The Unexpected G0/G1 Cell Cycle Status of Mobilized Hematopoietic Stem Cells From Peripheral Blood

Abstract Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit+ Thy-1.1lo Lin−/lo Sca-1+, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF ) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34+ Thy-1+ Lin−) and progenitor cells (CD34+ Thy-1− Lin−) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.

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Kinetics of normal hematopoietic stem and progenitor cells in a Notch1-induced leukemia model

Blood ◽

10.1182/blood-2009-06-227843 ◽

2009 ◽

Vol 114 (18) ◽

pp. 3783-3792 ◽

Cited By ~ 41

Author(s):

Xiaoxia Hu ◽

Hongmei Shen ◽

Chen Tian ◽

Hui Yu ◽

Guoguang Zheng ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Quiescent State ◽

Hematopoietic Stem ◽

Definitive Evidence ◽

Accelerated Proliferation ◽

Stem And Progenitor Cells ◽

Underlying Mechanisms ◽

The Impact

Abstract The predominant outgrowth of malignant cells over their normal counterparts in a given tissue is a shared feature for all types of cancer. However, the impact of a cancer environment on normal tissue stem and progenitor cells has not been thoroughly investigated. We began to address this important issue by studying the kinetics and functions of hematopoietic stem and progenitor cells in mice with Notch1-induced leukemia. Although hematopoiesis was progressively suppressed during leukemia development, the leukemic environment imposed distinct effects on hematopoietic stem and progenitor cells, thereby resulting in different outcomes. The normal hematopoietic stem cells in leukemic mice were kept in a more quiescent state but remained highly functional on transplantation to nonleukemic recipients. In contrast, the normal hematopoietic progenitor cells in leukemic mice demonstrated accelerated proliferation and exhaustion. Subsequent analyses on multiple cell-cycle parameters and known regulators (such as p21, p27, and p18) further support this paradigm. Therefore, our current study provides definitive evidence and plausible underlying mechanisms for hematopoietic disruption but reversible inhibition of normal hematopoietic stem cells in a leukemic environment. It may also have important implications for cancer prevention and treatment in general.

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Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.861.861 ◽

2006 ◽

Vol 108 (11) ◽

pp. 861-861 ◽

Cited By ~ 1

Author(s):

Viktor Janzen ◽

Heather E. Fleming ◽

Michael T. Waring ◽

Craig D. Milne ◽

David T. Scadden

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Caspase 3 ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

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Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint-Apoptosis Coupling.

Blood ◽

10.1182/blood.v110.11.3357.3357 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3357-3357

Author(s):

Sara Rohrabaugh ◽

Charlie Mantel ◽

Hal E. Broxmeyer

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Mitotic Spindle ◽

Flow Cytometric Analysis ◽

Spindle Checkpoint ◽

Embryonic Stem ◽

Hematopoietic Stem ◽

Mitotic Spindle Checkpoint

Abstract Cell cycle checkpoints guarantee that cells move through the events of the cell cycle in the appropriate manner. The mitotic spindle checkpoint, also known as the spindle assembly checkpoint (SAC), helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Our lab recently reported on the condition of the SAC in both mouse and human embryonic stem cells (ESCs). We found that ESCs do not initiate apoptosis when the SAC is activated, which allowed these cells to tolerate a polyploid state resulting from the aberrant mitosis (Mantel et al. Blood.109: 4518–4527. 2007). These results lead us to conclude that the spindle checkpoint is uncoupled from apoptosis in ESCs. Knowing whether adult tissue specific stem/progenitor cells, such as hematopoietic stem cells (HSCs), have checkpoints which are uncoupled from apoptosis is extremely important information. If HSCs were to manifest such checkpoint uncoupling as that which we defined for ESCs, this might present a problem for the ex-vivo expansion and transplantation of HSCs. Using multiparametric permeablized cell flow cytometric analysis, we found the mitotic spindle checkpoint to be functional in primary murine sca 1+/c-kit+/lin- cells (LSK cells), a population highly enriched in primitive hematopoietic stem/progenitor cells. Using nocodazole, which exerts its affect by depolymerizing microtubules, we were able to activate the spindle checkpoint in low density mononuclear cells collected from murine bone marrow. Through flow cytometric analysis of the LSK cells in the mononuclear fraction, we were able to determine that spindle checkpoint activation in LSK cells resulted in a cell cycle arrest in mitosis, which was determined by DNA content of the cells, and eventually this arrest lead to cell death via apoptosis, as indicated by caspase-3 activation. This behavior is unlike that of ESCs, which exit mitosis and become polyploidy after prolonged spindle checkpoint activation. Thus the mitotic spindle checkpoint appears to be coupled to apoptosis in this particular set of tissue specific stem/progenitor cells, which lessens the possibility that ex-vivo expansion of hematopoietic stem cells will result in abnormalities to these cells that may give rise to disease initiation or progression after their transplantation.

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Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.1799.1799 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1799-1799

Author(s):

Ingmar Bruns ◽

Sebastian Büst ◽

Akos G. Czibere ◽

Ron-Patrick Cadeddu ◽

Ines Brückmann ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Plasma Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Stem And Progenitor Cells

Abstract Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

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Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells

The Journal of Cell Biology ◽

10.1083/jcb.122.4.897 ◽

1993 ◽

Vol 122 (4) ◽

pp. 897-902 ◽

Cited By ~ 196

Author(s):

WH Fleming ◽

EJ Alpern ◽

N Uchida ◽

K Ikuta ◽

GJ Spangrude ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Significant Proportion ◽

Cell Activity ◽

Rhodamine 123 ◽

Hematopoietic Stem ◽

Cell Cycle Status

Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic cell lineages while retaining their capacity for self renewal. One of the predictions of this model is the existence of a heterogeneous pool of HSCs, some members of which are destined to become lineage restricted progenitor cells while others function to renew the stem cell pool. To test whether HSCs are heterogeneous with respect to cell cycle status, we determined the fraction of phenotypically defined murine HSCs (Thy1.1lo Lin-/lo Sca-1+) that contain > 2n amount of DNA as measured by propidium iodide staining, Hoechst dye uptake and [3H]thymidine labeling; that fraction is 18-22%. In contrast, in the developing fetal liver, 40% of HSCs are in the S/G2/M phases of the cell cycle. Those HSCs which exhibit a low level of staining with rhodamine 123 are almost exclusively in G0/G1 (97%) whereas only 70% of HSCs which stain brightly for rhodamine 123 are in G0/G1. The injection of 100 G0/G1 HSCs rescued 90% of lethally irradiated mice in contrast to 100 S/G2/M HSCs, which protected only 25% of lethally irradiated recipients. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of 100 G0/G1 HSCs compared to recipients of 100 S/G2/M cells. These data indicate that a significant proportion of HSCs are actively proliferating during steady state hematopoiesis and that this subpopulation of cells exhibits reduced stem cell activity.

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Change in SP Distribution with Age Reflects Intrinsic Age-Based Changes in Stem Cell Biology: Implications for the Mechanism of Aging.

Blood ◽

10.1182/blood.v106.11.4209.4209 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4209-4209

Author(s):

Daniel J. Pearce ◽

Catherine Simpson ◽

Kirsty Allen ◽

Ayad Eddaoudi ◽

Derek Davies ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Cell Biology ◽

Stem Cell Biology ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

The Absolute

Abstract It has been postulated that as we age, accumulated damage causes stem cells to die by apoptosis. This could lead to a diminished stem cell pool and consequently a reduced organ regeneration potential that contributes to somatic senescence. Hematopoietic stem cells have evolved many mechanisms to cope with their exposure to toxins during life. Cell surface transporters and anti-toxic enzymes are highly expressed in hematopoietic stem cells. If toxins do get the opportunity to damage the DNA of stem cells then the cell is more likely to die by apoptosis than attempt DNA repair and risk an error. Summarised below are our results from an investigation of the frequency, phenotype, cell cycle status and repopulation potential (in young recipients) of C57BL6 side population (SP) cells from mice with a range of ages. The absolute frequency of SP cells increases with age (Figure-A). The proportion of the lineage negative, Sca-1+, c-kit+ (KLS) cell population that is an SP stem cell increases from ~1% to over 30% during the murine lifetime (red bars in Figure-B). These SP cells from older mice have a reduced 4-month competitive repopulation potential when compared to SP cells from younger mice but contain a similarly low proportion of phenotypically-defined mature cells (blue bars in Figure-B) and have a similar cell cycle profile and progenitor cell output (2% of 3 x 96 well plates for each). SP cells from older mice contained a higher proportion of SP cells with the highest efflux ability (61 vs 414 days, p=<0.001, n=6) Engrafted cells derived from old SP cells 4 months previously still displayed an increased SP frequency when compared to engrafted cells derived from SP cells of young mice. Hence, more progenitors or committed cells have not gained the SP ability; rather this difference in SP distribution reflects an age-dependent change in hematopoietic stem cell biology that is independent of the microenvironment. Specifically, the proportion of stem and progenitor cells (KLS) that is a stem cell (SP fraction of KLS) increases with age. We hypothesize that this may be a progressive enrichment of primitive cells over time via selection. As we age, accumulative damage to hematopoietic stem and progenitor cells causes more cells to die by apoptosis. It may be that the stem/progenitor cells with the lowest hoechst efflux ability are most susceptible to damage and hence most likely to die by apoptosis. Since the HSCs with the highest efflux of hoechst are thought to be the most primitive, it may be that there is an enrichment of primitive cells. This could account for the increased SP proportion observed within KLS cells. As there may be cells with ABC/G2 activity that is undetectable via the SP technique, selection of cells with a higher pump activity could also explain the increased SP frequency we observed. This hypothetical mechanism would also be independent of microenvirinment. In summary, we surmise that HSCs have a mechanism that copes with cellular damage while compensating for the reduced cellular output of HSCs with age by increasing the absolute number of HSCs. Figure Figure

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Cell-Intrinsic and Cell-Extrinsic Involvement Of C/EBPβ In The Regulation Of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v122.21.1202.1202 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1202-1202

Author(s):

Akihiro Tamura ◽

Hideyo Hirai ◽

Yoshihiro Hayashi ◽

Asumi Yokota ◽

Atsushi Sato ◽

...

Keyword(s):

Stem Cells ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Peripheral Blood ◽

Leucine Zipper ◽

Conflicts Of Interest ◽

Chronic Phase ◽

Hematopoietic Stem ◽

The Difference

Abstract Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein β (C/EBPβ), a leucine zipper transcription factor, in emergency granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPβ is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem/progenitor cells (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPβ is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and leukemic stem cells exhaustion in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPβ plays important roles in normal hematopoietic stem cells (HSCs). Here we investigated the cell-intrinsic and -extrinsic function of C/EBPβ in the regulation of HSCs by analyzing C/EBPβ knockout (KO) mice. At steady state, no obvious defects have been reported in hematopoiesis of C/EBPβ KO mice. Accordingly, the frequencies of long-term and short-term HSCs and various kinds of progenitor cells in bone marrows (BM) of C/EBPβ KO mice were identical to those in BM of wild type (WT) mice. To examine the functional consequences of C/EBPβ deletion, competitive repopulation assay was performed. In brief, 5x105 BM cells from WT or C/EBPβ KO mice (CD45.2+) and the same number of competitor CD45.1+ BM cells were transplanted into lethally irradiated CD45.1+ mice and the chimerisms of CD45.2+ cells in the peripheral blood of the recipient mice were monitored monthly. The chimerisms of C/EBPβ KO cells were significantly lower than that of WT cell at 1 month after transplantation and the differences were maintained thereafter (Figure A). In order to elucidate the reason for the difference, homing ability of C/EBPβ KO cells were assessed. Lineage depleted CD45.2+ WT or C/EBPβ KO BM cells together with the equal number of lineage negative CD45.1+ BM cells were transplanted into lethally irradiated CD45.1+ mice and the frequencies of CD45.2+ cells were analyzed 16 hours after transplantation. The frequencies of CD45.2+ WT and C/EBPβ KO donor cells in the recipient BMs were identical and the data indicated that the differences in the chimerisms after primary BM transplantation were due to the difference in the initial expansion of transplanted cells after equivalent levels of homing. To see the roles of C/EBPβ in hematopoiesis under stressed conditions, CD45.1+ mice were transplanted with CD45.2+ WT or C/EBPβ KO BM cells with equal numbers of CD45.1+ BM cells and these mice were administered with 150mg/kg 5-fluorouracil (5-FU) once a month and the chimerisms of peripheral blood were monitored every time before the next 5-FU administration. In consistent with the results mentioned above, the frequencies of CD45.2+ C/EBPβ KO cells were significantly lower than those of CD45.2+ WT cells 1 month after transplantation. After repetitive administration of 5-FU, however, the chimerisms of CD45.2+ C/EBPβ KO cells gradually caught up with those of CD45.2+ WT cells, suggesting that C/EBPβ is involved in the exhaustion of HSCs under stressed conditions (Figure B). To explore the functions of C/EBPβ in hematopoietic microenvironments, 1x106 CD45.1+ BM cells from WT mice were transplanted into irradiated (5Gy or 7Gy) WT or C/EBPβ KO mice (CD45.2+). All the WT recipient mice survived after 5Gy or 7Gy irradiation (4/4 and 4/4, respectively). In contrast, only 2/4 and 1/4 C/EBPβ KO recipient mice survived after 5Gy or 7Gy irradiation, respectively. We are currently trying to identify the cells expressing C/EBPβ in BM microenvironments and investigating the mechanisms for the higher sensitivity of C/EBPβ KO mice to irradiation. In summary, these data suggested that C/EBPβ is required for initial expansion of hematopoietic stem/progenitor cells at the expense of HSCs under stressed conditions, while it is dispensable for maintenance of HSCs at steady state. We are now investigating the cellular and molecular targets of C/EBPβ in HSC regulation and would like to elucidate the cell-intrinsic and cell-extrinsic mechanisms in regulation of the homeostasis of hematopoietic system by C/EBPβ. Disclosures: No relevant conflicts of interest to declare.

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Polycomb group gene mel-18 modulates the self-renewal activity and cell cycle status of hematopoietic stem cells

Experimental Hematology ◽

10.1016/j.exphem.2004.03.001 ◽

2004 ◽

Vol 32 (6) ◽

pp. 571-578 ◽

Cited By ~ 64

Author(s):

Teruyuki Kajiume ◽

Yuichi Ninomiya ◽

Hiroto Ishihara ◽

Rieko Kanno ◽

Masamoto Kanno

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Polycomb Group ◽

The Self ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Cycle Status ◽

Polycomb Group Gene

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Acute Influenza A Virus Infection Affects Cell Cycle and Lineage Output of Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2019-126482 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2467-2467

Author(s):

Marcel E G Rommel ◽

Lisa Walz ◽

Saskia Kohlscheen ◽

Franziska Schenk ◽

Yvonne Krebs ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Influenza A Virus ◽

Inflammatory Cytokines ◽

Acute Phase ◽

Post Infection ◽

Influenza A ◽

Hematopoietic Stem ◽

The Impact

Long-term hematopoietic stem cells (LT-HSC) persist in quiescence to maintain their hematopoietic potential throughout life. In the case of need LT-HSC can be activated to replenish the pool of blood cells. We investigated the impact of acute influenza A virus (IAV) infection on hematopoiesis in C57Bl/6N mice, focusing on the most immature HSC and progenitors. Mice were infected with a lethal dose of IAV PR/8/1934 H1N1 (humane endpoints reached within 6 days post infection (dpi)). In two further groups, mice were treated daily with oseltamivir (antiviral neuraminidase inhibitor, dpi 0-4) or were vaccinated with single-cycle vesicular stomatitis virus replicon particles expressing a miss-matched neuraminidase from influenza A virus Yamaguchi/7/2004 H5N1 four weeks prior to infection. Both treatments rescued mice from infection-induced mortality. Every day 6-9 mice were analyzed for differences in the bone marrow (BM) and blood by flow cytometry and multiplex cytokine assays as well as in the lung to determine viral tissue titers and histopathology. HSC functionality was analyzed in a competitive BM transplantation of infected and non-infected mice. Irrespective of the treatment, high IAV lung tissue titers (≥5x106 tissue culture infectious dose 50) in the first days post infection (dpi 1-5) were associated with activation of HSC into the cell cycle. LT-HSCs (LSK, CD150+, CD34- and CD48-) were 50% less quiescent and shifted into the G1/S-G2-M phase (dpi 2-6) and returned to quiescence state after virus clearance (dpi 10). Furthermore, we detected 1.5-fold increase in proliferation of phenotypic LT-HSC. Differentiation was increased towards lymphoid progenitors (≥3-fold more compared to non-infected mice) during the acute phase of infection in untreated and oseltamivir treated mice and myeloid progenitors were reduced ~50% in all groups (dpi 4-8). We found the inflammatory cytokines IFNγ, IL-1α, IL-6, and TNFα to be significantly upregulated in the BM of untreated and oseltamivir treated mice but less in vaccinated animals (dpi 2-4). IL-1α or IL-6 stimulation of LT-HSCs was sufficient to initiate proliferation in cell culture. In all groups the initial drop of the peripheral platelet count (~30% lower compared to non-infected mice, dpi 2) was replenished with an excessive production of platelets (~45% increased, dpi 8-15). Histopathology and electron microscopy revealed the sequestration and accumulation of platelets in pulmonary capillaries and vessels. Subsequently, we detected twice as many mature megakaryocytes in the BM (dpi 2-4) and elevated CD41 expression on HSCs (LSK, CD150+ and CD34-; 3-fold more compared to non-infected mice dpi 2-6) indicating a myeloid/platelet-biased HSC compartment in response to infection. Competitive whole BM transplantation with activated LT-HSCs from the acute phase of infection vs non-infected mice showed delayed reconstitution of T-cells but a preferential differentiation towards platelets in recipient mice. Taken together, local IAV infection in the lung substantially affected LT-HSC quiescence and differentiation by inflammatory cytokines with systemic consequences and a myeloid/platelet-biased lineage output. Disclosures No relevant conflicts of interest to declare.

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Reversible Expansion of Hematopoietic Stem Cells (HSC) and CML-Like Disease in Transgenic Mice Expressing BCR-ABL under the Control of the SCL 3′ Enhancer.

Blood ◽

10.1182/blood.v104.11.715.715 ◽

2004 ◽

Vol 104 (11) ◽

pp. 715-715

Author(s):

Steffen Koschmieder ◽

Berthold Goettgens ◽

Pu Zhang ◽

Tajhal Dayaram ◽

Kristin Geary ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgenic Mice ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Peripheral Blood ◽

Molecular Mechanisms ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Double Transgenic Mice

Abstract Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hematopoietic stem cells (HSC) by the BCR-ABL oncogene. Using the tet-off system, we have generated double-transgenic mice in which BCR-ABL is expressed under the control of the murine SCL 3′ enhancer, which targets expression to the vast majority of HSC and progenitors. After induction of BCR-ABL, all mice developed progressive chronic neutrophilia and leukocytosis (20–40 K/ul), and the animals died or were sacrificed in moribund condition within 58+/−28 days. Upon necropsy, bone marrow granulocytic hyperplasia, splenomegaly as well as organ infiltration by leukemic cells (liver, kidney, lung, small intestine, skin) were found. In addition, 31% of the mice subsequently developed ALL or lymphomas. BCR-ABL mRNA and protein expression were demonstrated in the affected organs. Expression of the transactivating transgene tTA was high in HSC, CMP, and CLP, but low in GMP and MEP, as assessed by real-time PCR, suggesting that the SCL 3′ enhancer indeed directed BCR-ABL expression to the most primitive hematopoietic cells within the bone marrow. The percentage of HSC in the bone marrow was expanded 7- and 26-fold in double-transgenic as compared to single-transgenic or wild-type control mice within 12 and 21 days, respectively, after BCR-ABL induction. GMP were increased 2- and 3-fold while the number of CMP was decreased 2-fold after 12 days but was increased 1.5-fold after 21 days. MEP were decreased 3-fold at both time points. In keeping with these results, the percentage of Ter-119 positive erythroid cells was decreased while the percentage of Gr-1 positive granulocytic cells was increased in the bone marrow. To assess reversibility of the phenotype, we readministered tetracycline to abrogate BCR-ABL expression. Double-transgenic mice showed rapid clinical improvement, reversion of neutrophilia and leukocytosis, normalization of Gr-1/Mac-1 positive cells in the peripheral blood and spleen, and reversion of splenomegaly. In addition, in mice that had developed lymphoblastic disease, readministration of tetracycline led to disappearance of lymphomas and of B220/BP-1 positive lymphoblastic cells in the peripheral blood. Furthermore, expansion of the HSC compartment in the bone marrow was also reversible, and the percentage of HSC decreased to levels observed in control mice. Repeated induction of BCR-ABL expression by removal of tetracycline led to reappearance of the myeloid and lymphoid phenotype. Again, the disease was reversible, and none of the animals relapsed while on tetracycline, suggesting that the phenotype remained completely dependent on the expression of the oncogene. In conclusion, we present a model of BCR-ABL mediated CML-like disease with expansion of phenotypic hematopoietic stem cells and myeloid progenitor cells in the bone marrow. The target cell population in this model closely resembles the origin of transformation in patients with CML, allowing for in vivo monitoring of early molecular mechanisms of BCR-ABL transformation. We are currently studying the function of the expanded HSC and progenitor cells in transplantation experiments.

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