The Balance of Gfi-1 and Gfi-1b Maintains the Undifferentiated, Multipotent State of Hematopoietic Stem Cells and Regulates Lineage-Commitment

Abstract Abstract 1210 Gfi-1 and Gfi-1b are homologous transcriptional repressors that are expressed in hematopoietic stem cells (HSCs). Gfi-1 is crucial for the terminal maturation of neutrophils, and Gfi-1b is critical for erythropoiesis and thrombopoiesis. HSCs give rise to all mature blood lineages through a tightly regulated multistep differentiation process, but the mechanism of their early lineage specification remains largely elusive. Here, we have dissected the role of the Gfi-family factors in HSC maintenance and early lineage-commitment. To this end, we generated conditional targeted alleles for Gfi-1 and Gfi-1b that allowed for time controlled induced disruption of their genes. Acute disruption of Gfi-1 resulted in a rapid, severe decrease of HSCs numbers in the bone marrow and ablated their function in competitive repopulation assays. Surprisingly, and sharply contradicting recent claims to the opposite, acute disruption of Gfi-1b also led to decreased numbers of long-term repopulating HSCs in the bone marrow and decreased fitness in competitive transplantation. After induced, combined disruption of both factors, no HSC and progenitor cells were maintained in the bone marrow for more than 2 weeks. To elucidate the molecular mechanisms of the Gfi-family mediated HSC maintenance we performed global gene expression profiling of Gfi-1−/− and Gfi-1b−/− HSCs. Unexpectedly, both factors regulate highly distinct gene sets involved in differentiation of alternative lineages. Thus, surprisingly, their action in HSCs is not redundant but synergistic. Consistent with this, disruption of individual Gfi-family factors renders HSCs prone to differentiation to specific alternative lineages, while combined disruption is entirely incompatible with HSCs maintenance, in large part due to unchecked differentiation. Together, our data reveal that balanced expression of Gfi-1 and Gfi-1b is required for maintaining the undifferentiated, multipotent state of HSCs, while altering the balance is sufficient for inducing commitment to specific lineages. Disclosures: No relevant conflicts of interest to declare.

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Ang1 Functions as An Autocrine Activating Factor of Tie2 Signaling In Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1548.1548 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1548-1548

Author(s):

Haruka Momose ◽

Kazuya Takizawa ◽

Madoka Kuramitsu ◽

Takuo Mizukami ◽

Atsuko Masumi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Molecular Mechanisms ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Down Regulation ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 1548 Hematopoietic stem cells (HSCs) are clonogenic cells that possess the self-renewal capacity to produce more HSCs, as well as the multilineage potential that gives rise to a defined set of mature differentiated progeny for maintenance or repair of the whole blood system. HSCs lie in the hematopoietic niches located along the inner surface of the bone or the sinusoidal endothelium, and are in contact with niche cells. The cell-cell interactions with niche cells are believed to be an important prerequisite to trigger signaling events in HSCs, thereby controlling the balance between HSC self-renewal and differentiation. However, the precise molecular mechanisms regulating niche cell-HSC interactions are not well understood. One of the key molecules for those interactions is Angiopoietin-1 (Ang1). Ang1 is expressed by the niche cells and has been identified as an activating ligand for Tie2 (tyrosine kinase with Ig-like loops and epidermal growth factor homology domains 2). The expression of Tie2 is dominant in HSCs, and Tie2 in HSCs is supposed to be stimulated by Ang1 derived from niche cells. However, Ang1 is also expressed in HSCs. Detailed analysis has shown that Ang1 expression was found to be restricted in long-term HSCs (CD34-lineage-Sca-1+c-Kit+), indicating that Ang1 derived from HSCs plays a role in regulating HSCs. We attempted to elucidate a novel regulating system for HSCs through Ang1-Tie2 signaling by utilizing a hematopoietic cell line in which Tie2 was stably expressed (Ba/F3-Tie2). In Ba/F3-Tie2 cells, Tie2 was found to be phosphorylated on tyrosine residues, even without exogenous addition of Ang1. In the same cells, the expression level of endogenous Ang1 was increased four-fold. When Ang1 expression was down-regulated by transduction with a lentiviral vector expressing short hairpin RNA (shRNA) for Ang1 (shAng1), the phosphorylation of Tie2 was suppressed, suggesting that Tie2 expressed in Ba/F3-Tie2 cells could be stimulated by endogenous Ang1. To mimic the physiological circumstances of the bone marrow, Ba/F3-Tie2 cells were cultured on OP9 stromal cells. Under these culture conditions, the effect of endogenous Ang1 was investigated. Down-regulation of Ang1 by shAng1 demonstrated an approximate 50% reduction in the proliferation of Ba/F3-Tie2 cells on the OP9 cell layer. A HSC-rich population of cells prepared from bone marrow (lineage-Sca-1+c-Kit+; LSK) was also analyzed on OP9 cell layers. Similar to the results obtained from the analysis of Ba/F3-Tie2 cells, down-regulation of Ang1 by shAng1 resulted in an approximately 70% decrease in the proliferation of LSK cells cultured on OP9 monolayers. We confirmed that the suppressive effect on HSC proliferation was due to the lack of Ang1 from HSCs by culturing on Ang1-defective OP9 cells. Finally, we performed in vivo analysis to confirm the importance of endogenous Ang1 to HSCs. Ly5.2 LSK cells transduced with the shAng1 expressing vector were transplanted along with Ly5.1xLy5.2 bone marrow cells into lethally irradiated Ly5.1 mice. The Ly5.2 donor-derived cells in the recipient's peripheral blood were monitored every 2 weeks. As expected, shAng1-introduced donor cells were at decreased ratios at week four (mean ratios, 31.5% for control vs. 17.5% for shAng1), and were reduced to an even lower level at week 12 (mean ratios, 27.1% for control vs. 6.79% for shAng1). This phenomenon was also confirmed by histochemical results, where statistically fewer HSCs existed in the bone marrow of recipient mice in which shAng1-introduced HSCs were transplanted, as compared to the control. Altogether, our data suggested that Tie2 in HSCs could be stimulated by the Ang1 produced by the surrounding HSCs, and this possible autocrine regulation might control the functions of HSCs. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽

10.1182/blood-2020-137426 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-31

Author(s):

Maria Rosa Lidonnici ◽

Giulia Chianella ◽

Francesca Tiboni ◽

Matteo Barcella ◽

Ivan Merelli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Gene Set Enrichment Analysis ◽

Lineage Commitment ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Hematopoietic Stem ◽

Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

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Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2504.2504 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2504-2504

Author(s):

Russell Garrett ◽

Gerd Bungartz ◽

Alevtina Domashenko ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Poly I:C ◽

Hematopoietic Stem ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

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Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽

10.1182/blood.v120.21.sci-42.sci-42 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-42-SCI-42

Author(s):

Toshio Suda

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Metabolic Regulation ◽

Conflicts Of Interest ◽

Hypoxia Inducible Factor ◽

Ros Generation ◽

Hematopoietic Stem

Abstract Abstract SCI-42 Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed. Disclosures: No relevant conflicts of interest to declare.

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Impact of Infusion Rate on the Early Engraftment Following Allogeneic Intra Bone Marrow Infusion of Hematopoietic Stem Cells in a Canine DLA-Identical Reduced Intensity Transplantation Model

Blood ◽

10.1182/blood.v126.23.5406.5406 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5406-5406

Author(s):

Stephanie Schaefer ◽

Juliane Werner ◽

Sandra Lange ◽

Katja Neumann ◽

Christoph Machka ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Infusion Rate ◽

Conflicts Of Interest ◽

Conditioning Regimen ◽

Hematopoietic Stem ◽

Time Points ◽

The Impact ◽

Reduced Intensity

Abstract Introduction: Direct intra bonemarrow (IBM) infusion of hematopoietic stem cells (HSC) is assumed to improve the homing efficiency and to accelerate the early engraftment in comparison to the conventional intravenous application of HSC. Especially for transplantation of low cell numbers i.e. "weak grafts" that is generally associated with delayed engraftment. The direct infusion of HSC in close proximity to the HSC niche by intra bone marrow transplantation (IBMT) might be a promising way. Whether the HSC infusion rate might influence the homing process and therefore the outcome after IBMT is so far unknown. Aims: Herein, we analyzed in a canine DLA-identical littermate model the impact of different graft infusion rates on the hematopoietic recovery as well as on the engraftment kinetics after IBMT following reduced intensity conditioning. Methods: Recipient dogs received IBMT following a 4.5 Gy total body irradiation (TBI). From day (d) -1 until d+35 Cyclosporin A (15mg/kg) was administered orally twice a day as immunosuppression. For IBM transfusion the graft volume was reduced by buffy coat centrifugation and dogs obtained 2x25 ml simultaneously into the humerus and femur. The infusion rate of the graft was 25ml/10 min in group 1 (IBM10, n = 8) and 25 ml/60 min in group 2 (IBM60, n = 7). A 28 day follow-up is currently available for twelve dogs (IBM10 n = 7; IBM60 n = 5). The development of the peripheral blood mononuclear cell (PBMC) and granulocyte chimerism was tested weekly. Blood count, kidney and liver enzymes were monitored routinely. Results: All animals engrafted. One dog of the IBM10 group died at d+15 (infection) and was therefore not included into analysis. The median number of infused total nucleated cells were in IBM10 4.1*108/kg (range 2.3-6.0*108/kg) and in IBM60 3.2*108/kg (range 1.8-4.4*108/kg; p=0.4). The infused CD34+ numbers were median 3.2*106/kg (range: 1.2-10.0*106/kg; IBM10) and 3.6*106/kg (range: 1.5-6.8*106/kg; IBM60; p=0.7). Time of leukocyte recovery was median d+11 after IBMT in both groups (range: d+4 to d+11, IBM10; d+8 to d+14, IBM60; p= 0.5). Median leukocytes nadirs amounted to 0.2*109/l for IBM10 and 0.3*109/l for IBM60 (p= 0.08). The median duration of leukopenia (<1*109/l) were similar (6d, range: 4-11d, IBM10; 3-9d, IBM60) (p= 0.6). Median platelet nadir was 0*109/l for both cohorts (range: 0.0-7.0*109/l, IBM10; 0.0-1.0*109/l, IBM60). The period of thrombocytopenia (≤20.0*109/l) was significantly prolonged in the IBM60 group (median 10d, range) compared to 5d (range: 3-12d) in the IBM10 group (p=0.05). Donor PBMC chimerisms at d+7, d+14 and d+28 were median 22% (range: 8-34%), 50% (range: 29-53%) and 67% (range: 47-73%) in IBM10. The results of PBMC chimerism for IBM60 were 11% (range: 5-34%), 42% (range: 20-42%) and 59% (range: 44-66%) at these time points (p = n.s.). Donor granulocyte chimerisms of median 33% (range: 11-83%), 100% (range: 58-100%) and 100% (range: 82-100%) were detected at d+7, d+14 and d+28 after HSCT in IBM10, respectively. The granulocyte chimerism in IBM60 amounted to 34% (range: 3-87%), 96% (range: 94-100%) and 98% (range: 96-100%) at the above mentioned time points p=n.s. for all time points). Conclusion: Our data suggest that early granulocyte and PBMC engraftment is not influenced by modification of the HSC infusion rate. However, the period of thrombocytopenia seems to be prolonged following a 60 minutes application. Therefore, longer infusion times in an IBMT setting seem not to be beneficial following toxicity reduced conditioning regimen. Disclosures No relevant conflicts of interest to declare.

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JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Blood ◽

10.1182/blood.v128.22.2653.2653 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2653-2653 ◽

Cited By ~ 1

Author(s):

Yuichiro Nakata ◽

Norimasa Yamasaki ◽

Takeshi Ueda ◽

Kenichiro Ikeda ◽

Akiko Nagamachi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Epigenetic Mark ◽

M2 Macrophage ◽

Leukemic Stem Cells ◽

Cell Potential ◽

Hematopoietic Stem

Abstract Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive. To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs. We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. Disclosures No relevant conflicts of interest to declare.

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Establishment of Mouse PMF Model by the Introduction of Constitutive STAT5a Into Purified Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.3900.3900 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3900-3900

Author(s):

Takafumi Shimizu ◽

Akihiko Ito ◽

Akira Nakagawa ◽

Toshinobu Nishimura ◽

Satoshi Yamazaki ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Active Form ◽

Myeloproliferative Neoplasm ◽

Extramedullary Hematopoiesis ◽

Hematopoietic Stem ◽

Pmf Model ◽

Short Period

Abstract Abstract 3900 Poster Board III-836 Background Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofiblosis (PMF) are pathologically related and now classified under myeloproliferative neoplasm (MPN). Subsequent studies revealed that MPN is a group of clonal hematopoietic stem cell disorders characterized by proliferation of one or more of the myeloid lineages. The somatic activating mutation in the JAK2 tyrosine kinase, JAK2V617F, is now broadly recognized as a mutation responsible for MPN (Levine R.L. and Gilliland D.G. Blood 2008). Indeed, Most of PV patients, and half of patients with ET or PMF possess this mutation. Recent studies revealed that PV phenotype can be generated in homozygous JAK2V617F transgenic mice, while ET or atypical CML-like marked leukothrombocytosis with mild myelofibrosis can be observed in heterozygous JAK2V617F mice (Tiedt et al, Blood 2008, Shide et al Leukemia 2008). These results indicate that expression levels of JAK2V617F may influence PV and ET phenotypes. On the other hand, typical PMF phenotype has not been generated by the introduction of JAK2V617F. According to the WHO criteria, PMF could be defined as “spent phase of hematopoiesis” with fibrosis formation followed by increased bone marrow cellularity as consequences of granulocytic proliferation and megakaryocyte changes with ineffective hematopoiesis. In this study, we focused on STAT5a, a direct downstream molecule of JAK2, because we previously reported that upon transplantation, purified CD34- lineage- sca-1+ c-Kit+ (CD34-KSL) hematopoietic stem cells (HSCs) transduced with constitutive active form of STAT5A acted as MPN initiating cells causing granulocytosis without erythrocytosis/thrombocytosis (Kato Y. et al, J Exp Med 2005). Based on these observations, we attempted to make PMF model through mimicking typical PMF dynamics; hyper proliferation of HSCs by the introduction of constitutive active STAT5a and following early HSC exhaustion. Materials and Methods CD34-KSL HSCs or CD34+KSL hematopoietic progenitor cells (HPCs) were purified from bone marrow (BM) of C57BL/6 (B6)-Ly5.1 mice. Then, the cells were retrovirally transduced with STAT5a wild-type (wt) or its constitutive active mutant, STAT5a(1*6). The prepared cells were used for methylcellulose assay and were transplanted into lethally irradiated B6-Ly5.2 recipient mice together with 5 × 105 B6-Ly5.1/5.2 competitor BM cells. Peripheral blood (PB) of transplanted mice was monitored biweekly for donor chimerism and lineage deviation using flow cytometry. Subsequently, histrogical analyses of bone marrow and spleen were performed to determine myelofiblosis grade and detecting extramedullar hematopoiesis. Finally, immunohistochemical staining of bone marrow with anti-TGF-b antibody was performed to detect effector cells of myelofibrosis. Results Transplantation of STAT5a (1*6) transduced HSCs resulted in generation of 57 MPN mice (total 83 mice), while no MPN mouse was obtained by STAT5a (1*6) transduced HPCs (total 12 mice). Pathological analysis revealed that majority (70%) of MPN mice had PMF phenotype as defined by leukoerythroblastosis and dacryocytosis without leukothrombocytosis. These mice with PMF phenotype showed marked splenomegaly with extramedullary hematopoiesis, and granulocytic proliferation with megakaryocyte change. In BM, granulocytic proliferation advanced to severe myelofibrosis and osteomyelosclerosis in very short period of time (4 to 8 weeks). Those mice died of hemorrhage induced by pancytopenia within a few months, much faster than the mice with JAK2V617F based PV/ET models. Immunohistological analysis revealed that dominance of Gr-1 / Mac-1 positive granulocytes and CD41 positive small megakaryocytes strongly expressing TGF-beta, a putative inducer of fibroblastosis in BM of PMF mice. Conclusion By transplanting STAT5a(1*6) transduced HSCs, we were able to develop mice with phenotype closely resembling human PMF. Because PMF is rare disease, this animal model should be useful for understanding etiology of PMF, for evaluating existing treatment, and for developing therapeutics targeting STAT5a or its downstream pathway. Disclosures: No relevant conflicts of interest to declare.

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Competition In Engraftment of Normal Hematopoietic Stem Cells and Leukemic Stem Cells

Blood ◽

10.1182/blood.v116.21.4836.4836 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4836-4836

Author(s):

Gyeongsin Park ◽

Michael Heuser ◽

Tobias Berg ◽

R. Keith Humphries

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Leukemic Cell ◽

Fixed Number ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Abstract 4836 Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment. In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures: No relevant conflicts of interest to declare.

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Growth Factor Independence 1 b (Gfi1b) as a New Regulator of Hematopoietic Stem Cell Fate

Blood ◽

10.1182/blood.v116.21.837.837 ◽

2010 ◽

Vol 116 (21) ◽

pp. 837-837

Author(s):

Cyrus Khandanpour ◽

Lothar Vassen ◽

Marie-Claude Gaudreau ◽

Christian Kosan ◽

Tarik Moroy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Conflicts Of Interest ◽

Fold Increase ◽

Surface Proteins ◽

Expression Array ◽

Hematopoietic Stem

Abstract Abstract 837 Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times. We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation. Disclosures: No relevant conflicts of interest to declare.

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PDK1 Controls the Differentiation of Hematopoietic Stem Cells Via Modulating ROS Levels

Blood ◽

10.1182/blood.v126.23.896.896 ◽

2015 ◽

Vol 126 (23) ◽

pp. 896-896

Author(s):

Tianyuan Hu ◽

Cong Li ◽

Le Wang ◽

Yingchi Zhang ◽

Luyun Peng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Counts

Abstract Hematopoietic stem cells (HSCs) exist as a rare population with two essential properties of self-renewal and differentiation. HSCs can give rise to all hematopoietic progenitor and mature cells. While critical for a full understanding of the hematopoietic process and HSC-related clinical applications, the mechanisms of self-renewal and differentiation of HSCs remain elusive. The PI3K-Akt signaling pathway plays essential roles in the regulation of hematopoiesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activates multiple AGC kinases including Akt and is a pivotal regulator in this pathway. PDK1 phosphorylates Akt at its T308 residue and regulates the functional development of B and T cells during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. In this study, we generated PDK1 conditional knockout mice Vav-Cre;PDK1fl/fl (PDK1Δ/Δ) to explore the roles of PDK1 in HSCs. While PDK1Δ/Δ mice have reduced B and T cell counts as previously described, their LT-HSCs and ST-HSCs were significantly increased in comparison with WT mice while MPPs and CMPs were decreased after PDK1 deletion, indicating that the loss of PDK1 perturbed the steady-state hematopoiesis. Furthermore, although deletion of PDK1 increased the frequency of HSCs, PDK1-deficient HSCs fail to reconstitute the hematopoietic system when PDK1-deficient HSCs were used in bone marrow transplantation and competitive transplantation experiments in comparison to the WT HSCs, indicating that PDK1 is vital for hematopoiesis. To explore the mechanisms by which PDK1 regulates HSC function, we examined the cell cycle status and found the percentage of PDK1Δ/Δ HSCs was decreased significantly in G0 stage while increased in G1 and S/G2/M phases. This suggests an increase in HSC exit from a quiescent state. Since MPPs were significantly decreased in bone marrow, we examined the percentage of Annexin V+ DAPI- PDK1Δ/Δ and WT MPPs and found that they are comparable. This indicates that apoptosis did not cause the decrease in MPPs. In addition, a total of 300 LT-HSCs from PDK1Δ/Δ or WT mice and competitor cells were transplanted into lethally irradiated recipient mice to examine whether the decrease in MPPs is due to a defect in HSC differentiation. We found that less than 1% of MPPs arose from PDK1Δ/Δ HSCs 12 weeks after transplantation, indicating that PDK1 is required for the differentiation from LT-HSCs to MPPs. Because the full activation of Akt requires cooperative phosphorylation at its S473 and T308 residues by mTORC2 and PDK1, respectively, we also investigated the function of HSCs in RictorΔ/Δ PDK1Δ/Δ (DKO) mice in conjunction with RictorΔ/Δ or PDK1Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. The flow cytometric analyses of peripheral blood and bone marrow samples revealed very similar parameters of RictorΔ/Δ PDK1Δ/Δ and PDK1Δ/Δ mice. Interestingly, Rictor seemed to exert a minimal impact on HSCs and MPPs. More importantly, in contrast to RictorΔ/Δ, RictorΔ/Δ PDK1Δ/Δ HSCs failed to reconstitute the hematopoietic system after transplantation as PDK1Δ/Δ HSCs, suggesting that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. To explore the mechanism that leads to the defect in HSCs due to loss of PDK1, we assessed ROS levels in PDK1-deficient HSCs and found that PDK1-deficient LSKs and HSCs exhibit greatly reduced ROS levels when compared with the control HSCs. Treating PDK1-deficient BM cells with BSO in vitro increased cellular ROS levels and the colony counts of PDK1-deficient BM cells significantly. Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 mM. This suggests that ROS levels are precisely controlled in HSCs. Higher or lower ROS levels beyond the normal range are both harmful to normal HSC functions. Since increased SDFα expression is associated with cellular ROS levels in various cells including hematopoietic cells, we also treated PDK1Δ/Δ mice with SDFα and found that it couldpartially rescue the defective differentiation ability of PDK1-deficient HSCs. In addition, we found that PDK1 deletion could significantly prolong the life span and inhibit the leukemia development in murine T-ALL model via altering leukemic cell differentiation and proliferation. Taken together, PDK1 controls HSC differentiation via regulating cellular ROS levels and regulates malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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