Inflammageing of Hematopoietic Stem Cells Is Driven By IL-1

Introduction: Lifelong blood production is sustained through a stepwise differentiation program by a limited number of self-renewing hematopoietic stem cells (HSCs) in the bone marrow (BM). Hematopoietic cell development is tightly controlled by both cell-intrinsic and -extrinsic factors and its dysregulation can lead to aplasia or neoplasia. During ageing, HSCs increase in number, reduce self-renewal capacity on a per cell basis, skew towards myeloid differentiation, and show less efficient bone marrow (BM)-homing ability. We here evaluated how and to what extend HSC-extrinsic factors determine HSC behaviour during aging. Methods, Results and Discussion: To screen for aging-associated extrinsic factors, we performed antibody based protein arrays and transcriptome analysis with total BM of young (6-8 week old) versus aged (2 year old) animals. This demonstrated that RANTES, MIP-2, IL-1α and IL-1β are significantly upregulated in aged BM at both the protein as well as the RNA level. ELISA of peripheral blood (PB) serum and BM lysates indicated that IL-1α and IL-1β are locally increased and produced in BM, but are not significantly increased in PB serum. Further, qPCR of various BM cell types of hematopoietic (myeloid, lymphoid and progenitor cells) and non-hematopoietic/stromal origin indicated that multiple cell types upregulate Il1α and Il1β, with highest increase being derived from myeloid hematopoietic cells. This raised the possibility that elevated IL-1 is a result of an inflammatory response to circulating pathogen-derived compounds, possibly of bacterial origin. Indeed, we previously demonstrated that steady-state levels of granulopoiesis in young steady-state mice depend on heat-resistant microbiota-derived compounds (M.Balmer et al., The Journal of Immunology 2014). To prospectively test the role of IL-1-induced signalling and the microbiome during aging, we investigated the ageing-associated phenotype of HSCs in young and aged IL1RIKO mice and in young and aged germ-free mice. Both IL1RIKO and germ-free aged mice had lower counts of platelets and neutrophils in PB, and lower frequency of LT-HSCs (LKS Flt3-CD34-CD48-CD150+) in BM, compared to aged WT mice. Moreover, aged IL1RIKO LT-HSCs showed improved lymphoid lineage repopulation upon transplantation into lethally irradiated WT mice, compared to LT-HSCs of aged WT mice that demonstrated the known myeloid-biased lineage output. Interestingly, LT-HSCs from aged germ-free mice also demonstrated lymphoid-biased lineage differentiation as observed from young mice. In line with this finding, no difference was observed in IL-1α and IL-1β protein concentrations in BM lysates from young and aged germ-free mice. To test if IL-1 increase in aged steady-state mouse BM is indeed dependent on ligation of pattern recognition receptors and consecutive signalling, we analysed MyD88 and Trif KO mice, respectively. Both aged KO mice showed compared to WT mice reduced BM IL-1 levels and a reduced ageing-phenotype of HSCs, with the most profound difference in Trif KO mice. Interestingly, this correlates with our previous finding on pathogen-Induced TLR4-TRIF innate immune signaling in HSCs, inducing reduced competitive fitness (Takizawa et al., Cell Stem Cell 2017). Conclusions: Our data demonstrate that ageing associated phenotype and myeloid-biased differentiation of HSCs is a result of signals derived from the microbiome, that act through increased IL-1 signalling, locally in BM. 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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The Tao of Hematopoietic Stem Cells: Toward a Unified Theory of Tissue Regeneration

The Scientific World JOURNAL ◽

10.1100/tsw.2002.162 ◽

2002 ◽

Vol 2 ◽

pp. 983-995 ◽

Cited By ~ 4

Author(s):

Kevin D. Bunting ◽

Robert G. Hawley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Adult Stem Cells ◽

Developmental Plasticity ◽

Marrow Cell ◽

Cell Types ◽

Therapeutic Benefit ◽

Hematopoietic Stem ◽

The Individual

Hematopoietic stem cells (HSCs) are the best studied of the tissue-specific stem cells. By definition, HSCs have long been regarded as restricted to formation of blood cells of both the lymphoid and myeloid lineages. HSCs residing in the bone marrow microenvironment have self-renewal capacity and can repopulate the hematopoietic system of irradiated transplant recipients for the lifetime of the individual. Therefore, HSCs are extremely important targets for gene therapy applications aimed toward the treatment of inherited and acquired blood disorders. However, recent studies have suggested that a subpopulation of HSCs may have the ability to contribute to diverse cell types such as hepatocytes, myocytes, and neuronal cells, especially following induced tissue damage. Preclinical amelioration of liver disease and myocardial infarcts by HSC-enriched bone marrow cell populations raises the possibility that HSC transplants have the potential to provide therapeutic benefit for a wide variety of diseases. These surprising findings contradict the dogma that adult stem cells are developmentally restricted. Extrapolation of these findings to the clinic will be facilitated by prospective identification of the stem cells that possess this developmental plasticity. Furthermore, characterization of the signaling pathways and molecular determinants regulating the remarkable transdifferentiation capacity of these stem cells may provide insight into novel approaches for modulating frequency of differentiative potential.

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Hematopoietic Stem Cells Increase Quiescence during Aging

Blood ◽

10.1182/blood-2019-130668 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2484-2484 ◽

Cited By ~ 1

Author(s):

Larisa V. Kovtonyuk ◽

Peter Ashcroft ◽

Gianluca Spaltro ◽

Nageswara Rao Tata ◽

Radek C. Skoda ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Quiescent State ◽

Hematopoietic Stem ◽

Cfse Dilution ◽

Turn Over ◽

Old Mice

Introduction: Definitive hematopoietic stem cells (HSCs) sustain blood production from fetal development throughout life. In mice, most of steady state, young adult HSCs are in the G0 phase of cell cycle (quiescence), and are estimated to divide roughly once a month. Daily hematopoietic production is thus mainly sustained by highly proliferative downstream hematopoietic progenitor cells (HPCs). Aged haematopoiesis was demonstrated to be distinct from young haematopoiesis in various aspects such as i) a shift from lymphopoiesis to myelopoiesis, ii) functional decline of HSCs (self-renewal, homing), and iii) HSCs pool expansion. While several studies attempted to address whether changes in HSCs turnover during aging can account for the distinct aging associated phenotype and function, it remained to be determined whether aged HSCs overall cycle more or less frequently than young HSCs. Methods: To construct data-based, quantitative models, we measured turnover rates and compartment sizes of populations of HSCs, HSPCs and granulopoiesis/granulocytes, i.e. a post-mitotic mature hematopoietic linage with a short half-life. We examined four age groups: 3 week, 2 month, 1 year and 2 year old mice. Mice in each group were i.p. injected every 4 hours with 1 mcg EdU up to a maximum time of 48 hours. HSC, HSPC and granulopoiesis/granoulocyte compartment sizes and snapshot cell-cycle analysis was performed by FACS at multiple sampling points in BM and peripheral blood (PB), respectively. Based on this data, we built a mathematical model of HSC turn-over and HSPC differentiation during ageing. Moreover, we evaluated HSC cycling by CFSE dilution in steady-state transplantation experiments (as described before; Takizawa et al., J Exp Med 2011). Results: In line with previous reports, the HSCs compartment size gradually increased with age from 3wk old mice to 2 year old mice. In sharp contrast, cycling activity of HSCs as determined by EdU incorporation decreased gradually and significantly with increasing age. This was driven by decreased activation from the quiescent state, while the time that actively cycling HSCs require to progress through cell-division remains constant with age. Multipotent Progenitor (MPP) cycling showed a non-significant trend towards slower turn-over. These results were confirmed by complementary CFSE-dilution experiments. Mathematical modeling of HSC proliferation and differentiation revealed a higher probability of self-renewing divisions in 3 week old mice as compared to 2 month, 1 and 2 year old mice, with the latter both having nearly equal chances of self-renewing versus differentiating divisions. Conclusions: Our data clarifies the long-standing question, how the HSC pool increases with age. Instead of an increase in active cycling, an increase in HSC quiescence is responsible for the increased size of the HSCs pool in aging. 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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In Vivo divisional Tracking System Reveals Cycling Heterogeneity in Long-Term Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.814.814 ◽

2009 ◽

Vol 114 (22) ◽

pp. 814-814

Author(s):

Hitoshi Takizawa ◽

Markus G Manz

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Model Systems ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Tracking Model

Abstract Abstract 814 Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and give rise to all mature cells of hemato-lymphoid system for the lifetime of an individual. To ensure this, HSCs are kept at homeostatic levels in adult bone marrow. Steady-state HSC cycling kinetics have been evaluated by in vivo labeling assay using 5-bromo-2-deoxyuridine (BrdU) (Cheshier et. al., PNAS 1999; Kiel et al., Nature 2007), biotin (Nygren et. al., PLoS ONE 2008) and histon 2B-green fluorescent protein (H2B-GFP) transgenic model systems (Wilson et. al., Cell 2008; Foudi et. al., Nat. Biotech. 2008). Based on the latter, it was suggested that one HSC pool turns over faster than another, dormant pool with very limited divisions during a lifetime. However, the fast cycling HSCs did not have long-term multilineage reconstitution capacity in lethally irradiated animals in contrast to dormant HSCs (Wilson et. al., Cell 2008; Foudi et.al., Nat. Biotech. 2008). From these experiments remained unclear, whether the faster cycling HSC loose long-term repopulation potential according to divisional history, or whether they represent progenitors with limited self-renewal potential, sharing a long-term HSC phenotype. Therefore, the dynamics of steady-state long-term HSC homeostasis and blood production remains to be determined. To address this directly, we set up an in vivo HSC divisional tracking assay. Here we show i.v. transfer of CFSE (carboxyfluorescein diacetate succinimidyl ester) -labeled HSCs into non-conditioned CD45.1/2 congenic F1 recipient mice that allows evaluation of steady-state HSC dynamics as CFSE distributes equally to daughter cells upon each cellular division. Sorted naïve CD4+CD62L+ T cells were used as non-dividing control cell population to determine the zero division CFSE staining level over time. Upon transfer of Lin-c-kit+Sca-1+ cells (LKS) into sublethally irradiated mice, all donor derived Lin-c-kit+ cells had divided >5 times after 3 weeks. However, transfer of LKS cells into non-irradiated mice revealed non-divided LKS cells in recipient bone marrow over 20 weeks. FACS analysis with HSC or progenitor specific marker expression showed that most of 0-2 time-divided and few of >5x divided LKS cells maintained a long-term HSC phenotype (CD150+, c-mpl+, CD34-). In order to test HSC potential, non- or >5x divided cells were sorted based on divisional history from primary recipients at different time points after transplantation, and competitively transplanted into lethally irradiated secondary recipients. At 3 weeks post primary transfer, single non-divided LKS cell was able to multi-lineage repopulate recipients, while 50 of >5x divided LKS cells showed no engraftment. Interestingly, both non- and >5x divided LKS cells at 7 or 12-14 weeks after primary transfer had long-term multilineage repopulating potential. Limiting dilution transplantation experiments demonstrated that HSC with long-term multilineage capacity (LT-HSC) were maintained at constant numbers that fit the numbers of free bone marrow niche space, with non-divided LT-HSC decreasing and >5x divided LT-HSC increasing with a constant division rate. We next tested the effects of hemato-immunological challenge on HSC cycling dynamics. Upon i.p. LPS injection into mice, previously transplanted with CFSE-labeled LKS, almost all LT-HSCs entered cell cycle within one week after challenge. These findings directly demonstrate that some LT-HSCs are quiescent for up to one fifth of the life-time of a mouse, while other LT-HSCs divide more actively, thus proving asynchronous LT-HSC division and contribution to hematopoiesis in steady-state. In addition, the results demonstrate that quiescent LT-HSCs are driven into division in response to naturally-occurring hematopoietic challenges, such as systemic bacterial infection. The CFSE-tracking model established here now allows to directly test the role of intrinsic versus environmental cues on cycling-dynamics of HSCs as well as leukemia initiating cells in steady-state and upon challenge on multiple genetic and different species background. Disclosures: No relevant conflicts of interest to declare.

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Connexin-43 Expression Regulates the Migration of Hematopoietic Stem Cells and Progenitors towards and From Bone Marrow.

Blood ◽

10.1182/blood.v114.22.562.562 ◽

2009 ◽

Vol 114 (22) ◽

pp. 562-562

Author(s):

Daniel Gonzalez-Nieto ◽

Kyung-Hee Chang ◽

Anja Koehler ◽

Jorden Arnett ◽

Susan Dunn ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Hematopoietic Stem Cells ◽

Connexin 43 ◽

Cell Types ◽

Loss Of Function ◽

Hematopoietic Stem ◽

Hematopoietic Microenvironment ◽

Deficient Mice

Abstract Abstract 562 In the bone marrow (BM) cavity, the migratory traffic of hematopoietic stem cells and progenitors (HSC/P) from the endosteal niches to circulation and viceversa depends on their response to chemokine gradients and interaction with endothelial and mesenchymal pre-osteoblastic cells located at the endosteal niches, forming the hematopoietic microenvironment (HM). Several lines of evidence have pointed out the possible role of the gap junction-forming protein connexin-43 (Cx43) in the control of stem cell and progenitor migration. Our group previously demonstrated that Cx43 expression in the hematopoietic microenvironment (HM) is critical in the fetal liver and BM hematopoietic regeneration after administration of 5-fluorouracil (5-FU) and other investigators have shown that Cx43 is crucial controlling the migration of neural progenitors along radial glial during brain development. We hypothesized that Cx43 could regulate the bidirectional migration of HSC/P in the BM stroma. Since Cx43 is expressed by mesenchymal cells, endothelial cells and hematopoietic stem cells and progenitors, we decided to analyze the Cx43 contribution in the control of HSC/P migration in cell-specific conditional knock-out mice. To achieve this objective, we have used mice that were selectively deficient for Cx43 in the osteoblast/stromal cells (Collagen 1a-Creflox/flox; O/S-Cx43-deficient), in endothelial cells (Tek-Creflox/flox; E-Cx43-deficient) or in hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). O/S-Cx43-deficient mice have been shown to be a model of osteoblast loss of function (Chung DJ et al., J. Cell. Sci., 2006) and E-Cx43-deficient mice have been shown to be a model of arterial hypotension induced by both increase nitric oxide and angiotensin levels (Liao Y et al, PNAS 2001). Analysis with reporter crossings with Rosa-loxP-Stop-LoxP-LacZ mice showed anatomical specificity of the Cre recombinase expression in different cell types of BM, and western-blot and RT-PCR expression indicated practical abolishment of the expression of Cx43 in each of the specific cell types. First, we analyzed whether there were changes in the levels of circulating progenitors in O/S-, E- or H-Cx43-deficient mice. While H-Cx43-deficient mice did not show any change in the levels of circulating HSC/P, E-Cx43-deficient mice showed a 3.5-fold and 4.7-fold, respectively, increase of circulating CFU-C and competitive repopulating units while maintaining normal repopulation ability of BM HSC. O/S-Cx43-deficient mice showed a 30% reduction in basal conditions which was more accentuated when administered G-CSF (50% reduction on day +6), compared with their WT counterparts. Interestingly, while osteoblast loss-of-function was induced in O/S Cx43-deficient mice, the intramarrow expression levels of CXCL12a/b and mesenchymal progenitor content (CFU-F) were increased (4- and 2-fold, respectively). In correlation with the increased levels of CXCL12, the distance to endosteum of transplanted CFSE+/lin-/c-kit+ BM cells into non-myeloablated O/S-Cx43-deficient mice was dramatically decreased (36.1±4.3 vs 23.2±2.1 mm, p<0.01), suggesting a major change in the cellular composition and chemokinesis within the hematopoietic microenvironment “in vivo”. Interestingly, the 16-hour homing of HSC/P transplanted into lethally irradiated O/S-Cx43KO recipient mice showed a ∼60% reduction and a significantly decreased survival in a limiting-dose transplantation radioprotection assay (50% survival in WT mice vs 0% survival in O/S Cx43-deficient recipients). The homing/engraftment defect of these mice correlated with a reversal of the increased levels of CXCL12 in irradiated BM and a 50% reduction of the migration of WT HSC/P through O/S-Cx43-deficient stroma in response to CXCL12. Altogether, these data indicate that intercellular communication through Cx43 shares distinct functions between the different cell components of the hematopoietic microenvironment, and mediates CXCL12-dependent and CXCL12-independent mechanisms in control of the BM homing and retention of HSC/P. 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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