate the molecular mechanisms underlying the dnIK6 effects on cell output, we compared the levels of activated signaling proteins by intracellular flow cytometry. This showed increased activation of STAT5 (80±14% higher median fluorescent intensity (MFI), p In summary, IKAROS disruption in patients’ CP CD34+ CML cells recapitulates cardinal features of AP disease, including an increased cell output associated with an enhanced production of basophils and an eventual accumulation of primitive cells. We further identify dnIK6-mediated suppression of SOCS2 and the β-catenin destruction complex, with enhanced activation of STAT5 and β-catenin in progenitor cells, as a likely key mechanism contributing to the altered biology of AP cells. Disclosures: No relevant conflicts of interest to declare.

The critical human cells that produce neutrophils and platelets within 3 weeks in recipients of hematopoietic transplants are thought to produce these mature blood cells with the same kinetics in sublethally irradiated immunodeficient mice. Quantification of their numbers indicates their relative underrepresentation in cord blood (CB), likely explaining the clinical inadequacy of single CB units in rescuing hematopoiesis in myelosuppressed adult patients. We here describe that exposure of CD34(+) CB cells ex vivo to growth factors that markedly expand their numbers and colony-forming cell content also rapidly (within 24 hours) produce a significant and sustained net loss of their original short-term repopulating activity. This loss of short-term in vivo repopulating activity affects early platelet production faster than early neutrophil output, consistent with their origin from distinct input populations. Moreover, this growth factor-mediated loss is not abrogated by published strategies to increase progenitor homing despite evidence that the effect on rapid neutrophil production is paralleled in time and amount by a loss of the homing of their committed clonogenic precursors to the bone marrow. These results highlight the inability of in vitro or phenotype assessments to reliably predict clinical engraftment kinetics of cultured CB cells.