Competitive Repopulating Units Research Articles

Deciphering the Boundaries of KBTBD4-CoREST1 Axis Modulation to Maximally Expand Human HSCs

Background: The UM171 technology has led to major progress in the expansion of cord blood units (CBUs) with low cellularity (Fares et al., Science 2014). This breakthrough has notably increased availability of better HLA-matched CBUs and improvements in clinical outcome (Cohen et al., Lancet Hematol 2020 and Blood Advances 2023, PMID: 37467030; see also Cohen et al. and Milano et al., this meeting). Enhanced HLA matching is a direct consequence of the selection of CBUs which are otherwise too small without expansion. Indeed, with this technology, approximately 50% of units in CB banks are eligible for clinical use, versus 5% without expansion. As recently reported, UM171 promotes the activation of the novel CUL3-KBTBD4 E3 ligase complex leading to the ubiquitylation and degradation of its natural substrate, CoREST1, a repressor complex that includes RCOR1, LSD1, HDAC1/2. This complex is upregulated in human HSC upon their ex vivo culture where it compromises their potential in the absence of UM171 (Chagraoui et al., Cell Stem Cell, 2021). Aims and Methods: The goal of this research is to understand the boundaries of KBTBD4-CoREST1 axis modulation that determine the optimal expansion of human HSCs in vitro and in vivo. More precisely, we monitored the relationship between CoREST1 levels in primary CD34+CD45RA-CD90+ CB cells and determined the impact of maximal CoREST1 degradation on the yield of expanded HSC using a series of functional in vitro and in vivo approaches along with transcriptomic data. Results: Using flow cytometry, we noticed a gradual degradation of RCOR1 with increasing concentrations of UM171 with a plateau at 125nM suggesting that maximal RCOR1 degradation was reached at this dose which represented a 4-fold increase above the concentration of UM171 used in current clinical manufacturing. Of interest, at high concentration of UM171 (i.e.,125nM), total nucleated cell expansion was reduced by approximately 30% while that of primitive CD34+CD45RA-CD90+EPCR+ HSC was maximal and significantly above that measured at 35nM suggesting a preferential impact of CoREST1 degradation on the more primitive cell compartment along with improvement in graft quality. CITE-Seq experiments confirmed the preferential expansion of primitive HSPC including HSCs and LMPPs with a reduction of more mature cellular subsets when cells were exposed to 125nM of UM171 in comparison to 35nM. The in vivo functionality of the 7-day expanded grafts (35nM vs 125nM UM171) were compared by transplanting the outcome of 100 Day 0 CD34+ CB cells in NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF) mice, a cell dose representing less than 1 competitive repopulation unit (CRU) of unexpanded cells in this model. Using this low cell dose (limit dilution), we observed that the 125nM expanded graft provided 10-times greater engraftment than observed in mice transplanted with cells exposed to 35nM UM171 with values reaching a median of 5±5% vs. 0.4±0.13%(n=10 mice per condition, p=0.01; Mann Whitney test) of human engraftment at 12 weeks post-transplantation, respectively. Conclusion: Maximal stimulation of CUL3-KBTBD4 by higher concentration of UM171 leads to much better expansion of functional human HSC and opens new avenues for optimal expansion of very small cord blood units and possibly adult bone marrow-derived HSCs which require more than minimal manipulation (e.g., gene therapy). We believe this new data represents a novel milestone in HSC expansion with obvious subsequent clinical benefits.

Mitigating Detrimental Influences of Ambient Air Collection of Bone Marrow By Hypoxic Collection Results in Detection of Functionally Enhanced Aged Mouse Hematopoiesis: Reflecting Stem Cell Expression Levels of CXCR4, CCR5 and ROS

Bone marrow (BM) of aged mice have significantly increased phenotypically-defined hematopoietic stem cells (HSCs), but greatly decreased competitive engrafting capability with a shift to decreased lymphoid capability when compared to young mice. HSCs and HPCs reside in vivo in a grossly hypoxic (e.g. ~3% O2) environment compared to that of ambient air (~21% O2). Yet, until recently all knowledge of HSCs and HPCs was based on cells initially collected in ambient air, which allows for the gross underestimation of the numbers of phenotypic and functional HSCs while overestimating HPCs. Within minutes of exposure to ambient air, levels of reactive oxygen species (ROS) are increased, inducing rapid differentiation, not death, of HSCs (Mantel, et. al., Cell 16:1553,2015). We hypothesized that BM HSCs and HPCs from aged mice collected/processed in ambient air does not reflect their true numbers and functional capabilities. We thus re-evaluated the aged hematopoietic system of mouse BM collected/processed in more physiologically relevant conditions of 3% O2 in comparison to the artificially induced extra physiological oxygen shock stress (EPHOSS) situation associated with collecting/processing cells in ambient air in 6-10 week old and 20-28 month old C57Bl/6 mice (N = 8-15 mice/group in a total of 3-4 experiments). BM cells from old, compared to young, mice collected in air had significant increases in LT-HSC, ST-HSC, MPP, CMP, GMP, MEP, but decreases in CLP numbers per femur. When BM was collected under hypoxic conditions, LT-HSC numbers were increased in both young and old mice, with enhanced numbers of CLP in the older mice. When collected in air, old BM cells had ~50% decreased chimerism in a competitive repopulating assay over six months of primary engraftment compared to young BM cells with respective young vs. old competitive repopulating units (CRUs) of 19.4 vs. 4.5 per 106 cells (p<0.02) based on limiting dilution analysis, and lymphoid/myeloid ratios of 3.54 vs. 0.95 (p<0.002). Secondary transplants over 4 months showed 75% reduced chimerism of old vs. young BM. However, when compared to BM collected in air, hypoxia collected cells from young mice demonstrated over a 50% increase in chimerism in 1° transplants (up to 6 months), with old and young hypoxia collected BM having much less drastic changes in primary chimerism and lymphoid:myeloid ratio (2.2±0.6 vs. 3.3.±0.47; p>0.05). CRUs of hypoxia collected old cells (14.2) and air collected young cells (19.4) were similar (p>0.05). There were 2-3 fold fewer CFU-GM, BFU-E, and CFU-GEMM from air collected old BM when compared to young BM. Old BM HPCs were in a slow/non-cycling state compared to the rapid cycling of the young BM HPCs. In contrast, when collected in hypoxia, there were fewer HPCs in the BM of young mice and they were in a slow cycling state when compared to young BM cells collected in air. In old BM collected under hypoxic conditions, there were more HPCs then when collected in air and these cells were in rapid cycle. Results for the numbers of phenotypic HSC and HPC and functional characteristics of HPC have been duplicated in CB6 and BALB/c mice. Hence, there are more phenotypic and functional HSCs and HPCs when old BM cells are collected/processed in hypoxia compared to that of ambient air, numbers equal or close to those when young BM is processed in ambient air. Mechanistically, the enhanced numbers and function of old HSCs/HPCs when BM is collected/processed in hypoxia is associated with changes in expression of CXCR4, CCR5, and ROS. CXCR4 signaling mediates HSC homing. CCR5 is a receptor for RANTES, a chemokine implicated in increasing lymphoid-biased HSCs. ROS is implicated in EPHOSS-mediated differentiation of LT-HSC. We found that expression of CXCR4 and CCR5 was lower in old vs. young LT-HSCs collected in air but was enhanced in hypoxia-collected old LT-HSCs to the level of air collected young cells. Total and mitochondrial ROS were higher in old vs. young LT-HSC from air collected cells, but were decreased in old cells collected in hypoxia to the level of young air collected cells. Therefore, although cells from older BM are more sensitive than younger BM cells to the stress of EPHOSS, enhanced numbers of functional HSCs and HPCs can be obtained from old BM by hypoxic collection and processing. DisclosuresNo relevant conflicts of interest to declare.

Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

AbstractPlant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

Latexin Inhibition Mitigates Irradiation Induced Hematopoietic Injury

Radiation-associated bone marrow (BM) injury is one of the most serious limiting factors of radiotherapy. Radiation-induced hematopoietic injury, no matter how transient or long lasting, can ultimately impair HSC function and decrease the HSC reserve, leading to increased risk for the development of BM failure or cancer. However, molecular mechanisms underlying radiation-induced HSC functional decline are largely unknown.We previously identified a stem cell regulatory gene, latexin (Lxn), as a novel negative regulator of HSCs in mice. HSCs in Lxn knockout mice (Lxn-/-) had increased self-renewal and survival. In our new findings, we surprisingly found that Lxn-/- mice had the significant survival advantages under lethal dose of total body irradiation (TBI). We further found that HSCs and hematopoietic progenitor cells (HPCs), measured by immunophenotypes and colony assay, recovered much faster in Lxn-/- mice than wild-type mice (WT) within one month after sub-lethal dose of TBI. The better preserved HSC/HPC pool was due to the decreased apoptosis in which the percentage of Annexin V + PI- apoptotic HSCs/HPCs cells was significantly lower in Lxn-/- mice than WT mice. These data suggest that Lxn inactivation protects HSCs and HPCs from radiation-induced cell death, thus mitigating acute hematopoietic suppression and conferring a survival advantage.To determine the long-term effect of TBI on Lxn-/- HSCs, we performed limiting dilution competitive repopulation unit assay (CRU), and found that Lxn-/- CRU was significantly higher than WT CRU. Moreover, we performed serial transplantation experiment, and found that Lxn-/- HSC continuously regenerated blood and bone marrow cells even at the 4th round of transplantation whereas WT HSCs were exhausted. These data provide robust evidence that Lxn inactivation protects functional long-term HSCs from radiation-induced injury. Radiation can increase the risk of hematological malignancy later in the life. We thus maintained a group of mice that were subject to either a single dose of 6.5Gy TBI or split low doses of TBI (2 Gy daily for 6 days), and monitored their gross condition and blood cell counts for 20 months. At 20 month post-radiation, we performed bone marrow analysis and histopathology analysis. We found that Lxn-/- mice did not spontaneously develop hematopoietic malignancies, their bone marrow HSCs/HPCs had normal population size, and bone marrow had normal histopathology. These data suggest that Lxn inactivation mitigates radiation-induced short-term myelosuppression and long-term HSC functional impairment without induction of hematologic malignancy.At the molecular level, we previously reported that Lxn sensitized leukemogenic cells to gamma-irradiation-induced cell-cycle arrest and cell death through Rps3 pathway, and Rps3 was a binding protein of Lxn. Rps3 has been shown to be involved in the NFkB pathway. We found that Rps3 bound Lxn in primary hematopoietic stem and progenitor cells (HSPCs) using Co-IP assay. Lxn-/- HSPCs had the increased expression of Rps3 and NFkB p65 before or post-irradiation. Knockdown of Rps3 in Lxn-/- HSPCs decreased NFkB p65 and increased radiation-induced apoptosis. Moreover, when Lxn-/- HSPCs were treated with NFkB p65 specific inhibitor, the similar phenotypes were also shown, suggesting that Lxn functions through Rps3-NFkB-mediated pro-survival pathway in primary HSPCs. We are currently proving this molecular pathway using the in vivo model by crossing p65 knockout mice with Lxn-/- mice.In conclusion, latexin inhibition mitigates irradiation induced hematopoietic injury via Rps3-NFkB-mediated pro-survival pathway. DisclosuresNo relevant conflicts of interest to declare.

A Conserved Cis-Regulatory Retinoic Acid Responsive Element Is Essential for Maintenance of Primitive Hematopoietic Stem Cells through Regulation of Hoxb Cluster

Hematopoietic stem cells (HSCs) sustain lifelong production of multiple blood cell types through a finely-tuned balance between stem cell maintenance and activation to prevent bone marrow exhaustion or overgrowth. The highly conserved Hox family of homeodomain containing transcription factors have been identified as key regulators and contributors in both normal hematopoiesis and leukemogenesis. Most previous work has focused on individual Hox genes; however, it remains largely unknown whether and how multiple Hox genes in a cluster are regulated and function in hematopoiesis. We initiated a study to perform systematic, high-throughput transcriptome analysis in the following 17 cell types from the bone marrow (BM) of C57BL/6J mice: 4 hematopoietic stem and progenitor cells (CD49blo long-term (LT)-HSC, CD49bhi intermediate-term (IT)-HSC, short-term (ST)-HSC, and MPP); and 4 committed progenitors (CLP, CMP, GMP and MEP); and 9 mature lineage cells (B cell, T cell, NK cell, dendritic cell, monocyte, macrophage, granulocyte, megakaryocyte and nucleated erythrocyte). Intriguingly, as part of a unique fingerprint observed in the most primitive CD49blo LT-HSCs, we detected expression from the Hoxb cluster. Further analysis on all the four Hox clusters revealed that most of the genes from the Hoxb cluster, and not from the other Hox clusters, were predominantly expressed in the CD49blo LT-HSCs. This suggests that they might function as a cluster to maintain CD49blo LT-HSCs. A previous study has shown that one cis -regulatory retinoic acid responsive element (RARE), is conserved among vertebrate species and regulates multiple Hoxb gene expression in central nervous system development. Thus, we asked whether RARE is essential for maintenance of primitive CD49blo LT-HSCs by regulation of Hoxb cluster. To test this hypothesis, we utilized a RAREΔ knockout mouse model and assayed for HSC numbers in BM. We observed that homozygous deletion of RARE led to 2-fold reduction in both the frequency and absolute number of CD49blo LT-HSCs. Functionally, we first conducted limiting dilution, competitive repopulating unit (CRU) assays by transplanting 2.5×104, 7.5×104 or 2×105 of BM cells from RAREΔ mutants and their control littermates, together with 2×105 recipient BM cells derived from the Ptprc mutant strain, into lethally irradiated recipient mice. Our data showed a 2.5-fold decrease in functional HSCs in RAREΔ HSCs (1/20,326) compared to control (1/50,839). To further evaluate the long-term effect of RARE on HSCs, we performed serial BM transplantation and observed a 12.9-fold reduction of reconstitution ability after secondary transplantation. These data indicate that deletion of RARE compromised HSC long-term reconstitution capacity. Collectively, our work provides evidence showing that RARE is essential for maintenance of the primitive HSCs by regulation of Hoxb cluster genes. DisclosuresNo relevant conflicts of interest to declare.

NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool

Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated.NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipako/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipako/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipako/ko vs. wt animals (11 vs. 14.5 days).To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipako/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response.To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population.Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool. DisclosuresNo relevant conflicts of interest to declare.

Opposite Effects of M1 and M2 Macrophages on Hematopoietic Stem Cell Self-Renewal and Ex Vivo Expansion

Macrophages (MΦ) are professional phagocytes in the innate immune system. They are not only involved in regulation of various immune functions and inflammation, but also exhibit plasticity in modulation of tissue regeneration and repair after being polarized into M1 and M2 MΦ by different inflammatory cytokines. In addition, several recent studies show that MΦ are a new constituent of the hematopoietic stem cells (HSCs) niche and play a role in regulation of HSCs maintenance and mobilization in bone marrow (BM). However, it is not known whether MΦ can regulate HSCs self-renewal and whether the effects of MΦ on HSCs can be influenced by differential MΦ polarization. This was investigated using an ex vivo HSCs expansion model consisting of mouse bone marrow LSK (Lin-sca-1+c-Kit+) cells cultured with or without MΦ in a mouse HSCs expansion medium (StemSpanTM serum-free medium supplemented with 20ng/ml of stem cell factor [SCF] and thrombopoietin [TPO]). We found that LSK cells were expanded about 20-, 15-, and 30-fold after 6 days of co-culture with MΦ harvested from mouse BM, spleen, and peritoneal cavity, respectively, whereas there was no significant expansion after culture without MΦ or with BM Gr-1high or Gr-1low monocytes. In addition, we found that M1-MΦ polarized by INFγ were more effective than IL4-polarized M2-MΦ in promoting LSK cells expansion ex vivo (45-fold vs. 15-fold). However, the promotion of LSK cells expansion by M1-MΦ resulted in about 88% reduction in HSCs as judged by 5-week cobblestone area forming cell (CAFC) assay. In contrast, M2-MΦ significantly promoted HSCs expansion. A greater expansion of HSCs was achieved after LSK cells were co-cultured with M2-MΦ for 9 days than for 6 days (20-fold vs. 6-fold). These findings suggest that M1-MΦ are more effective than M2-MΦ in promoting LSK cells or hematopoietic progenitor cells (HPCs) expansion, at the expense of HSCs self-renewal, whereas M2-MΦ can promote HPCs expansion as well as HSCs self-renewal. This suggestion is supported by results of serial transplantation and competitive repopulation unit (CRU) assays. CRU assay showed that LT-HSCs (e.g. 4-month CRU) were increased about 13 folds relative to the starting numbers of CRU in the input after LSK cells were co-cultured with M2-MΦ for 9 days, but were barely detectable after the cells were cultured without MΦ or with M1-MΦ. The inhibitory effect of M1-MΦ on HSCs self-renewal and expansion was attenuated by inhibition of inducible nitric oxide synthase (iNOS) activity with an inhibitor or knockout iNOS. Inhibition of arginase and/or cyclooxygenase activities with an inhibitor attenuated the promotion of HSCs self-renewal and expansion by M2-MΦ. More importantly, we found that human CD34+ cells, 8-week CAFC, and SCID mice repopulating cells (SRCs) were increased 42±14, 8±2.1, and 4 folds over the input values, respectively, after human cord blood CD34+ cells were co-cultured with M2-MΦ generated from human cord blood CD34- cells for 7 days in a human HSCs expansion medium (StemSpanTM serum-free medium supplemented with 50 ng/ml of SCF, TPO, and FLT-3 ligand). These findings demonstrate that M1-MΦ and M2-MΦ have opposite effects on HSCs self-renewal, which may be important for regulation of hematopoiesis under various pathological conditions in which MΦ are differentially polarized to M1 or M2 by diverse inflammatory cytokines. In addition, M2-MΦ may be used to promote human cord blood HSCs ex vivo expansion to make human cord blood transplantation available to more patients. DisclosuresNo relevant conflicts of interest to declare.

Mitigation of a Newly Discovered Phenomenon, Extra Physiologic Oxygen Shock/Stress (EPHOSS), Mediated By the Mitochondria Permeability Transition Pore, Greatly Improves Stem Cell Collection and Transplantation

Hematopoietic stem cells (HSCs) reside in hypoxic niches within the bone marrow (BM). Yet, all HSC studies have been performed to date with cells immediately isolated in non-physiologic ambient air, whether or not they are subsequently processed in low oxygen tension. By collecting/manipulating BM in physiologically native conditions of hypoxia where all procedures are performed inside a hypoxic chamber, we demonstrate that brief exposure of mouse BM or human cord blood (CB) to ambient oxygen decreases recovery of phenotypically-defined and functional self-renewing long-term repopulating HSC and concomitantly increases numbers of progenitor cells, a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS). This new phenomenon is exquisitely sensitive to oxygen and great care must be taken to ensure all reagents, solutions, plastics, and anything that will come into contact with the cell suspension, is extensively pre-equilibrated in hypoxia. Up to 5-fold greater numbers of long-term (LT)-HSCs (CD34-CD150+Lin-Sca1+c-kit+CD41-CD48- or CD34-CD135-Lin-Sca1+c-kit+) could be recovered from mouse BM harvested in 3% O2 compared to BM harvested in air, or even BM harvested in 3% O2 and then exposed to air for as little as 30 min before analysis, even if subsequently returned to hypoxia. There was a concomitant decrease in short term-HSCs and multipotent progenitors when BM was harvested in hypoxia, an effect associated with decreased functional cytokine-stimulated colony formation of hematopoietic progenitor cells (HPC: CFU-GM, BFU-E, and CFU-GEMM). Similarly, if human CB was harvested under similar low oxygen conditions, a 3-fold increase in recovered HSCs (Lin-CD34+CD38-CD45RA+CD90+CD49f+) could be achieved compared to CB harvested in air. Using a custom mouse respirator to conduct competitive repopulating transplant experiments completely in a 3% O2 environment revealed an increase in competitive repopulating units (CRUs) up to more than 42 fold was recovered when BM is harvested in hypoxic conditions compared to air harvested BM in primary recipients, thus demonstrating the beneficial effects of hypoxic harvest on functional/transplantable HSCs with secondary transplant capability. These data strongly support the surprising conclusion that, until now, the true numbers of HSCs and the transplantation potency of BM and CB has been routinely and consistently underestimated because of rapid initiation of differentiation of LT-HSCs in ambient air. We present evidence linking mitochondrial function and cyclophilin D to EPHOSS. Genetically or pharmacologically suppressing cyclophilin D function, or p53 gene deletion link production of reactive oxygen species (ROS) to induction of the mitochondrial permeability transition pore (MPTP) as a molecular mechanism of EPHOSS, where rapid ROS generation in HSCs after exposure to “hyperoxic” room air initiates an irreversible cascade of differentiation signals (see illustration). We present additional evidence from gene knock-out model studies implicating roles for miR210 and Hif-1a in EPHOSS. The MPTP inhibitor, cyclosporine A, protects phenotypically-defined as well as functional and transplantable HSCs from EPHOSS during collection in air resulting in at least a 3-fold increase in HSC recovery as well as increased transplantation potency. Thus, pharmacological mitigation of EPHOSS during HSC collections for use in patient transplantation procedures may be clinically advantageous. Because cyclosporine A is already in use clinically, this EPHOSS-reducing strategy may be readily and easily tested for efficacy in a hospital setting. Because many different adult stem cells exist naturally in hypoxic niches, EPHOSS is likely relevant to other stem cells routinely harvested in air. Evidence suggests that aged HSCs may be more sensitive to the deleterious effects of EPHOSS than young HSCs. We propose that metabolic profiling of stem cells, including cancer stem cells, may not accurately represent the metabolism, behavior, and responses of these cells as they exist in their native hypoxic environments because they are harvested and studied in air. Thus, experimental designs that include a consideration of EPHOSS effects may be required to obtain a more complete understanding of stem cell metabolism and biology especially as it relates to stem cell aging or responses of cancer stem cells to chemotherapy. [Display omitted] DisclosuresBroxmeyer:CordUse: Membership on an entity’s Board of Directors or advisory committees.

Low-dose insulin-like growth factor binding proteins 1 and 2 and angiopoietin-like protein 3 coordinately stimulate ex vivo expansion of human umbilical cord blood hematopoietic stem cells as assayed in NOD/SCID gamma null mice

IntroductionInsulin-like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin-like proteins (ANGPTLs) can enhance the ex vivo expansion of hematopoietic stem cells (HSCs) when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3 ligand (FL). In order to determine the optimal dose and combination of IGFs, IGFBPs and ANGPTLs, serial dilution and full permutation of IGFBP1, IGFBP2, IGF2 and ANGPTL3 were applied on a cryopreserved umbilical cord blood mononuclear cell (UCB-MNC) ex vivo expansion system.MethodsIn this system, 4 × 105 cells/ml of UCB-MNCs were inoculated in serum-free Stemspan® medium (Stemcell technologies, vancouver, BC, Canada) supplied with standard basal cytokine combination of 100 ng/ml SCF, 50 ng/ml FL and 100 ng/ml TPO and supported by a bone marrow mesenchymal stromal cell layer.ResultsParadoxically, experiment results showed that the highest expansion of CD34+CD38−CD90+ primitive progenitor was stimulated by cytokine combination of SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3 at a low dose of 15 ng/ml IGFBP1 and 20 ng/ml IGFBP2 and ANGPTL3. This ex vivo expansion was further validated in 8-week-old to 10-week-old nonobese diabetic/severe combined immunodeficiency interleukin 2 gamma chain null (NOD/SCID-IL2Rγ−/−) mice. Limiting dilution assay showed excellent correlation between the HSC ex vivo surface marker of CD34+CD38−CD90+ and the in vivo competitive repopulating unit (CRU) functional assay.ConclusionIGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34+CD38−CD90+ primitive progenitor at low dose. The optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. The CD34+CD38−CD90+ phenotype can serve as a surrogate ex vivo surface marker for HSCs due to consistency with the in vivo CRU functional assay.

Hepatic Leukemia Factor Is An Important Regulator Of Hematopoietic Stem Cell Activity and Identity

The transcription factor Hepatic Leukemia Factor (HLF) was originally identified in a chromosomal translocation with the gene E2A causing a subset of childhood B-lineage acute lymphoid leukemia. Moreover, HLF has been described as a regulator of circadian rhythm and recent findings have implicated HLF as a candidate “stemness” gene in both normal and malignant stem cells. Accordingly, overexpression of HLF in human hematopoietic stem cells (HSC) results in an enhanced reconstitution capability in NOD-SCID mice. However, little is known about HLF's physiological role in hematopoiesis and HSC regulation. Using quantitative PCR, we found that HLF is highly expressed in mouse (C57Bl/6) HSC and is downregulated upon differentiation (HSC 3.2 (±0.95) fold (p<0.001), LSK 1.9 (±0.47) fold (p<0.05), CMP, GMP MEP all less then 0.1 fold, all values are compared to HPRT). This encouraged us to further investigate HSC function in the absence of HLF.The conventional HLF knockout (KO) mice (C57bl/6 background) were viable, born at normal Mendelian ratios and showed normal hematopoietic parameters (bone marrow cellularity: WT 2.7x107 (±5.4 x106), KO 3.3x107 (±6.4 x106), p>0.2 n=9). In addition, the HLF KO mice demonstrated normal lineage distribution of both mature cells in the peripheral blood and bone marrow as well as the frequency of immunophenotypic HSC (Lin-Sca1+ckit+CD34-Flt3-: WT 0.0005 (±0.5x10-4)%, KO 0.0005 (±0.1x10-3)%; n>10). However, in a serial competitive transplantation assay using whole bone marrow (200 000 cells 1:1 ratio), HLF KO cells demonstrated a significant reduction in reconstitution capacity in primary recipients (WT 56 (±15)%, KO 40.2 (±16)%, p=0.028, n>10), which was further increased in the secondary recipients (WT 87.2 (±26)%, KO 8.7 (±5.8)%, p<0.001, n>10). Almost no engraftment was detected from the HLF KO cells in tertiary recipients. To further evaluate stem cell activity in the absence of HLF, we next enumerated the number of competitive repopulating units (CRU) by limiting dilution assay, which revealed a 2.6 fold reduction, of CRU in the HLF KO mice compared to WT controls (WT 1.6 (±0.4)/105 bone marrow cells, KO 0.6 (±0.2)/105 bone marrow cells). Similarly, transplantation of sorted HSC (Lin-Sca1+ckit+CD34-Flt3-) also showed a 2.4 fold (WT 47.3 (±24)%, KO 19.4 (±25)%, p=0.16, n=9) reduced engraftment of total cells but with enhanced T cell frequency in peripheral blood (WT 19.5 (±6.2)%, KO 40.8 (±7.4)%, p=0.01, n=9).Since we also found that HLF was highly expressed in fetal liver derived HSC, we transplanted fetal liver HLF KO cells from E14.5 in a competitive repopulation setting. In line with the phenotype seen in the adult HLF KO mice, the fetal liver HLF KO cells demonstrated impaired reconstitution ability (WT 52.8 (±16)%, KO 0.9 (±1.4)%, n>10). Intriguingly, the phenotype was stronger than in the adult HLF KO HSC, indicating that HLF is particularly important during the expansion phase of HSC in embryonic development.The underlying mechanism of the reduced HSC activity is still unclear, but preliminary findings show that HLF KO HSC have enhanced ROS levels (WT 337 (±33), KO 510 (±55), p<0.05, n=3) and increased cycling HSC (G0: WT 66.5 (±6.4)%, KO 58.5 (±4.7)%; G1/S/G2/M: WT 33.6 (±6.6)%, KO 41.7 (±4.9)%, n=3). We are currently performing global gene expression analysis to further understand the mechanism of HLF in HSC regulation.Interestingly, we also found that HLF appears to regulate the identity of HSC by modulating the expression of the SLAM code on the cell surface of the HLF KO HSC. In contrast to the normal frequency of LSK Flt3-CD34- cells, the HLF KO mice displayed a 3.5 fold reduction in the frequency of LSK CD150+CD48- cells (WT 1.94x10-4 (±4.4x10-5)%, KO 0.56x10-4 (±1.5x10-5)%, p<0.001 n>10). Strikingly, transplantation of as many as 150 LSK CD150+CD48-HLF KO cells showed a complete lack of repopulating capacity in vivo. This did not correlate to the number of functional HSC seen when transplanting whole bone marrow and indicates that HLF affects the identity of HSC by modulating the expression of the SLAM markers.Taken together, we show here for the first time that HLF has a fundamental role in HSC biology during both fetal and adult hematopoiesis by regulating HSC activity and identity. Disclosures:No relevant conflicts of interest to declare.

Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice.Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals.Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state.Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures:No relevant conflicts of interest to declare.

Runx1 Induced Chromatin Folding of the PU.1 Gene Locus Is Necessary for Adult Long-Term Hematopoietic Stem Cell Maintenance

Abstract 2364 Background:PU.1 is a hematopoietic transcription factor that is essential for normal hematopoiesis. PU.1 gene expression is mainly regulated through the proximal promoter (PrPr), and an upstream regulatory element (URE) located –14kb or –17kb upstream of the transcription start site in mice and humans, respectively. We previously demonstrated that PU.1 is a downstream target of core binding factor Runx1 (also called AML1) for megakaryocytic, lymphoid, and myeloid differentiation. Recent reports identified Runx1 to restrict proliferation of hematopoietic stem cells (HSCs) and to prevent HSC exhaustion. Approach:By employing conditional (Mx1-Cre) Runx1 knockout mice (Runx1Δ/Δ) and knock-in mice with targeted disruption of all three Runx1 binding sites at the −14kb URE of PU.1 (mRunxki/ki) we aimed to genetically dissect a potential functional role and mechanisms by which Runx1 might regulate PU.1 expression in HSCs. Results:Both, Runx1Δ/Δ and mRunxki/ki mice exhibited decreased PU.1 levels of approximately 65% in purified phenotypic HSCs (CD150+CD48-LSKs). In addition, activating histone marks H3K4me3 and H3Ac were significantly decreased at the −14kb enhancer and the PrPr. Mechanistically, we found by quantitative chromosome conformation capturing (3C) that Runx1 binding established a physical interaction of the −14kb enhancer with the PrPr thereby forming an active chromosomal conformation for proper PU.1 transcription. In concordance to Runx1Δ/Δ, phenotypic HSCs of mRunxki/ki mice were significantly increased in number. However, limiting dilution transplantations using the congenic Ly5.1/Ly5.2 system revealed a 25-fold decrease of competitive repopulating units (CRU) in phenotypic HSCs of mRunx-ki/ki mice compared to controls. Long-term reconstitution in serial transplantation assays was also severely impaired indicating HSCs exhaustion in mRunxki/ki mice. HSC homing and engraftment was not affected. Importantly, restoration of PU.1 levels by crossing to a human PU.1 transgenic fully rescued long-term HSC functions, thus demonstrating that HSC maintenance was strictly related to PU.1 levels. Conclusion:Runx1 binding to the −14kb enhancer of PU.1 is necessary to maintain an active chromosomal conformation for proper PU.1 transcription in order to prevent HSC exhaustion. Disclosures:No relevant conflicts of interest to declare.

P190-B RhoGAP Is Critical for Hematopoietic Stem Cell Niche Regulation

Abstract 221Hematopoiesis is regulated by components of the stromal microenvironment, so-called niche. Although the concept of hematopoietic stem and progenitor cell (HSC/P) niche is well known, its molecular regulation remains ill-defined. Here, we provide evidence that p190-B GTPase Activating Protein (p190-B), a negative regulator of Rho activity, is a regulator of mesenchymal/stromal cell functions necessary for normal hematopoiesis during fetal development. Mice lacking p190-B die before birth. At day 14.5 post coitum, p190-B−/− embryos are paler than WT embryos with a 40% lower hematocrit. Cellularity and numbers of burst forming-unit (BFU) erythroid (E), colony forming-unit (CFU)-granulo-monocytic and CFU-erythroid per p190-B−/− fetal livers (FL) were 50% lower than WT. In addition, the frequency of LongTerm-HSC (LinnegScaposKitposCD150posCD48neg), ShortTerm-HSC (LSK-CD150posCD48pos), and progenitors (LK) appeared significantly reduced in the bone marrow of e17.5/18 embryos. These data suggest a defect in fetal hematopoiesis. However, p190-B−/− FL cells were able to fully reconstitute hematopoiesis of adult irradiated recipients (Xu, Blood 2009). Mice reconstituted with p190-B−/− FL cells exhibited bone marrow content, white blood count, red blood count and hematocrit similar to those reconstituted with WT cells. Furthermore, hematopoietic recovery of p190-B−/− reconstituted animals following 5-Fluorouracil or phenylhydrazine-induced stress was comparable to that of WT. Therefore, FL HSC/P retain hematopoietic potential, which suggests a non-cell autonomous defect in p190-B−/− embryos. In the present study, we examined in more detail this possibility. The numbers of CFU-fibroblast (CFU-F) were 2-fold lower in p190-B−/− FL compared to WT FL. We next examined the hematopoietic supportive capacity of p190-B−/− FL microenvironment using stromal cell cultures derived from e14.5 FL. No noticeable differences were observed during the establishment of the stromal cell cultures. p190-B−/− stromal cells exhibited a spindle-like shape and expressed CD90, CD44 and Sca-1 but not CD45, CD31 or CD11b similarly to WT stromal cells. Cobblestone Area Forming Cell assays (CAFC) was performed in co-culture between various number of WT BM cells and p190-B−/− and WT stromal cells. We examined 5 independent stromal cell cultures from both genotypes. Frequency of CAFC was 75% lower on p190-B−/− stroma than WT stroma at both 1 week (1 in 5 × 10 3 versus 1 in 21 × 10 3, n = 5 p < 0.01) and 2 weeks (1 in 21 × 10 3 versus 1 in 87 × 10 3, n = 4 p < 0.01) in culture. CFU with cells recovered from one week coculture was also performed. Cocultures with p190-B−/− stroma gave rise to 2-fold less CFU than with WT stroma (538+90 versus 255+81, p < 0.01, one representative experiment of 4). Using competitive repopulation assay, we then assessed the in vivo repopulation ability of CD45.2+ hematopoietic cells co-cultured for one week on each stroma. Fifteen weeks following transplantation, the frequency of CD45.2+ cells in the peripheral blood of mice that received cells cocultured on p190-B−/− stroma was dramatically reduced compared to mice that received cells cocultured on WT stroma (0.43% + 0.37 versus 32.4% + 7.5, n = 6, p < 0.01), equivalent to a 100-fold difference in calculated competitive repopulation unit. Therefore, p190-B−/− stroma exhibited defective hematopoietic supportive activity. Interestingly, p190-B−/− stromal cells, like WT, showed characteristic of mesenchymal stem cells (MSC), i.e. ability to give rise to CFU-F and to differentiate to adipocytes and osteoblasts. They express relatively high level of nestin and osteopontin. At a mechanistic level, gene expression analysis of molecules known to play a critical role in HSC maintenance in the niche indicated 200-fold downregulation of Kit-ligand and Wnt3a in p190-B−/− stroma compared to WT stroma (p<0.05). Surprisingly, BMP4, angiopoietin and CXCL12 were upregulated in p190-B−/− stroma, 35-fold, 14-fold and 16-fold, respectively (p<0.05). Hence, p190-B expression in MSC/stromal cell microenvironment fine-tunes niche regulatory pathways to maintain normal hematopoiesis. Therefore, p190-B may be a critical regulator of the mesenchymal stem/hematopoietic stem and progenitor niche. Disclosures:No relevant conflicts of interest to declare.

Bmi1 Confers Stress Resistance to Self-Renewing Hematopoietic Stem Cells.

AbstractAbstract 1607The polycomb group (PcG) proteins form chromatin-modifying complexes that implement transcriptional silencing. There are two major PcG complexes, polycomb repressive complex (PRC) 1 and PRC2. PRC1 ubiquitylates histone H2A at lysine 119 and PRC2 trimethylates lysine 27 of histone H3. Among PcG proteins, Bmi1, a core component of PRC1, plays an essential role in the self-renewal and maintenance of various kinds of stem cells including hematopoietic stem cells (HSCs), neural stem cells, and leukemic stem cells. We previously reported that forced expression of Bmi1 using a Bmi1 retrovirus promotes symmetrical cell division of HSCs, resulting in a marked ex vivo expansion of multipotent progenitors and an enhancement of repopulating capacity of HSCs in vivo. However, the impact of overexpression of Bmi1 in HSCs remains to be precisely addressed. To this end, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the Rosa promoter in a tissue-specific fashion by the Cre-LoxP system. We crossed the mice to Tie2-Cre mice (Tie2-Cre; Rosa-Bmi1fl/+) and induced overexpression of Bmi1 in hematopoietic cells in vivo. Real-time PCR analysis demonstrated that expression of Bmi1 inpurified bone marrow (BM) c-Kit+Sca-1+Lineage-marker- (KSL) cells was 6-fold higher in Tie2-Cre; Rosa-Bmi1fl/+ mice than control Tie2-Cre mice mice. Overexpression of Bmi1 did not significantly affect steady state hematopoiesis. The number of HSCs and progenitors (multipotent progenitors, CMPs, GMP, MEPs, and CLPs) and lineage composition (myeloid cells, B cells, and T cells) in BM of Tie2-Cre; Rosa-Bmi1fl/+ mice was not significantly changed compared to those in control mice. We then performed serial transplantation assay. The repopulating capacity of Tie2-Cre; Rosa-Bmi1fl/+ BM cells was comparable to those of the control cells in primary recipients. However, Tie2-Cre; Rosa-Bmi1fl/+ cells retained higher repopulating capacity during serial transplantation compared to those of the control cells. Moreover, ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs for 10 days contained approximately 2-fold more high proliferative potential colony-forming cells (HPP-CFCs; colony diameter>1mm) and colony-forming unit-neutrophil/macrophage/erythroblast/megakaryocyte (CFU-nmEM) which retain multi-lineage differentiation potential along myeloid lineage than the control. Of note, cells in ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs exhibited significantly augmented repopulating capacity in recipient mice and we are now engaged in competitive repopulation unit (CRU) assays to determine the net HSC expansion by overexpression of Bmi1 during ex vivo culture. These results indicate that overexpression of Bmi1 confers stress resistance to HSCs during ex vivo culture and serial transplantation. Our findings also provide Bmi1 as a potential target for efficient manipulation of HSCs ex vivo. Disclosures:No relevant conflicts of interest to declare.

Intramarrow injection of β-catenin-activated, but not naïve mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naïve MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with beta-catenin-activated MSCs. These results reveal that naive MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/beta-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.

Notch2 Signaling Inhibits Differentiation and Promotes Self Renewal of Hematopoietic Stem and Progenitor Cells During Marrow Regeneration.

Abstract 2544Poster Board II-521Notch regulates numerous lineage choices during vertebrate development, and although ex vivo studies suggest that Notch regulates hematopoietic stem cell (HSC) and multipotential progenitor (MPP) differentiation, a functional role for Notch in HSC/MPP self renewal in vivo remains controversial. We previously reported a Notch2 signaling role during bone marrow (BM) recovery following injection with chemotherapeutic agent 5-fluorouracil (5FU), where Notch2 signaling impedes myeloid differentiation, allowing for generation of sufficient numbers of progenitor cells. Herein, we examine a Notch2 signaling role in HSC as well as progenitor cell self renewal by enumerating generation of HSC and short term repopulating cells in lethally irradiated recipients (Ly5.1+) transplanted with a limiting number (5 × 105) of BM cells from either control mice or from mice bearing Cre-LoxP-inducible Notch2 deletions (Ly5.2+). In recipient mice transplanted with control BM, recovery was evident from Day11 to Day13 post transplant when significantly more than the initial post-irradiation number of 9.0 × 106 BM cells was seen in the recovering marrow. In recovering mice, recipients receiving control cells generated more BM cells than did recipients receiving Notch2-deficient cells. Furthermore, mice receiving control cells generated significantly more donor Sca-1+c-kit+ (SK+) cells than recipients receiving Notch2-deficient BM cells [44.4×103 (s.e.m.+/− 14×103) vs 8.2×103 (s.e.m.+/−1.5×103), respectively, p=0.001]. To quantitate the generation of short term repopulating cells, secondary radioprotection assays were performed. Irradiated secondary recipient mice received 1×106 BM cells from the primary recipients previously transplanted with either control cells or Notch2-deficient cells. Secondary recipients receiving cells from primary control transplants survived significantly longer than those receiving cells from primary Notch2-deficient transplants or than irradiated mice receiving no cells (n=4, p=0.01), indicating Notch2 is required to generate sufficient numbers of cells to provide radioprotection. To quantitate long term HSC generated in the recovering marrow, competitive repopulating units (CRU) were enumerated by performing secondary transplants in which 4-doses of BM cells ranging from 4 × 104 to 5 × 106 cells from primary transplants were injected into secondary recipients along with 1 × 105 Ly5.1+ competing cells. Enumeration of CRU at 2 weeks post transplant confirmed the number of short term repopulating cells was significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(1.3 CRU vs 8.8 CRU / 1×106 BM cells, respectively), p=0.0004)]. Enumeration of CRU at 9 weeks post transplant indicated HSC numbers were also significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(0.1 CRU vs 0.7 CRU / 1×106 BM cells, respectively), p=0.02]. Taken together, our results demonstrate a role for Notch2 in enhancing generation of long term HSC as well as short term repopulating cells and suggests that Notch2 signaling regulates a hierarchy of events to assure the initial repopulation by HSC and MPP, while delaying myeloid differentiation during hematopoietic regeneration. Disclosures:No relevant conflicts of interest to declare.

Tie2+ Bone Marrow Endothelial Cells Regulate Hematopoietic Reconstitution In Vivo.

Abstract 248Radiation and chemotherapy cause myelosuppression in patients via damage to bone marrow (BM) hematopoietic stem cells (HSCs) and progenitor cells. It has been shown that BM HSCs reside in close association with osteoblasts and that BM osteoblasts regulate the maintenance of quiescent HSCs in vivo. HSCs also reside in proximity to BM sinusoidal vessels but the function of BM endothelial cells (BM ECs) in regulating HSC fate in vivo remains less well understood. We hypothesized that BM ECs play a critical role in regulating BM hematopoietic reconstitution following stress. In order to test this hypothesis, we utilized Cre-LoxP recombination to generate mice bearing targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ BM ECs (Tie2Cre;Bak−/− ;BaxFl/− mice) and measured their hematopoietic response to total body irradiation (TBI). Tie2Cre;Bak−/−;BaxFl/− mice (EC-Bak;Bax knockouts) were compared with Tie2Cre;Bak−/−;BaxFl/+ mice (EC-Bak;Bax (+) mice) which have constitutive Bak deletion but retain Bax in Tie2+ BM ECs. EC-Bak;Bax knockout mice had no detectable Bak or Bax by Western Blot in vascular tissues. After exposure to 100 cGy total body irradiation (TBI), EC-Bak;Bax knockout mice displayed a 2-fold increase in total viable BM cells (p=0.04), a 3-fold increase in BM ckit+sca+lineage- (KSL) progenitor cells, a 3-fold increase in colony-forming unit-spleen day 12 (CFU-S12) content (p=0.0003) and a 2-fold increase in 4 week competitive repopulating units (CRUs) compared to EC-Bak;Bax (+) mice. After 300 cGy TBI, comparable radioprotection was observed in the EC-Bak;Bax knockout mice, with a 1.9-fold increase in total BM cells, a 2.4-fold increase in CFU-S (p=0.01) and a 3.6-fold increase in 4 week CRU compared to EC-Bak;Bax (+) mice. Taken together, these results suggested that targeted protection of Tie2+ BM ECs from the intrinsic pathway of apoptosis mediated protection of BM hematopoietic stem/progenitor cells following TBI. Since Tie2 is expressed by BM ECs and a small subset of quiescent BM HSCs, we carried out experiments to determine whether the radioprotection we observed in EC-Bak;Bax knockout mice was caused autonomously by protection of Tie2+ BM ECs or Tie2+ HSCs. We transplanted 4 × 106 BM cells from EC-Bak;Bax knockout mice into lethally irradiated (950 cGy) wild type (WT) B6.SJL mice such that, after 12 weeks post-transplant, the recipient mice were chimeric for Bak and Bax deletions only in hematopoietic cells while retaining a wild type BM microenvironment (HSC-Bak;Bax knockout;EC-wild type), verified by qRTPCR. At 16 weeks post-transplant, the chimeric recipient mice were then exposed to 100 and 300 cGy TBI and we compared the hematopoietic response of these mice to that of EC-Bak;Bax knockout mice. Interestingly, after 100 cGy TBI, the hematopoietic response of the chimeric mice was comparable to that of wild type C57Bl6 mice and revealed a significant reduction in total viable BM cells (1.3-fold), BM KSL cells (7.4 fold) and BM CRU-4 weeks (9-fold) compared to 100 cGy-irradiated EC-Bak;Bax knockout mice. Significant reductions in total BM cells and BM CRU content were also observed in chimeric mice compared to EC-Bak;Bax knockout mice following 300 cGy TBI. These results suggest that deletion of Bak and Bax-mediated apoptosis in Tie2+ BM ECs protects BM hematopoietic stem and progenitor cells from ionizing radiation damage and this protection is largely autonomous to Tie2+ BM ECs. More generally, these data demonstrate that protection of BM ECs from the deleterious effects of ionizing radiation results in augmented hematopoietic reconstitution following TBI. This study demonstrates that Tie2+ BM ECs have an essential role in regulating hematopoietic regeneration following injury and reveals that BM ECs are a novel and attractive therapeutic target to augment hematopoietic reconstitution in vivo. Disclosures:No relevant conflicts of interest to declare.

Increasing C/Ebpα Expression Regulates the Transition of Hematopoietic Stem Cells During Development.

Abstract 1506Poster Board I-529During ontogeny, hematopoietic stem cells (HSCs) undergo a change from rapid dividing cells with high reconstitution ability to mainly quiescent cells with lower repopulation capacity. However, little is known about how this switch is regulated. Here we report that levels of C/EBPα, a transcription factor that is frequently disrupted in human acute myeloid leukemia, regulate the proliferation and self-renewal transition of HSCs during development. Loss of C/EBPα in adult mice resulted in a profound expansion of phenotypic HSCs and elevated proliferation rates. Limiting dilution transplantation to measure the frequency of competitive repopulation units (CRU) revealed an increase in the number of functional HSC in C/EBPα-/- mice. Serial transplantation of C/EBPα-/- bone marrow showed advanced reconstitution ability, indicating enhanced self-renewal ability. Interestingly, levels of C/EBPα in HSCs were significantly up-regulated 3 weeks after birth during which HSCs change from an actively cycling state to quiescence in bone marrow. When we conditionally inactivated C/EBPα in mice of different age, we observed a tight correlation between the age-specific levels of C/EBPα expression and the expansion of HSCs. Gene profiling analysis of C/EBPα-/- adult HSCs showed the up-regulation of oncogenes c-myc and n-myc, whose expression can regulate pluripotency and self-renewal of stem cells, as shown by recent induced pluripotent stem cell studies. Knocking down n-myc and c-myc expression in C/EBPα-/- HSCs using shRNA, we observed reduced proliferation and decreased colony formation in serial replating assay, which assesses the preservation of “self-renewal” in the progenitor cell compartment. Consistently, we observed down-regulation of n-myc in HSCs during the transition time, which is reciprocal to C/EBPα expression. Together, our data indicate C/EBPα as a key regulator of HSC self-renewal and proliferation during development, whose levels of expression might control the fetal to adult switch of HSC properties through regulating myc genes. DisclosuresNo relevant conflicts of interest to declare.

Autoregulation of the Transcription Factor PU.1 Via Its Evolutionarily Conserved Upstream Regulatory Element Is Critical in Adult Mouse Hematopoiesis.

Abstract 1468Poster Board I-491 Background:Levels of the Ets transcription factor PU.1 control normal hematopoietic differentiation and even modest alterations can lead to leukemia and lymphoma. Regulation of PU.1 levels at different stages of hematopoiesis requires multiple interactions between several regulatory elements and transcription factors. Our previous studies identified a potential autoregulatory mechanism of the PU.1 gene through the combined activity of the proximal promoter and an evolutionarily conserved upstream regulatory element (URE), located at –14 kb relative to the transcription start site in mice. PU.1 binds to a conserved PU.1 site in the PU.1 URE both in vitro and in vivo. Approach:To ask at which stages of hematopoietic differentiation autoregulation of PU.1 via binding to its URE might play a role, we developed a mouse model with targeted disruption of the PU.1 binding site in the PU.1 URE. Results:Targeted mutation of the PU.1 autoregulatory site in PU.1 URE abolished PU.1 binding as verified by Chromatin Immuno-precipitation (ChIP). PU.1 URE activity was manifestly reduced resulting in a variety of lineage-specific abnormalities. As shown here in adult mice, the absence of the autoregulatory PU.1 site affected PU.1 expression in a lineage dependent manner. PU.1 expression was markedly decreased in phenotypic long term hematopoietic stem cells (LT-HSC: CD150+/CD48−/ c-kit+/sca-1+/lin−) and short term HSCs (ST-HSCs: CD150−/CD48+/ c-kit+/sca-1+/lin−) and, to a lesser extent, in Common Myeloid Progenitors (CMPs: lin−/c-kit+/Sca-1−/CD34+/FcrRlow), and Megakaryocyte/Erythrocyte Progenitors (MEPs: lin−/c-kit+/Sca-1−/CD34−/FcrRhigh). Within the lymphoid linage, PU.1 levels were unchanged in Common Lymphoid Progenitors (CLPs: lin−/c-kitlow/Sca-1low /IL-7Ra+/Thy1.1−) and pre-B-cells (B220+/ CD43−), up in pro-B-cells (B220+/CD43+), and down in mature B cells. Myeloid cells appeared to be unaffected. Interestingly, while PU.1 levels were decreased in LT- and ST-HSC populations, only phenotypic LT-HSCs were reduced in number. To further analyze HSC function of PU.1 site mutated mice we performed limiting dilution transplantation assays and measured the frequency of competitive repopulation units (CRU) using the congenic Ly5.1/Ly5.2 system. Our preliminary data indicated a decrease of LT-HSC function in PU.1 site mutated mice, although their homing and engraftment functions were not affected. This was also observed in mice with targeted disruption of all three AML-1 sites that are in close proximity of the PU.1 site at the PU.1 URE. While AML-1 itself appeared not to influence LT-HSC function (M. Ichikawa, T. Asai et al. Nature Medicine, 2004), we found that the conformational changes of the URE present in mice with disrupted AML-1 binding sites, as measured by Quantitative Chromosome Conformation Capture, impede PU.1 binding to its autoregulatory site. Conclusion:PU.1 indeed autoregulates its expression via binding to the -14kb URE in a lineage specific manner in vivo. Our data point to a critical role of PU.1 autoregulation especially for long-term hematopoietic stem cell function. Disclosures:No relevant conflicts of interest to declare.

Systemic Administration of Pleiotrophin Induces Hematopoietic Stem Cell Regeneration In Vivo.

Abstract 1498Poster Board I-521Significant progress has been made toward delineating the intrinsic and extrinsic signaling pathways that regulate hematopoietic stem cell (HSC) self-renewal. However, much less is known regarding the process of HSC regeneration or the extrinsic signals that regulate hematopoietic reconstitution following stress or injury. Elucidation of the microenvironmental signals which promote HSC regeneration in vivo would have important implications for the treatment of patients undergoing radiation therapy, chemotherapy and stem cell transplantation. We recently reported that pleiotrophin, a soluble heparin-binding growth factor, induced a 10-fold expansion of murine long-term repopulating HSCs in short term culture (Himburg et al. Blood (ASH Annual Meeting Abstracts), Nov 2008; 112: 78). Based on this observation, we hypothesized that PTN might also be a regenerative growth factor for HSCs. Here we tested the effect of systemic administration of PTN to non-irradiated and irradiated C57Bl6 mice to determine if PTN could promote HSC regeneration in vivo. C57Bl6 mice were irradiated with 700 cGy total body irradiation (TBI) followed by intraperitoneal administration of 2 μg PTN or saline x 7 days, followed by analysis of BM stem and progenitor cell content. Saline-treated mice demonstrated significant reductions in total BM cells, BM c-kit+sca-1+lin- (KSL) cells, colony forming cells (CFCs) and long term culture-initiating cells (LTC-ICs) compared to non-irradiated control mice. In contrast, PTN-treated mice demonstrated a 2.3-fold increase in total BM cells (p=0.03), a 5.6-fold increase in BM KSL stem/progenitor cells (p=0.04), a 2.9-fold increase in BM CFCs (p=0.004) and an 11-fold increase in LTC-ICs (p=0.03) compared to saline-treated mice. Moreover, competitive repopulating transplantation assays demonstrated that BM from PTN-treated, irradiated mice contained 5-fold increased competitive repopulating units (CRUs) compared to saline-treated, irradiated mice (p=0.04). Taken together, these data demonstrate that the administration of PTN induces BM HSC and progenitor cell regeneration in vivo following injury. Comparable increases in total BM cells, BM KSL cells and BM CFCs were also observed in PTN-treated mice compared to saline-treated controls following 300 cGy TBI, demonstrating that PTN is a potent growth factor for hematopoietic stem/progenitor cells in vivo at less than ablative doses of TBI. In order to determine whether PTN acted directly on BM HSCs to induce their proliferation and expansion in vivo, we exposed mice to BrDU in their drinking water x 7 days and compared the response to saline treatment versus PTN treatment. PTN-treated mice demonstrated a significant increase in BrDU+ BM KSL cells compared to saline-treated controls (p=0.04) and cell cycle analysis confirmed a significant increase in BM KSL cells in S phase in the PTN-treatment group compared to saline-treated controls (p=0.04). These data indicate that PTN serves as a soluble growth factor for BM HSCs and induces their proliferation and expansion in vivo while preserving their repopulating capacity. These results suggest that PTN has therapeutic potential as a novel growth factor to accelerate hematopoietic reconstitution in patients undergoing myelosuppressive radiotherapy or chemotherapy. Disclosures:No relevant conflicts of interest to declare.