Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

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Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽

10.1182/blood.v90.6.2148 ◽

1997 ◽

Vol 90 (6) ◽

pp. 2148-2159 ◽

Cited By ~ 118

Author(s):

Harshal H. Nandurkar ◽

Lorraine Robb ◽

David Tarlinton ◽

Louise Barnett ◽

Frank Köntgen ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Bone Marrow Cells ◽

White Blood Cells ◽

Null Mutation ◽

Wild Type ◽

Normal Numbers ◽

Interleukin 11 ◽

Marrow Cells

Abstract Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

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Impaired neutrophil maturation in truncated murine G-CSF receptor–transgenic mice

Blood ◽

10.1182/blood.v101.8.2990 ◽

2003 ◽

Vol 101 (8) ◽

pp. 2990-2995 ◽

Cited By ~ 26

Author(s):

Tetsuo Mitsui ◽

Sumiko Watanabe ◽

Yoshihiro Taniguchi ◽

Sachiyo Hanada ◽

Yasuhiro Ebihara ◽

...

Keyword(s):

Bone Marrow ◽

Blood Cells ◽

Bone Marrow Cells ◽

White Blood Cells ◽

Acute Myelocytic Leukemia ◽

Wild Type ◽

Maturation Arrest ◽

Mutant Receptor ◽

Cell Counts ◽

Marrow Cells

Abstract Severe congenital neutropenia (SCN) is a hematopoietic disorder characterized by neutropenia in peripheral blood and maturation arrest of neutrophil precursors in bone marrow. Patients with SCN may evolve to have myelodysplastic syndrome or acute myelocytic leukemia. In approximately 20% of SCN cases, a truncation mutation is found in the cytoplasmic region of the granulocyte colony-stimulating factor receptor (G-CSFR). We then generated mice carrying murine wild-type G-CSFR and its mutants equivalent to truncations at amino acids 718 and 731 in human G-CSFR, those were reported to be related to leukemic transformation of SCN. Although numbers of peripheral white blood cells, red blood cells, and platelets did not differ among mutant and wild-type G-CSFR transgenic (Tg) mice, both of the mutant receptor Tg mice had one third of peripheral neutrophil cell counts compared with wild-type receptor Tg mice. The mutant receptor Tg mice also showed impaired resistance to the infection with Staphylococcus aureus. Moreover, bone marrow of these Tg mice had an increased percentage of immature myeloid cells, a feature of SCN. This maturation arrest was also observed in in vitro cultures of bone marrow cells of truncated G-CSFR Tg mice under G-CSF stimulation. In addition, clonal culture of bone marrow cells of the truncated G-CSFR Tg mice showed the hypersensitivity to G-CSF in myeloid progenitors. Our Tg mice may be useful in the analysis of the role of truncated G-CSFR in SCN pathobiology.

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Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells

Blood ◽

10.1182/blood.v95.3.829.003k18_829_836 ◽

2000 ◽

Vol 95 (3) ◽

pp. 829-836 ◽

Cited By ~ 8

Author(s):

Timothy W. Austin ◽

Suzan Salimi ◽

Gabor Veres ◽

Franck Morel ◽

Heini Ilves ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Transgene Expression ◽

Bone Marrow Cells ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Marrow Cells

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR+ cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin−/loSca-1+ and c-kit+Sca-1+ subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR+c-kit+Sca-1+bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR+ cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

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Canonical NF-κB Signalling Is a Potential Target in FLT3/ITD AML.

Blood ◽

10.1182/blood.v120.21.2447.2447 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2447-2447 ◽

Cited By ~ 2

Author(s):

Jing Zhang ◽

Li Li ◽

Alan D. Friedman ◽

Donald Small ◽

Ido Paz-Priel

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Myeloproliferative Neoplasm ◽

Initial Response ◽

Wild Type ◽

Normal Numbers ◽

Toxic Dose ◽

Marrow Cells

Abstract Abstract 2447 Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor is common in acute myeloid leukemia (AML) and is associated with a dismal outcome. Despite initial response, FLT3/ITD AMLs often relapse early, suggesting a residual population of resistant leukemia stem cells (LSC). Clinically, FLT3 inhibitors asmonotherapy have yet to improve outcome significantly and therefore, targeting additional pro-survival pathways may be necessary for this group of AML patients. Mice genetically egineered to express a hemizygous FLT3/ITD mutation develop a progressive, fatal, myeloproliferative neoplasm. Lin−cells isolated from the bone marrow of FLT3/ITD or control mice were subjected to gel shift analysis using a radio-labeled NF-kB binding site. This analysis demonstrated high levels of nuclear activation of NF-kB in the FLT3/ITD-expressing cells, suggesting its activation downstream of mutant FLT3 signaling. MV4–11 is a human AML-derived cell line harboring a homozygous FLT3/ITD mutation. Cells expressing high levels of aldehyde dehydrogenase (ALDH) have been shown to be enriched for LSC in primary AML samples and cell lines. High ALDH expressing MV4–11 cells were isolated using FACS and analyzed for NF-kB activation. Western blot analysis demonstrated preferential phosphorylation of NF-kB p65 by activated IKK on Ser536 in this subpopulation, compared with cells with low ALDH activity. These findings indicate activation of NF-kB in MV4–11 LSCs. We wanted to next test the requirement for NF-kB signaling in transformation by FLT3/ITD mutations. NF-kB p65 null mice die in utero. We therefore established C57BL/6 p65(flox/flox);Mx1-Cre mice. Intra-peritoneal injection of pIpC every other day for 7 doses efficiently deletes the RelA/p65 gene, resulting in expression of <1% of the corresponding RNA or protein. Despite effective excision of p65, the mice survive. Bone marrow cells harvested from control or p65(del/del) mice were transduced with a FLT3/ITD-expressing lentivirus and seeded in methylcellulose without cytokines. Equal transduction rate was verified by measurement of GFP expression by flow cytometry. Reproducibly, p65(del/del) marrow transduced with FLT3/ITD was ineffective in forming cytokine independent colonies, in contrast to wild-type marrow (5 +/− 0.6 vs. 55 +/− 6 colonies per 1E5 cells, P<0.001), and the few p65(del/del) colonies that resulted were smaller than those from p65 expressing wild-type marrow cells. Cells transduced with a lentiviral vector expressing GFP but not FLT3/ITD did not form colonies without cytokines, and p65(del/del) marrow formed normal numbers of colonies of normal size and distribution in the presence of IL-3, IL-6, and SCF. Sorafenib inhibits FLT3 signaling and kills MV4–11 cells with an IC50 of approximately 10 nM. Reproducibly, a sub-toxic dose of sorafenib (5 nM) combined with sub-toxic levels of the IKKb inhibitor IMD-0354 (400 nM) resulted in synergistic cell killing as indicated by the calculated combination index of 0.55. Currently, clinical efforts in FLT3/ITD leukemia concentrate on FLT3 inhibition alone. Our data suggest that canonical NF-kB may be an important pathway in FLT3/ITD AML and that simultaneously targeting FLT3 and NF-kB in this disease may be an effective approach. Disclosures: No relevant conflicts of interest to declare.

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Evaluation of the genotoxic potential of Austroplenckia populnea (Reiss) Lundell chloroform fraction from barkwood extract in rodent cells in vivo

Brazilian Journal of Biology ◽

10.1590/s1519-69842009000500019 ◽

2009 ◽

Vol 69 (4) ◽

pp. 1141-1147 ◽

Cited By ~ 2

Author(s):

JC. Ribeiro ◽

SF. Andrade ◽

JK. Bastos ◽

EL. Maistro

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Chromosome Aberrations ◽

Blood Cells ◽

Bone Marrow Cells ◽

Chloroform Fraction ◽

Peripheral Blood Cells ◽

Swiss Mice ◽

Marrow Cells

The genotoxic effect of the Austroplenckia populnea chloroform fraction from barkwood extract was tested in vivo on peripheral blood cells of Swiss mice with the comet assay (SCGE), and the clastogenic effect was investigated on peripheral blood cells of Swiss mice and bone marrow cells of Wistar rats, with the micronucleus and chromosome aberrations tests. The animals were treated by gavage with 3 concentrations of the extract: 300, 600 and 900 mg.kg-1. Peripheral blood cells of Swiss mice were collected 4 and 24 hours after the treatment to the SCGE assay and 48 and 72 hours to the micronucleus test. Bone marrow cells of Wistar rats were collected 24 hours after the treatment to the micronucleus and chromosome aberration tests. The results showed that the A. populnea chloroform fraction induced an increase in the average number of DNA damage in peripheral blood cells at the three concentrations tested, but this increase was not statistically significant. In the micronucleus and chromosome aberrations test, no significant increase was observed in the mean number of micronucleated polychromatic erythrocytes (MNPCE) of Swiss mice or MNPCE or chromosome aberrations for the rat bone marrow cells, for any of the tested doses. Our findings enable us to conclude that by the comet assay, A. populnea chloroform fraction from barkwood extract showed no genotoxic effects, and by the micronucleus and chromosome aberration tests, the extract fraction showed no clastogenic/aneugenic effects on the rodent cells tested.

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Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow

Blood ◽

10.1182/blood.v88.1.89.89 ◽

1996 ◽

Vol 88 (1) ◽

pp. 89-97 ◽

Cited By ~ 114

Author(s):

DM Bodine ◽

NE Seidel ◽

D Orlic

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Blood Cells ◽

Bone Marrow Cells ◽

Peripheral Blood Cells ◽

Equivalent Number ◽

High Level ◽

Stimulating Factor ◽

Marrow Cells

Abstract We have examined the repopulating ability of bone marrow and peripheral blood cells collected immediately and at intervals after treatment of donor mice with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Using a competitive repopulation assay we showed that the repopulating ability of peripheral blood cells was highest immediately after cytokine treatment and declined to normal levels within 6 weeks of the termination of treatment with G-CSF and SCF. In contrast the repopulating ability of bone marrow cells was low immediately after cytokine treatment and increased to levels that were 10-fold or more greater than marrow from untreated mice by 14 days after termination of treatment with G-CSF and SCF. This high level of repopulating activity declined to normal levels by 6 weeks after termination of treatment with G-CSF and SCF. The high level of repopulating ability was confirmed by injecting cells from G- CSF- and SCF-treated donors into unconditioned recipients. Peripheral blood cells collected immediately after treatment with G-CSF and SCF engrafted into unconditioned mice sevenfold better than an equivalent number of bone marrow cells from untreated mice. Likewise, bone marrow cells collected 14 days after treatment of the donor animal with G-CSF and SCF engrafted at 10-fold higher levels than an equivalent number of bone marrow cells from untreated mice. We conclude that the treatment of donor mice with G-CSF and SCF causes a transient increase in the repopulating ability of peripheral blood and later of bone marrow. These observations may have applications to clinical hematopoietic stem cell transplantation.

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R-Ras Regulates Hematopoietic Stem/Progenitor Cell Adhesion, Migration, and Mobilization through Rac GTPase-Mediated Signals.

Blood ◽

10.1182/blood.v110.11.221.221 ◽

2007 ◽

Vol 110 (11) ◽

pp. 221-221

Author(s):

Xun Shang ◽

Lina Li ◽

Jose Concelas ◽

Fukun Guo ◽

Deidre Daria ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Wild Type ◽

Rac Gtpase ◽

Hematopoietic Stem ◽

And Migration ◽

Marrow Cells

Abstract Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p<0.001). Fourthly, isolated R-Ras−/− HSPCs displayed a constitutively assembled cortical actin cytoskeleton structure in the absence of cytokine or chemokine stimulation, similar to that of activated wild-type HSPCs. The R-Ras−/− HSPCs were defective in adhesion of cobblestone area-forming cells to a bone marrow-derived stroma cell line (FBMD-1) and in adhesion to fibronectin CH296 fragment, and showed a drastically increased ability to migrate toward a SDF-1a gradient (WT: 16% vs. KO: 38%, p<0.001). These data point to a HSPC-intrinsic role of R-Ras in adhesion and migration. Finally, the functional changes of R-Ras−/− cells were associated with a ∼3 fold increase in Rac-GTP species and constitutively elevated Rac downstream signals of phsopho-PAK1 and phospho-myosin light chain. Partial inhibition of Rac activity by NSC23766, a Rac GTPase-specific inhibitor, readily reversed the migration phenotype under SDF-1a stimulation. Taken together, these studies demonstrate that R-Ras is a critical signal regulator for HSPC adhesion, homing, migration, and mobilization through a mechanism involving Rac GTPase-regulated cytoskeleton and adhesion machinery.

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Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.

Blood ◽

10.1182/blood.v114.22.1492.1492 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1492-1492

Author(s):

Akil Merchant ◽

Anju Singh ◽

Giselle Joseph ◽

Qiuju Wang ◽

Ping Zhang ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Bone Marrow ◽

Transcription Factor ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Cytokine Signaling ◽

Wild Type ◽

Increased Sensitivity ◽

Marrow Cells

Abstract Abstract 1492 Poster Board I-515 Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2wt and Nrf2null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2null HSC. However, Nrf2null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H2O2 resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2wt or Nrf2null HSC. Mice receiving Nrf2null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2wt and Nrf2null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2null HSC by treating mice with exogenous G-CSF. Nrf2wt and Nrf2null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2wt or Nrf2null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling. Disclosures: No relevant conflicts of interest to declare.

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Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow

Blood ◽

10.1182/blood.v88.1.89.bloodjournal88189 ◽

1996 ◽

Vol 88 (1) ◽

pp. 89-97 ◽

Cited By ~ 6

Author(s):

DM Bodine ◽

NE Seidel ◽

D Orlic

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Blood Cells ◽

Bone Marrow Cells ◽

Peripheral Blood Cells ◽

Equivalent Number ◽

High Level ◽

Stimulating Factor ◽

Marrow Cells

We have examined the repopulating ability of bone marrow and peripheral blood cells collected immediately and at intervals after treatment of donor mice with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Using a competitive repopulation assay we showed that the repopulating ability of peripheral blood cells was highest immediately after cytokine treatment and declined to normal levels within 6 weeks of the termination of treatment with G-CSF and SCF. In contrast the repopulating ability of bone marrow cells was low immediately after cytokine treatment and increased to levels that were 10-fold or more greater than marrow from untreated mice by 14 days after termination of treatment with G-CSF and SCF. This high level of repopulating activity declined to normal levels by 6 weeks after termination of treatment with G-CSF and SCF. The high level of repopulating ability was confirmed by injecting cells from G- CSF- and SCF-treated donors into unconditioned recipients. Peripheral blood cells collected immediately after treatment with G-CSF and SCF engrafted into unconditioned mice sevenfold better than an equivalent number of bone marrow cells from untreated mice. Likewise, bone marrow cells collected 14 days after treatment of the donor animal with G-CSF and SCF engrafted at 10-fold higher levels than an equivalent number of bone marrow cells from untreated mice. We conclude that the treatment of donor mice with G-CSF and SCF causes a transient increase in the repopulating ability of peripheral blood and later of bone marrow. These observations may have applications to clinical hematopoietic stem cell transplantation.

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Correction of Murine Hemophilia A by Hematopoietic Stem Cell Gene Therapy.

Blood ◽

10.1182/blood.v106.11.1291.1291 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1291-1291

Author(s):

Robert G. Hawley ◽

Morvarid Moayeri ◽

Teresa S. Hawley

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Hemophilia A ◽

Bone Marrow Cells ◽

Sublethal Dose ◽

Peripheral Blood Cells ◽

Hematopoietic Stem ◽

Fviii Inhibitor ◽

Marrow Cells

Abstract A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally-delivered FVIII transgene offers the possibility to permanently correct the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based oncoretroviral vector encoding a secretion-enhanced B domain-deleted human FVIII transgene linked to a downstream EGFP reporter gene into immunocompetent hemophilia A mice (FVIII exon 16 knockout mice on a C57BL/6 background) that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy) or a nonmyelablative preparative regimen involving busulfan (two intraperitoneal doses of 10 mg/kg). Both groups of irradiated mice were transplanted with 2 × 106 sorted EGFP+ bone marrow cells. At 26 weeks, 48 ± 24% (n = 10) and 18 ± 11% (n = 12) EGFP+ nucleated peripheral blood cells were detected in mice conditioned with 800 and 550 cGy irradiation, respectively. The busulfan-conditioned mice (n = 4) were transplanted with 15–20 × 106 transduced unsorted bone marrow cells. One mouse died at 4 weeks posttransplant due to an unknown cause. The reconstitution kinetics of the remaining three mice was very similar to the group of mice conditioned with 550 cGy irradiation (18 ± 7% of their nucleated peripheral blood cells were EGFP+ at 26 weeks posttransplant). Broad transcriptional activity of the vector was observed in cells belonging to both the myeloid and lymphoid lineages in peripheral blood, and in donor-derived cells residing within the bone marrow, spleen and thymus. Importantly, therapeutic levels of FVIII (42%, 18% and 11% of normal, respectively, by COATEST assay) were detected in the plasma of all recipients 22–26 weeks posttransplant. When the mice were subsequently challenged with high doses of recombinant human FVIII (up to eight intravenous injections of 5–10 IU of recombinant full-length human FVIII at weekly intervals) to investigate the durability of tolerance induction, only minimal levels of inhibitor antibodies were detected in a subset of the corrected animals (0.8 ± 0.6, 0.7 ± 0.5 and 3 ± 4 Bethesda units per ml) in contrast to the robust anti-FVIII inhibitor response seen following immunization of naive hemophilia A mice (98 ± 48 Bethesda units per ml; n = 11). Suppression of the immune response to human FVIII was specific, as gene-treated mice mounted a normal humoral immune reaction to an unrelated antigen, tetanus toxoid. While we make no claims as to the nature of the hyporesponsive states elicited in each case, the results obtained in the small cohort of busulfan-conditioned animals are particularly exciting since the experimental protocol more closely approximates a clinically-acceptable situation, both in terms of a mild conditioning regimen as well as the lack of a preselection step for transduced bone marrow cells. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.

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