Murine Bone Marrow Niche Research Articles

15 Burn Injury Reveals Myeloid Priming at the Progenitor Level in the Murine Bone Marrow Niche

15 Burn Injury Reveals Myeloid Priming at the Progenitor Level in the Murine Bone Marrow Niche

Biomimetic Prussian blue nanozymes with enhanced bone marrow-targeting for treatment of radiation-induced hematopoietic injury

There is an urgent medical need to develop effective therapies that can ameliorate damage to the radiation-exposed hematopoietic system. Nanozymes with robust antioxidant properties have a therapeutic potential for mitigating radiation-induced hematopoietic injury. However, enhancing nanozyme recruitment to injured tissues in vivo while maintaining their catalytic activity remains a great challenge. Herein, we present the design and preparation of a biomimetic nanoparticle, a mesenchymal stem cell membrane camouflaged Prussian blue nanozyme (PB@MSCM), which exhibits biocompatible surface properties and demonstrates enhanced injury site-targeting towards the irradiated murine bone marrow niche. Notably, the constructed PB@MSCM possessed redox enzyme-mimic catalytic activity and could scavenge overproduced reactive oxygen species in the irradiated bone marrow cells, both in vitro and ex vivo. More importantly, the administration of PB@MSCM significantly mitigated hematopoietic cell apoptosis and accelerated the regeneration of hematopoietic stem and progenitor cells. Our findings provide a new targeted strategy to improve nanozyme therapy in vivo and mitigate radiation-induced hematopoietic injury.

Key Subsets of Immune Cells in Bone Marrow Myeloid Compartment Indicate Degree of Tumor Infiltration and Avenues for Successful Immunotherapy in Multiple Myeloma

With the onset of single-cell omics, the complete gene expression landscape of immune cells can be leveraged to differentiate distinct subsets with inherently diverse functions. By analyzing the sample-specific composition of these distinct cellular groups, we can decipher the hallmarks of healthy and fully tumor-infiltrated bone marrow microenvironments as well as compositional modulations during effective immunotherapy. Unique to our approach, our analyses leverage over 500,000 existing RNA-Seq experiments packaged in the ARCHS4 repository to strengthen the biological signal and quality of our tertiary analyses by informing us in an unbiased way about lesser known immune-typologies. We applied this technique to our well-established immunocompetent mouse model of multiple myeloma (MM) which we rendered sensitive to IMiDs by introducing a human CRBN transgene: Vk*MYChCRBN. In multiple single cell experiments measuring effectiveness of Iberdomide in combination with Dexamethasone and an additional experiment comparing aged and untreated Vk*MYC mice with multiple myeloma (MM), key compositional patterns emerge in the murine bone marrow niche. Two distinct polarities of C1q expressing macrophages are present within murine bone marrow and spleen-one expressing Fcgr4 and present in untreated, tumor-bearing samples and a separate population elevated in Apoe, Vcam1, and MHC-II markers that can be found in healthy and tumor-bearing mice. Among splenocytes, we observed that the proportion of C1q macrophages correlates strongly with tumor burden and that BM and spleen samples in the process of tumor debulking via effective immunotherapy dramatically reduce relative frequencies of C1q macrophages. We hypothesize that the C1q macrophages without MHC-II features represent a tumor-associated macrophage (TAM) phenotype that is MM dependent and promotes tumor progression within the BM niche.1 Additional analyses of murine splenocytes have revealed that effective immunotherapy via an IAP antagonist induces the presence of immature or migratory dendritic cells (high expressers of Ccl5 and likely key immune recruiters) and that tumor-bearing splenocytes typically maintain large segments of immature neutrophils (high expressers of Ngp and Camp) without other neutrophils in more advanced stages of development2-while both immature and mature neutrophils are found in healthy controls. When comparing the BM composition of an aged, tumor-bearing mouse with that of a young, healthy mouse, key differences emerge. While multiple subsets of progenitors in murine BM niche can be identified via single cell, a unique population of cells over-expressing Cxcl12 that resemble Lepr+ mesenchymal stem cells from existing experiments (GSM3676085) are nearly absent in the young mouse, highly represented in the aged mouse, and may be a critical in creating a BM niche that supports tumor expansion.3 Curiously, a sizeable population of basophils is evident in the young, non-tumor bearing mouse but not found in the adult, tumor-bearing mouse. In addition, both types of C1q macrophages have expanded frequencies in the aged mouse. In total, single-cell analyses provide a snapshot of the compositional landscape of immune cell types thus illustrating hallmarks of tumor-infiltrated, healthy, and therapy modulated BM niches, providing potential new therapeutic targets.

MECHANISM OF IMPROVED HUMAN HEMATOPOIETIC STEM CELL ENGRAFTMENT IN NON-CONDITIONED NSGW41 MICE

Humanized mouse models enable the study of human hematopoietic stem cell (HSC) function in vivo. However, human HSC engraftment and self-renewal are still hampered in xenotransplantation settings restricting the study of mechanisms regulating human HSC function. Further, irradiation conditioning limits the study of interactions between human HSCs and niche cells. We previously described NOD/SCID Il2rg-/- KitW41/W41 (NSGW41) mice, which combine an impaired endogenous HSC compartment, due to a loss-of-function KIT receptor, with immunodeficiency and support stable long-term engraftment of human HSCs without the need for preconditioning. By transplanting titrated numbers of human HSCs in primary and secondary recipients, we could show enhanced pick-up, maintenance and expansion of human HSCs in vivo compared to KIT-proficient NSG mice. Thus, human HSCs efficiently engraft in NSGW41 mice whereupon their self-renewal capacity und functionality are conserved. We hypothesized that human KIT-proficient HSCs have a competitive advantage over murine KIT-deficient HSCs and thus can stably engraft in NSGW41 mice. In fact, the endogenous HSC pool is significantly decreased in NSGW41 mice after engraftment of human HSCs, indicating that endogenous HSCs are replaced from their niche. The remaining murine KitW41/W41 HSCs express wildtype levels of KIT receptor, suggesting competition between endogenous and human donor HSCs for the KIT-ligand stem cell factor (SCF). Importantly, analysis of the murine bone marrow niche revealed a significant expansion of CD51+ PDGFRα+ mesenchymal stromal cells (MSCs) upon engraftment of human cells. The transcriptional profile of murine MSCs is significantly altered after humanization. We suggest that human HSCs actively modify the murine bone marrow niche after xenotransplantation and that MSCs provide part of the niche for human HSCs in mice.

Evaluation of Extrinsic and Intrinsic Cues Involved in BCR-ABL-Induced Leukemogenesis

Intrinsic and extrinsic signals together contribute to determine self renewal, quiescence or the specific metabolic status of leukemic stem cells (LSC) in BCR-ABL mediated chronic myeloid leukemia (CML). Our previous studies have shown that expression of BCR-ABL together with the polycomb repression complex 1 member BMI1 in human CD34+ cells is sufficient to induce a serially transplantable lymphoid leukemia in vivo while a myeloid phenotype was never observed. Yet in vitro, both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in CML patients. Since NSG models are typically lymphoid biased due to the absence of species-specific myeloid growth factors, we hypothesized that extrinsic factors might dictate lineage fate. Using a “humanized” NSG mouse model in which scaffolds seeded with human mesenchymal stromal cells were implanted we observed that, in contrast to the murine niche, BCR-ABL overexpression alone was sufficient to induce a serially transplantable leukemia of both the lymphoid and myeloid lineage. Using myeloid blast-crisis CML patient cells, engraftment was also observed whereby the immature blast-like phenotype was predominantly maintained in the humanized scaffold niche, and to a much lesser extent in the murine niche. This distinction could also be demonstrated functionally by using in vitro long-term self-renewing cultures. Blast cells retrieved from the human scaffold niche could readily be established while no long-term cultures could be initiated from cells retrieved from the murine bone marrow niche. Genome-wide transcriptome analyses of leukemic cells retrieved from the mouse BM niche and from the human scaffold niche revealed striking differences in gene expression imposed on BCR-ABL+ cells by these different environments. For example, endogenous BMI1 levels were significantly higher in BCR-ABL cells retrieved from human scaffold niche as compared to murine BM harvested cells suggesting that BMI1 might still be required as additional factor to prevent oncogene-induced senescence.Apart from epigenetic modifiers, we hypothesized that the hypoxic microenvironment might play an important role in maintaining CML LSCs and studied that in detail. Hypoxia inducible factor 1α (HIF1) and HIF2 act as transcription factors that are stabilized under hypoxic conditions. HIF1 has been characterized as an important factor that controls cellular metabolism while the role of HIF2 is still less clear. Earlier we identified HIF2 as downstream target of STAT5 and observed elevated glucose uptake in STAT5 activated HSCs. Several genes associated with glucose metabolism were upregulated by STAT5 in an HIF2 dependent manner, including SLC2A1 and GYS2. Here, we investigated metabolic changes in BCR-ABL expressing human stem/progenitor cells and focused on the role on HIF1 and HIF2. Genome-wide transcriptome analyses were performed on human CB CD34+ cells transduced with BCR-ABL as well as on BCR-ABL-positive CML and B-ALL patient samples. GSEA analyses indicated that these transcriptome changes were strongly enriched for STAT5 and MYC signatures as well as for hypoxia, embryonic stem cell and glucose metabolism gene signatures which included upregulation of e.g. SLC2A3, SLC2A1 and HIF1 and HIF2. These data suggest that BCR-ABL imposes hypoxic signaling under normoxic conditions. Moreover, downregulation of HIF1 and HIF2 using a shRNA approach impaired proliferation and reduced progenitor frequencies of BCR-ABL+ cells.Next we studied metabolic changes in BCR-ABL+ cells using NMR spectroscopy. We observed striking differences in uptake and secretion of metabolites when BCR-ABL CB CD34+ cells were compared to normal CB CD34+ cells under normoxia and hypoxia. As expected, BCR-ABL cells exhibited enhanced glycolysis as determined by an increased production and secretion of lactate under both normoxic and hypoxic conditions. Interestingly, glutamine levels were strongly enhanced in BCR-ABL+ cells, in a HIF1/2-dependent manner, possibly via enhanced glutamine import or glutamine production via upregulation/activation of Glutamine Synthase. Our current hypothesis is that BCR-ABL+ cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells, and studies focus on whether targeting this pathway might provide alternative means to eradicate LSCs. DisclosuresNo relevant conflicts of interest to declare.

Mouse Versus Human Extrinsic Cues Dictate Transformation Potential In BCR-ABL/BMI1-Induced Leukemia In Humanized Xenograft Models

The NSG [NOD/Lt-scid/IL2Rγnull] xenotransplantation mouse model is currently the model of choice to evaluate human hematopoietic engraftment and to study development of human leukemia. Indeed, we have previously shown that co-expression of BCR-ABL together with the polycomb repression complex 1 (PRC1) member BMI1 in human cord blood (CB) derived CD34+ cells was sufficient to induce a serially transplantable lymphoid leukemia (Rizo et al., Blood 2010). This leukemia was characterized by high levels of CD34+/CD19+/CD20-/IgM-/CD33-/CD15- lymphoid blasts in the bone marrow and a high degree of infiltration of blasts in spleen and liver. Clonal analysis revealed that similar clones gave rise to leukemia in primary and secondary recipients. Although in vivo no myeloid leukemias were observed, in vitro both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in chronic myeloid leukemia patients whereby a chronic myeloid phase can egress into a myeloid or lymphoid blast crisis. It is very plausible that differences between murine and human hematopoietic stem cell niches underlie these observed differences. Human engraftment in NSG mice is typically lymphoid biased, and since many growth factors and cytokines are species-specific it is clear that the murine niche is not ideal to evaluate human hematopoietic engraftment and leukemic transformation potential.In our current study we have evaluated the in vivo leukemic transformation potential of human CB derived CD34+ cells expressing BCR-ABL and/or BMI1 in NSG mice in which scaffolds coated with culture-expanded human mesenchymal stromal cells (MSCs) were implanted subcutaneously 8 weeks prior to injection of transduced cells, to allow the development of a humanized niche containing mineralized bone-matrix, osteoblasts, stromal cells, as well as appropriate vascularization (Groen et al., Blood 2012). BCR-ABL/BMI1 transduced human CB derived CD34+ cells or primary blast crisis CML patient cells were injected either intravenously or directly into the humanized scaffolds, and leukemia development was evaluated.Our data indicate that in a humanized niche, in contrast to a murine niche, BCR-ABL was sufficient to induce leukemia as a single hit without overexpression of exogenous BMI1. Furthermore, both ALL as well as erythro/myeloid leukemias could be induced. The ALL could be transplanted to secondary recipients and besides the lymphoid marker CD19, the cells also expressed CD33 and CD15, but not CD11b or GPA. These data are in sharp contrast to results obtained in xenograft mouse models without human niches, where BCR-ABL expression alone in human cells was not sufficient to induce leukemia, and secondary hits such as BMI1 were essential.Efficient engraftment of a blast-crisis CML patient sample was also observed in the human niche model, whereby the immature blast-like phenotype was maintained in the human scaffold niche, while more differentiated cells were observed in the mouse bone marrow niche. In vitro, long-term self-renewing cultures could readily be established with cells retrieved from the human scaffold niche of these leukemic mice, while no long-term cultures could be initiated with cells retrieved from the murine bone marrow niche, from the same mouse. These data indicate that a human niche is required to maintain appropriate in vivo self-renewal of human BC CML cells. Interestingly, the endogenous BMI1 levels were significantly higher in cells retrieved from the human scaffold niche as compared to the mouse BM niche.In conclusion, our data indicate that BCR-ABL transformed cells needs secondary event such as over expression of oncogene like BMI1 for its full transformation potential, most likely to overcome or repress oncogene-induced senescence. The mouse environment is not able to provide these secondary events in human cells, whereas the human niche is able to provide signals that together with BCR-ABL are sufficient to fully transform human cells in xenograft models. Disclosures:No relevant conflicts of interest to declare.

Proteomic analysis of murine bone marrow niche microenvironment identifies thioredoxin as a novel agent for radioprotection and for enhancing donor cell reconstitution

Hematopoiesis is regulated by the bone marrow (BM) niche microenvironment. We recently found that posttransplant administration of AMD3100 (a specific and reversible CXCR4 antagonist) enhanced donor cell engraftment and promoted recovery of all donor cell lineages in a congeneic mouse transplant model. We hypothesized that AMD3100 enhances donor cell reconstitution in part by modulating the levels and constitution of soluble factors in the niche microenvironment. In the current study, the effects of the BM extracellular fluid (supernatant) from AMD3100-treated transplant recipient mice on colony-forming units (CFUs) were examined. A semiquantitative, mass spectrometry-based proteomics approach was used to screen for differentially expressed proteins between the BM supernatants of PBS-treated transplant mice and AMD3100-treated transplant mice. A total of 178 proteins were identified in the BM supernatants. Thioredoxin was among the 32 proteins that displayed greater than a twofold increase in spectral counts in the BM supernatant of AMD3100-treated transplant mice. We found that thioredoxin increased CFUs in a dose-dependent manner. Thioredoxin improved hematopoiesis in irradiated mice and protected mice from radiation-related death. Furthermore, ex vivo exposure to thioredoxin for 24 hours enhanced the long-term repopulation of hematopoietic stem cells. Additionally, combined posttransplant administration of thioredoxin and AMD3100 improved hematologic recovery in primary and secondary transplant recipient mice. Our studies demonstrated that factors in the BM niche microenvironment play a critical role in hematopoiesis. Identifying these factors provides clues on potential novel targets that can be used to enhance hematologic recovery in hematopoietic stem cell transplan`tation.

Detection and isolation of human disseminated tumor cells in the murine bone marrow stem cell niche.

The presence of disseminated tumor cells (DTCs) in the bone marrow is associated with poor prognosis of cancer patients. However, little is known about the biology of DTCs due to lack of relevant animal models. Here, we describe the methods for detecting and isolating human DTCs from the murine bone marrow niche by PCR using human Alu sequences and by fluorescence-activated cell sorting and immunohistochemistry using anti-HLA antibody. These strategies could be useful for exploring the biology of DTCs.

Tle4 Is Critical for Proper B- Cell Differentiation and Maintenance of the Murine Bone Marrow Niche

AbstractAbstract 1202Transducin-like/Enhancer of Split 4 (TLE4), a homolog of the master developmental regulator in Drosophila, Groucho, is a co repressor previously described by our group as the putative tumor suppressor lost in a subset of t(8:21) AML patients also harboring 9q deletion. TLE4 has been found to be of fundamental importance in the repressive function of multiple highly conserved transcriptional complexes, including Hes1, Tcf/Lef, CSL, Runx1-3, and Pax5, all of which have been shown to be intimately connected to normal steady state hematopoiesis and deregulated in malignancy. Owing to its deletion in leukemia and its ability to confer functionality to a variety of signaling networks, our lab sought to investigate its role in hematopoiesis by generating a murine knockout model for Tle4.While normal at birth, within 2–3 weeks of age knockout (KO) animals become runted and eventually moribund by the time of weaning. Gross examination of the spleen and thymus in 3 week old animals reveals significant atrophy of these organs with severe abnormalities in normal tissue architecture. Tle4−/− mice lack clear demarcations between medullary and cortical regions of the thymus, in addition to having a significant increase in apoptosis seen via TUNEL staining. FACS analysis of the thymus shows CD4/CD8 double positive cells are heavily reduced in KOs, with an increase in both single positive mature populations. Additionally, there is a partial, but not complete, block in T cell progenitor development through TCR rearrangement (DN2-DN3 stages). Structural defects are seen in the spleen as well, with a failure in the formation of follicular zones. B cells are significantly reduced with an increase in CD11b+ cells in the spleen, in addition to a shift in the percentages of B1, B2, and marginal zone B cells in some animals. Histological examination of the long bones reveals dramatic abnormalities, both in hematopoietic and bone lineages. Tle4−/− mice exhibit a reduction in calcified bone, a dysplastic and reduced hypertrophic zone, and reabsorption of the trabecular region. These mice also have a gradual clearing of the marrow itself, with 2 week old animals first showing evidence of cytopenia that then progresses to full marrow failure by 3–3.5 weeks of age. The bone marrow exhibits a marked leukocytopenia, with a clear B lymphocytopenia as in the spleen. Furthermore, within the bone marrow, there is an increase in the number of CD4 and decrease in the number of CD8 T cells. ProB staging of developing B cells also trends towards a partial arrest in the transitions from the A through C fractions. When analyzed for progenitor populations, KO mice show severe reductions in the number of common lymphoid progenitors, while myeloid progenitors show no particular shifts in population frequency. Finally, there is a significant decrease in HSCs, defined as lin-Sca1+Ckit++ (LSK).In order to assess whether the hematological phenotype observed in these mice are of a cell intrinsic or extrinsic nature, transplants were performed using embryonic day 13.5 fetal liver and 2 week old bone marrow. Analysis of recipient animals 16 weeks after transplant revealed leukocytopenia with a specific reduction in B cells in the blood, however the T cell and LSK defects seen in donor animals was not observed. Furthermore, when LSK CD34+ Flt3+ or – cells from KO or WT 2 week old animals were plated on OP9 cells over-expressing Deltex1 or GFP, there were no defects in T, B, or CD11b+ cell production.The unique spectrum of phenotypic consequences concomitant to Tle4 deletion argues for a complex yet fundamental role in the maintenance of the hematopoietic niche. As illustrated by the ability to recapitulate some yet not all of the KO hematological aberrations in transplants, it appears that Tle4's involvement in lineage commitment is heterotypic in nature, an unsurprising finding considering its direct involvement in multiple highly conserved signaling pathways. Considering its principal role in those pathways, continued efforts to investigate the role the TLE family of corepressors play in the bone marrow niche may prove vital in reworking our paradigms for both normal and malignant hematopoiesis. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Bleeding Defect Exhibited by Aryl Hydrocarbon Receptor-Null Mice Is Due to Defective Collagen-Dependent Outside-in Signaling

Abstract 3294We previously identified AHR as a novel regulator of megakaryocytic (Mk) polyploidization and differentiation (Lindsey et al. Brit J of Haem, 2011). Best known as a mediator of toxicological signals, we propose that AHR influences multiple aspects of normal hematopoietic differentiation (Lindsey et al. Stem Cell Rev, 2012), including unpublished data suggesting a role for AHR in mediating platelet function. AHR-null mice had 9% fewer platelets and 10.4% fewer reticulated, young RNA-containing platelets than WT mice. Abnormal Mk maturation played a role in this phenotype, as AhR-null mice had ca. 25% fewer high ploidy (= 32n) Mks residing within the murine bone marrow niche compared to WT mice. While investigating if AHR influenced platelet function, we found that AHR-null mice bleed 5.3 times longer (8 minutes for AHR-null mice compared to 1.5 minutes for WT mice) and lose 3 times as much blood as WT mice during bleeding time assays. Although significant, we felt that the decreased Mk polyploidization and resulting reduced platelet counts were not enough to explain the drastic bleeding phenotype in AHR-null mice. In agreement with our hypothesis that AHR impacts platelet function, others have suggested AHR is critical for blood clotting during Oryzias latipes embryogenesis (Kawamura et al. Zoolog Sci, 2002). We previously showed that treatment of bone marrow-derived progenitor cells with AHR ligands such as TCDD (dioxin, a prototypic AHR ligand and activator) during ex vivo expansion could produce polyploid CD41-expressing cells in the absence of any cytokines. Here, we show that ex vivo expansion of murine progenitor cells with 10 mM of the AHR inhibitor 6’,2’,4’-trimethoxyflavone (TMF) resulted in 37% fewer highly polyploid (≥32n) Mks by day 7 (n=3, p=0.017), effectively blocking the effects of TPO on Mk differentiation and suggesting that AHR activation is downstream of TPO signaling.To examine the dramatic bleeding phenotype present in AHR-null mice, we next turned our attention toward platelet function, mediated by both outside-in and inside-out signaling. Defects in either or both of these signaling cascades could result in the bleeding defect present in AHR-null mice. In our initial experiments, we found that platelets from AHR-null mice bind fibrinogen equivalently to WT platelets (n=3), suggesting that AHR is not involved in inside-out platelet signaling. As we investigated other measures of platelet activation, we found that although platelets from AHR-null mice efficiently aggregated in response to ADP and the PAR-4 agonist AYPGKF (n=3 p=0.897 and 0.914, respectively), only 20 percent of AHR-null murine platelets (compared to 60 percent for WT platelets) aggregated in response to collagen (n=3, p=0.013). Spreading assays further demonstrated defective collagen-dependent outside-in signaling in AHR-null mice. We found that 5 times as many AHR-null platelets remained round (lacking filipodia or lamellipodia) as WT platelets after resting on collagen-coated slides (100 ug/mL) for 5 minutes. Over 60 percent of WT platelets were fully spread after a 20 minute incubation on collagen, compared to only 35 percent of AHR-null platelets. Additionally, roughly 20 percent of AHR-null platelets failed to respond and maintained a round morphology, representing 8 times as many unresponsive platelets as WT mice. The extent of spreading also appeared altered in AHR-null platelets, as the surface area of AHR-null platelets spreading on collagen was reduced by 42% after 5 minutes and 39% after 20 minutes compared to WT (n=3; p<0.001 for both experiments). Similar responses were seen when AHR-null platelets were allowed to spread on fibrinogen (100 ug/mL) after activation by 1 ug/mL collagen, with a 39% and 28% reduction in the surface area of AHR-null platelets after 5 and 20 minutes, respectively (n=3; p<0.001 for both experiments). Based on these findings, we are now investigating the molecular mechanisms of the collagen signaling defects present in AHR-null platelets, beginning with known interactions between AHR and vav genes, critical mediators of collagen-dependant platelet outside-in signaling. Our work is significant in that it builds upon our previously reported data and provides evidence that AHR is a critical component of the physiologic response platelets undergo in response to collagen. This information may provide novel treatment options for patients with bleeding disorders. Disclosures:No relevant conflicts of interest to declare.

Hes1 Expression Impacts Megakaryocytic Polyploidization and May Contribute to the Platelet Defects Found In Aryl Hydrocarbon Receptor (AhR)-Null Mice.

Abstract 2612 Microarray analyses (n = 3) from our lab uncovered that Aryl hydrocarbon receptor (AhR) mRNA increased 4–6 fold in Megakaryocytes (Mks) compared to isogenic granulocytic cultures and identified the Aryl hydrocarbon receptor as a novel transcriptional activator of megakaryocytic differentiation and maturation (Lindsey et al. Blood, 2009). (Q)-RT-PCR and Western Blot analyses confirmed these findings in differentiating Mks derived from primary human CD34+ cells and differentiating CHRF cells (human megakaryoblastic leukemia cell line). Unpublished data from our lab found that AhR-null mice (Schmidt et al. Proc Natl Acad Sci, 1996) exhibited a 9% decrease in platelet counts compared to WT mice. There were also 10.4% fewer reticulated young, RNA-containing platelets in AhR-null mice compared to WT mice, indicating a defect in either platelet generation or Mk maturation. We examined platelet functionality and found that while the average bleeding time for WT mice was approximately 1.5 minutes, AhR-null mice bled on average 8 minutes, or roughly 5.3 times longer. We also calculated the volume of blood loss and found that AhR-null mice lost 3 times as much blood during these assays as WT mice. To investigate if these platelet defects were due to abnormal Mk maturation, we examined the level of steady-state polyploidization of Mks residing within the murine bone marrow niche and found that compared to WT mice, AhR-null mice had ca. 25% fewer high ploidy (≥ 32n) Mks, but more Mks in the lower ploidy classes of 8n and 16n. Together, these data suggest that AhR-null mice exhibit platelet defects of both number and function, and steady-state Mks from AhR-null mice are less polyploid than WT mice. Previous EMSA experiments (n = 4) had found that AhR transcriptional activity, measured by the binding of AhR to a consensus DNA binding site, increased during Mk polyploidization and that decreased Mk ploidy correlated to a 2.7 fold decrease in expression of Hes1, an AhR target gene (n = 3; p= 0.018). Therefore, we hypothesized that AhR impacts Mk ploidy through transcriptional regulation of Hes1. In our current work, EMSA experiments (n = 3) showed a 3.5 fold increase of AhR binding to oligonucleotides corresponding to a putative AhR binding site within the Hes1 promoter, indicating that AhR could transcriptionaly regulate Hes1 during megakaryopoiesis. As an important component and regulator of the Notch signaling cascade, Hes1 regulates the cell cycle and cellular quiescence (Sang et al. Science, 2008), but its role during normal Mk differentiation remains unexplored. Interestingly, mRNA expression of Dlk1, a Hes1 transcriptional target, is increased in CD34+ cells isolated from MDS patients compared to CD34+ cells from normal individuals (Sakajiri et al. Leukemia, 2005), suggesting that Hes1 target genes may be deregulated in megakaryoblastic leukemia. To determine if Hes1 could be involved in Mk differentiation, we performed (Q)-RT-PCR experiments on differentiating Mks, granulocytes, and erythrocytes ex vivo expanded from primary human CD34+ cells. We found that by day 12 Hes1 expression increases 2.8 fold in Mks, but decreases more than 2.9 fold in isogenic granulocytes (n = 2 for each; p = 0.044). Hes1 expression appears to remain unchanged during erythroid development (n = 2). To investigate the functional impact of Hes1 during megakaryopoiesis, we used RNAi-mediated knockdown of Hes1 to generate two independent KD-Hes1 cell lines. These cell lines express 68 and 59 percent less Hes1 mRNA (n = 3; p = 0.008 for Hes1-A and p = 0.015 for Hes1-B), 31 and 47 percent less Hes1 protein (n = 3), without impacting AhR expression (n = 3; p = 0.46). Similar to knocking down AhR, Mk-ploidy distributions in KD-Hes1 cells were shifted towards lower ploidy classes, and were incapable of reaching higher ploidy classes (i.e., ≥ 32n) seen in control cells. Although there was little impact in undifferentiated CHRF cells, ploidy levels on day 7 (maximal ploidy in control cells) were between 25.3 and 26.7 percent less than control cells depending on the cell line (n = 3; p &lt; 0.05 for both cell lines). These experiments genetically link Hes1 and AhR and suggest that AhR regulation of Hes1 during megakaryopoiesis mediates megakaryocytic polyploidization. As such, we provide a novel molecular model of endomitotic entry and Mk polyploidization, while also providing a mechanistic explanation for the platelet defects found in AhR null mice. Disclosures: No relevant conflicts of interest to declare.