Erythropoietin Signaling Inhibits Long Term Marrow Reconstitution.

Abstract Murine hematopoietic stem cells (HSC) transfected with a gain-of-function human erythropoietic receptor (EPOR) transgene were reported to have a competitive advantage over wild type mouse hematopoietic stem cells in a bone marrow transplantation (BMT) model (Kirby, Blood95(12): 3710, 2000). However, EPOR transgenes may not be normally expressed in early progenitor/stem cells. Moreover, whether Epo/EpoR signaling plays a role in hematopoietic stem cell engraftment is unknown. Our lab previously created mouse models harboring the wild type human EPOR (wthEPOR) or the mutant human gain-of-function EPOR (mthEPOR) gene knocked into the mouse EPOR locus (Divoky, PNAS 98(3): 986, 2001). This animal model has augmented Epo signaling in all tissues that express EpoR, thus the wthEPOR mice are anemic while the mthEPOR mice are polycythemic. We compared the relative engraftment efficiency of mthEPOR vs. wthEPOR HSCs in a competitive bone marrow transplantation (BMT) assay using C57/Bl6 congenic mice. Bone marrow from wthEPOR (CD45.1) and mthEPOR (CD45.2) mice were co-transplanted (1:1) into lethally irradiated (137Cs > 11Gy split) normal recipients (CD45.1/CD45.2). At 7 months after transplantation, peripheral blood chimerism demonstrated skewing towards wthEPOR rather than mthEPOR origin in the granulocyte, macrophage, T cell, and B cell compartments (Data Table). Bone marrow chimerism paralleled peripheral blood chimerism (not shown). Examination of the stem cell compartment by Hoechst 33342 staining demonstrated similar skewing towards wthEPOR origin (Data Table). Because unequal numbers of HSC may result in skewed chimerism, we examined the relative proportions of HSC to total bone marrow cells. In wthEPOR mice, the Flt3− Rh123low subset of cKit+Sca1+ cells (KLS-FS) cells represented 0.011±0.003% of total bone marrow cells while in mthEPOR mice these cells represented 0.023±0.006% of total bone marrow cells (p=0.025). Since equal numbers of wthEPOR and mthEPOR total bone marrow cells were co-transplanted, relatively fewer wthEPOR HSC than mthEPOR HSC were transferred. Taken with the above chimerism data showing skewing towards wthEPOR, these results suggest that wthEPOR HSCs have a significant engraftment advantage over mthEPOR HSCs. Furthermore, enhanced Epo/EpoR signaling may interfere with the long term repopulation of hematopoietic progenitors. Hematopoietic stem cells undergo self renewal or differentiation/proliferation; in the presence of erythropoietin, a cytokine with proliferative and differentiating properties, it may be that self renewal is suppressed leading ultimately to the observed skewed chimerism. These data suggest that erythropoietin administration to patients during and immediately after marrow transplantation may be detrimental and should be used judiciously. Peripheral Blood and Marrow Chimerism Compartment wthEPOR (CD45.1) mthEPOR (CD45.2) Endogenous control (CD45.1/CD45.2) All p values for wthEPOR vs mthEPOR < 0.01 Neutrophil (blood) 72.7% 18.8% 8.5% Macrophage (blood) 76.8% 14.7% 8.5% T cell (blood) 78.6% 9.3% 12.2% B cell (blood) 72.8% 17.7% 9.5% HSC (marrow) 66% 15.1% 18.9%

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Role Of Sf3b1 On Hematopoiesis

Blood ◽

10.1182/blood.v122.21.600.600 ◽

2013 ◽

Vol 122 (21) ◽

pp. 600-600

Author(s):

Manabu Matsunawa ◽

Ryo Yamamoto ◽

Masashi Sanada ◽

Aiko Sato ◽

Yusuke Shiozawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Transplantation ◽

Hematopoietic Stem Cells ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Ring Sideroblasts ◽

Marrow Cells

Abstract Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown. The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice. In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance. To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule

Blood ◽

10.1182/blood.v78.7.1706.bloodjournal7871706 ◽

1991 ◽

Vol 78 (7) ◽

pp. 1706-1712 ◽

Cited By ~ 5

Author(s):

S Okada ◽

H Nakauchi ◽

K Nagayoshi ◽

S Nishikawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Flow Cytometric Analysis ◽

Tyrosine Kinase Receptor ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Marrow Cells

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK- 2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr- 1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c- kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

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Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow.

Journal of Experimental Medicine ◽

10.1084/jem.175.1.175 ◽

1992 ◽

Vol 175 (1) ◽

pp. 175-184 ◽

Cited By ~ 250

Author(s):

N Uchida ◽

I L Weissman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Cell Activity ◽

Hematopoietic Stem ◽

Stem Cell Activity ◽

Marrow Cells

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

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Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule

Blood ◽

10.1182/blood.v78.7.1706.1706 ◽

1991 ◽

Vol 78 (7) ◽

pp. 1706-1712 ◽

Cited By ~ 195

Author(s):

S Okada ◽

H Nakauchi ◽

K Nagayoshi ◽

S Nishikawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Flow Cytometric Analysis ◽

Tyrosine Kinase Receptor ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK- 2) against the murine c-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)-, TER119(erythroid)-, Thy1negative-low, and WGA+. A small number of Mac-1(macrophage)+ or Gr- 1(granulocyte)+ cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)-c-kit+ cells were rhodamine-123low and this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin-WGA+c-kit+ cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin-c-kit+ cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 micrograms ACK-2, but a small number of day-12 colonies were spared. Our data show that the c- kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

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Pharmacologic Control of HOXB4-Mediated Expansion of Murine Hematopoietic Stem Cells; Evidence for a Restricted Time Interval for Expansion.

Blood ◽

10.1182/blood.v104.11.1689.1689 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1689-1689 ◽

Cited By ~ 1

Author(s):

Yan Shou ◽

Lilia Stepanova ◽

Brian Sorrentino

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Blood Cells ◽

Bone Marrow Cells ◽

Retroviral Vector ◽

Time Interval ◽

Hematopoietic Stem ◽

Marrow Cells

Overexpression of the homebox transcription factor HOXB4 can enhance self-renewal of murine hematopoietic stem cells (HSCs) and thereby result in an increased number of HSCs in vivo. In mice transplanted with bone marrow cells transduced with a retroviral vector expressing HOXB4, HSC expansion stopped after HSC numbers regenerated to a normal level. Furthermore, when transduced bone marrow cells from primary transplant recipients were transplanted into secondary recipients, HSCs failed to recover to normal numbers (G. Sauvageau et al, Genes and Dev, 9:1753, 1995). One possible explanation for these results is that HSC expansion could be limited to an early time interval in the primary transplant recipient. In order to determine if a time-window exists for HOXB4-mediated HSC expansion, and to develop a method to control HSC expansion for gene therapy applications, we generated a retroviral vector expressing a HOXB4 protein that was fused to a variant estrogen responsive binding element (ERT2). This HOXB4-ERT2 protein allowed HOXB4 function to be regulated with 4-hydroxytamoxifen (TAM). Murine bone marrow cells were transduced with the MSCV- HOXB4-ERT2-GFP vector and transplanted into lethally irradiated recipients. A 3 week course of daily TAM treatment was started either immediately after transplant, or in a second cohort, 12 weeks after transplant. When TAM treatment was administered for the first 3 weeks after transplant, there was a 7-fold increase in the percentage of GFP positive peripheral blood leukocytes compared to the cohort transplanted with the same cells but not receiving TAM treatment (15% +/−8, n=7, VERSUS 2 % +/− 2, n=9). In contrast, an identical 3-week course of TAM treatment beginning at 12 weeks post-transplant had no effect on the proportion of GFP+ cells in the peripheral blood (3% +/−2, n=5 VERSUS 2% +/−2, n=4). Bone marrow cells from mice in each of these cohorts were harvested at 21 weeks after transplant, and infused into secondary recipients. The proportion of GFP+ blood cells noted in the primary recipients that were treated with TAM for weeks 1 through 3 was maintained in untreated secondary recipients, confirming that early TAM treatment had resulted in amplification at the level of HSCs. The other half of these secondary recipients was treated immediately after transplant with the same 3 week course of daily TAM treatment. TAM treatment in secondary recipients did not lead to a further increase in the proportion of GFP+ blood cells compared to values in the untreated secondary recipients (9% +/−7, n=9 VERSUS 10% +/−3, n=6). These results show that the early 3 week time interval for HSC expansion was not reset with secondary transplantation and suggest that there is a HSC intrinsic mechanism that limits HOXB4-mediated expansion based on past replication history. This model would explain the physiologic limitation on HSC expansion that has been noted with wildtype HOXB4 vectors. Experiments are now in progress to further elucidate this putative mechanism, including further refinement of the time limits for expansion and microarray analysis of downstream target genes at different time points relative to transplantation.

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Long-Term Maintenance of Hematopoiesis in Irradiated Mice by Retrovirally Transduced Peripheral Blood Stem Cells

Blood ◽

10.1182/blood.v89.5.1811 ◽

1997 ◽

Vol 89 (5) ◽

pp. 1811-1817 ◽

Cited By ~ 21

Author(s):

Nina Drize ◽

Joseph Chertkov ◽

Elena Sadovnikova ◽

Stefan Tiessen ◽

Axel Zander

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Peripheral Blood Stem Cells ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Mobilized peripheral blood stem cells (PBSC) are used as a source of hematopoietic stem cells for transplantation and gene therapy. It is still unclear, however, whether the PBSC are fully equivalent to normal bone marrow hematopoietic stem cells and whether they are able to provide long-term function of transgene in reconstituted mice. In the present study, mobilized PBSC from male mice were transduced with human adenosine desaminase gene (hADA) and were used for reconstitution of lethally irradiated female mice. At 112, 3, 6, 9, and 12 months after reconstitution, the bone marrow cells were repeatedly collected from each mouse under light anesthesia and the number of colony-forming unit-spleen (CFU-S), spleen repopulating ability (SRA), and the integration of human ADA gene were studied in CFU-S–derived colonies by polymerase chain reaction (PCR) and Southern blot hybridization analyses. After 9 months, the proportion of donor CFU-S detected by PCR with a Y-chromosome–specific probe in mice reconstituted with mobilized PBSC was 75.3% ± 6.0%, which is similar to the concentration of donor CFU-S seen after bone marrow transplantation. Similarly, there was no difference in the concentration of CFU-S in mice reconstituted with transduced mobilized PBSC or bone marrow cells. However, in both cases the CFU-S content in the bone marrow was reduced fivefold to 10-fold compared with the concentration of CFU-S in mice transplanted with nontransduced bone marrow. The SRA of CFU-S in mice reconstituted with peripheral blood and bone marrow cells was the same 1.5 months posttransplantation, but after an additional 4 months, SRA of mice reconstituted with bone marrow cells was fivefold higher as compared with those engrafted by PBSC. The integration of the human ADA gene was observed during 9 months in about 60% of studied CFU-S. The proportion of marked colonies sharply decreased 1 year following reconstitution. One to 9 individually labeled clones could be shown simultaneously by Southern blot hybridization in the same reconstituted mice during the whole period of observation. The time of clone existence was about 3 months. We conclude that long-term marrow repopulating cells mobilized into circulation by treatment with granulocyte colony-stimulating factor (G-CSF ) and stem cell factor (SCF ) are capable of maintaining lifelong polyclonal hematopoiesis in reconstituted mice.

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Long-Term Maintenance of Hematopoiesis in Irradiated Mice by Retrovirally Transduced Peripheral Blood Stem Cells

Blood ◽

10.1182/blood.v89.5.1811.1811_1811_1817 ◽

1997 ◽

Vol 89 (5) ◽

pp. 1811-1817

Author(s):

Nina Drize ◽

Joseph Chertkov ◽

Elena Sadovnikova ◽

Stefan Tiessen ◽

Axel Zander

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Blot Hybridization ◽

Southern Blot Hybridization ◽

Peripheral Blood Stem Cells ◽

Hematopoietic Stem ◽

Marrow Cells

Mobilized peripheral blood stem cells (PBSC) are used as a source of hematopoietic stem cells for transplantation and gene therapy. It is still unclear, however, whether the PBSC are fully equivalent to normal bone marrow hematopoietic stem cells and whether they are able to provide long-term function of transgene in reconstituted mice. In the present study, mobilized PBSC from male mice were transduced with human adenosine desaminase gene (hADA) and were used for reconstitution of lethally irradiated female mice. At 112, 3, 6, 9, and 12 months after reconstitution, the bone marrow cells were repeatedly collected from each mouse under light anesthesia and the number of colony-forming unit-spleen (CFU-S), spleen repopulating ability (SRA), and the integration of human ADA gene were studied in CFU-S–derived colonies by polymerase chain reaction (PCR) and Southern blot hybridization analyses. After 9 months, the proportion of donor CFU-S detected by PCR with a Y-chromosome–specific probe in mice reconstituted with mobilized PBSC was 75.3% ± 6.0%, which is similar to the concentration of donor CFU-S seen after bone marrow transplantation. Similarly, there was no difference in the concentration of CFU-S in mice reconstituted with transduced mobilized PBSC or bone marrow cells. However, in both cases the CFU-S content in the bone marrow was reduced fivefold to 10-fold compared with the concentration of CFU-S in mice transplanted with nontransduced bone marrow. The SRA of CFU-S in mice reconstituted with peripheral blood and bone marrow cells was the same 1.5 months posttransplantation, but after an additional 4 months, SRA of mice reconstituted with bone marrow cells was fivefold higher as compared with those engrafted by PBSC. The integration of the human ADA gene was observed during 9 months in about 60% of studied CFU-S. The proportion of marked colonies sharply decreased 1 year following reconstitution. One to 9 individually labeled clones could be shown simultaneously by Southern blot hybridization in the same reconstituted mice during the whole period of observation. The time of clone existence was about 3 months. We conclude that long-term marrow repopulating cells mobilized into circulation by treatment with granulocyte colony-stimulating factor (G-CSF ) and stem cell factor (SCF ) are capable of maintaining lifelong polyclonal hematopoiesis in reconstituted mice.

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MEIS1 Is Required for the Maintenance of Long Term Hematopoietic Stem Cells Wherein It Regulates Quiescence

Blood ◽

10.1182/blood.v118.21.551.551 ◽

2011 ◽

Vol 118 (21) ◽

pp. 551-551

Author(s):

Zeenath Unnisa ◽

Jason P Clark ◽

Elizabeth Wojtowicz ◽

Lino Tessarollo ◽

Neal G. Copeland ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Bone Marrow Analysis ◽

Marrow Cells

Abstract Abstract 551 Normal hematopoiesis is maintained by long-term hematopoietic stem cells (LT-HSCs) that are defined by their extensive self-renewal and multipotency. Self-renewal of LT-HSCs in turn is regulated by a complex network of intrinsic and extrinsic factors. The transcription factor MEIS1 is highly expressed in hematopoietic stem and progenitor cells and also in several leukemias, suggesting that MEIS1 might be important in regulating self-renewal. However, the role of MEIS1 in normal hematopoiesis has not been defined. To determine the role of MEIS1 in hematopoiesis, we studied conditional knockout mice. We generated transgenic mice bearing loxp sites flanking the homeodomain of MEIS1. The MEIS1-floxed mice were then bred to Rosa26-CreERT2 mice, the latter expressing cre-recombinase ubiquitously, that can be activated by estrogen or its analog Tamoxifen (Tam). Efficient, complete recombination was achieved in vivo by treating MEIS1-f/f-Cre (homozygous for MEIS1-flox) mice with Tam and in vitro by treating bone marrow cells with 4-hydroxy tamoxifen. Loss of MEIS1 expression was detected by QRT-PCR and western blotting. To determine the role of MEIS1 in the maintenance of adult hematopoiesis, MEIS1-f/f-Cre and control mice were treated with Tam and MEIS1 deletion confirmed by PCR. At three weeks post deletion, bone marrow analysis showed a significant reduction in the number of LT-HSCs defined as lin-/c-Kit+/Sca1+/CD48−/CD150+ in the MEIS1-depleted mice compared to controls (0.012% compared to 0.037%, N=6, p<0.05, t-test). However, the progenitor populations were unaffected by MEIS1 deletion. Over a period of 12 weeks of observation, the mice did not show any signs of distress and the peripheral blood counts of the experimental and control mice remained normal, indicating that short term hematopoiesis was not affected. Cell cycle analysis of LT-HSCs showed that MEIS1 deletion resulted in a significant shift of cells from G0 to G1 phase (G0 and G1 proportions respectively, 81.75±3.25% and 9.40±3% for control and 56.10±0.873% and 31.17±1.5% for MEIS1-deleted). To determine the effects of MEIS1 loss on intrinsic hematopoietic stem cell function, we performed competitive repopulation assays. Bone marrow cells harvested from MEIS1-f/f-Cre or MEIS1-f/+-Cre (control) mice were combined with equal numbers of bone marrow cells from BoyJ mice and transplanted via tail vein injection into lethally irradiated BoyJ mice. Four weeks after transplant, recipients were treated with Tam or vehicle for 5 days and deletion of MEIS1 confirmed by PCR on peripheral blood. Peripheral blood of recipient mice was analyzed at 1, 4, 8, 12 and 16 weeks after treatment and relative chimerism assessed by flow cytometry. At 1 and 4 weeks after treatment, the chimerism in the MEIS1 deleted group (Tam treated MEIS1-f/f-CreER) and the control groups (Tam treated MEIS1-f/+-CReER and vehicle treated MEIS1-f/f-CreER) was comparable (41%, 40.5% and 41.5% respectively, average, N=5 to 8). However, by 8 weeks after treatment, the MEIS1 deleted group showed a significant decline in chimerism compared to controls (18.2% compared to 43.1% and 35.1% respectively, p<0.02, t-test) and at 16 weeks the chimerism in the MEIS1-deleted group declined further (11.1% compared to 40.2% and 35.0% respectively, p<0.001). Subpopulation analysis showed loss of chimerism in granulocytes and in B and T lymphocytes. The latency and breadth of the effect of MEIS1 loss suggested an effect on the hematopoietic stem cell population. Indeed, bone marrow analysis of transplant recipients showed near complete loss of LT-HSC chimerism (3% compared to 70.25% and 75.6% respectively, p<0.001). Finally, we performed gene expression profiling on lineage negative bone marrow cells with and without MEIS1 deletion. Results showed that loss of MEIS1 was associated with decreased expression of hypoxia-responsive genes. Collectively, these results indicate that MEIS1 is required for the maintenance of the pool of LT-HSCs. Loss of MEIS1 promotes cycling and exhaustion of LT-HSCs. Further, we propose that activation of the hypoxia-response pathway may be one of the mechanisms by which MEIS1 exerts its effects on hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽

10.1182/blood.v84.1.74.74 ◽

1994 ◽

Vol 84 (1) ◽

pp. 74-83 ◽

Cited By ~ 36

Author(s):

SJ Szilvassy ◽

S Cory

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Bone Marrow Cells ◽

High Titer ◽

Marker Gene ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

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Reversible Expression of CD34 by Murine Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v94.8.2548.420k38_2548_2554 ◽

1999 ◽

Vol 94 (8) ◽

pp. 2548-2554 ◽

Cited By ~ 291

Author(s):

Takashi Sato ◽

Joseph H. Laver ◽

Makio Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Cell Populations ◽

Hematopoietic Stem ◽

Adult Mice ◽

Cd34 Expression ◽

Marrow Cells

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34−. We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34− and CD34+ cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage−c-kit+ Sca-1+ CD34− bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34− and CD34+ cell populations and tested for their engraftment capabilities, only CD34+ cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34+ stem cells to CD34− state. We transplanted Ly-5.1 CD34+post–5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34− cell fraction, indicating the reversion of CD34+ to CD34−stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.

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