Thrombopoietin-Mimetic Romiplostim Confers the Complete Survival Rate to Mice Exposed to Lethal Ionizing Radiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2390-2390 ◽  
Author(s):  
Masaru Yamaguchi ◽  
Tokuhisa Hirouchi ◽  
Mitsuru Chiba ◽  
Satoru Monzen ◽  
Hironori Yoshino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Peripheral Blood Cells ◽  
Blood Cell Count ◽  
Hematopoietic Progenitor ◽  
Γ Irradiation ◽  
Radiation Induced ◽  
Marrow Cells

Abstract Radiation-related casualties following exposure to a lethal dose of ionizing radiation show severe acute radiation syndromes (ARS) involving bone marrow death and gastrointestinal death. ARS cause decreases in red blood cell count, white blood cell count, platelet count and gastrointestinal dysfunction, finally leading to death caused by systemic bleeding. Therefore, reconstitution and restoration of hematopoiesis is a top priority. Although bone marrow transplantation (BMT) is also available for recovery from radiation-induced bone marrow damage, BMT for victims in radiation accidents has many limitations, including histocompatibility, age constraints, HLA type and the fact that immunosuppression would be required to reduce the risk of graft versus host rejection. In contrast, pharmacological approaches can accommodate a large number of victims with few limitations. Our previous study showed that the combined administration of erythropoietin, granulocytecolony stimulating factor and nandrolone decanoate after lethal ionizing irradiation resulted in the survival of approximately 50% of irradiated mice at day 30. When a c-Mpl agonist (Romiplostim: RP) was added to this protocol, 100% survival was obtained. Finally, we found that RP play a key role in the survival of irradiated mice. In the present study, we examined the effects of RP alone on mice exposed to lethal radiation. RP was administered at a dosage of 50 μg/kg of body weight/day to 8-weekold female C57BL/6JJcl mice for 1, 3, or 5 days immediately following exposure to a lethal 7 Gy dose of 137Cs γ-rays. The condition of each animal was analyzed via morphological evaluations of the small intestine and various parameters such as the numbers of peripheral blood cells, bone marrow cells, and hematopoietic progenitor cells along with cell surface antigen expression. By day 30, all untreated irradiated control mice died, whereas RP administration for 3 or 5 consecutive days after irradiation led to a 100% survival rate among the irradiated mice. At this time, the numbers of peripheral blood cells, bone marrow cells and hematopoietic progenitor cells were not significantly different between RP-untreated non-irradiated and RP-treated irradiated mice. In addition, the expression of macrophages, granulocytes and erythroid progenitors-related cell surface antigens on the bone marrow cells was significantly recovered in RP-treated irradiated mice compared to RP-untreated irradiated mice until day 20 after γ-irradiation. And, to estimate the effects of RP on gastrointestinal tissues in each individual, morphological evaluation H&E stain of the small intestine was performed until day 20 after γ-irradiation. As a result, RP promoted the recovery of gastrointestinal tissues damages in RP-treated irradiated mice compared to RP-untreated irradiated mice. Regarding cell death, radiation-induced gamma-H2AX expression in the nuclear of bone marrow cell was significantly decreased in RP-treated irradiated mice compared to RP-untreated irradiated mice immediately and after a period of 24 hours following a lethal 7 Gy dose of X-irradiation, indicating that the rate of apoptotic bone marrow cells was significantly decreased by RP-treatment. Meanwhile, 53BP1, which is well known as non-homologous end joining (NHEJ) factor, was significantly increased, showing that RP promoted NHEJ DNA repair in bone marrow cells treated with RP. These results demonstrate that c-Mpl agonist RP promotes the recovery of serious damages caused by lethal irradiation to the hematopoietic and gastrointestinal systems, and RP might be a useful radiomitigator in the case of ARS. Disclosures No relevant conflicts of interest to declare.

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

2009 ◽  
Vol 69 (4) ◽  
pp. 1141-1147 ◽  
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.

scholarly journals 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 ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 89-97 ◽  
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.

scholarly journals Interleukin 6 is a permissive factor for monocytic colony formation by human hematopoietic progenitor cells.

1992 ◽  
Vol 175 (4) ◽  
pp. 1151-1154 ◽  
Author(s):  
J H Jansen ◽  
J C Kluin-Nelemans ◽  
J Van Damme ◽  
G J Wientjens ◽  
R Willemze ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Interleukin 6 ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Marrow Cells

Since monocytes and macrophages that arise during the culture of bone marrow progenitor cells are potential sources of interleukin 6 (IL-6), we investigated whether auto- or paracrine production of this factor is involved in colony formation by normal hematopoietic progenitor cells. We added a polyclonal anti-IL-6 antiserum and a monoclonal anti-IL-6 antibody to cultures of monocyte- and T cell-depleted bone marrow cells. Colony formation was stimulated with granulocyte/monocyte-colony-stimulating factor (GM-CSF), monocyte-CSF, or IL-3. Addition of anti-IL-6 antibody resulted in decreased numbers of monocytic colonies to 40-50% of control values, whereas the numbers of granulocytic colonies were not altered. The inhibitory effect was preserved in cultures of CD34(+)-enriched bone marrow cells. As a second approach, we added a monoclonal antibody directed against the IL-6 receptor to cultures of monocyte- and T cell-depleted bone marrow cells. This antibody almost completely inhibited the growth of monocytic colonies, again without decreasing the number of granulocytic colonies. Finally, the importance of IL-6 in monocytopoiesis was demonstrated in serum-deprived bone marrow cultures: addition of exogenous IL-6 to cultures stimulated with GM-CSF resulted in increased numbers of monocytic colonies. Our results indicate that the permissive presence of IL-6 is required for optimal monocytic colony formation by bone marrow progenitor cells.

scholarly journals 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 ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 89-97 ◽  
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.

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

Blood ◽  
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.

Expression of PML-RARα by the Murine PML Locus Leads to Myeloid Self-Renewal, Clonal Expansion and Morphologic Promyelocytic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 932-932 ◽  
Author(s):  
John S. Welch ◽  
Wenlin Yuan ◽  
Timothy James Ley
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Clonal Expansion ◽  
Promyelocytic Leukemia ◽  
Peripheral Blood Cells ◽  
Self Renewal ◽  
Marrow Cells

Abstract Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation that leads to expression of a fusion protein, PML-RARα, and haploinsufficiency for both RARα and PML. We have generated a novel murine model of APL using homologous recombination to place a pathogenic human PML-RARα cDNA into the murine PML locus (mPML-PRflox). Expression of PML-RARα is initially prevented by stop codons within the loxP-flanked PGK-neo cassette. This new model recapitulates key elements of human APL lacking in other models; it provides PML locus appropriate regulation of PML-RARα expression, haploinsufficiency of PML, and somatic acquisition of the fusion protein. We have exposed mPML-PRflox mice to a conditionally-active Cre transgene (ER-T2- Cre, which activates Cre only during Tamoxifen treatment), and a compartmentally restricted Cre transgene (Lysozyme M-Cre, which has low activity in early myeloid precursors and increasing activity with myeloid differentiation). Bone marrow and spleen cells doubly heterozygous (DH) for mPML-PRflox and an activated Cre allele express PML-RARα mRNA, display neutrophil POD disruption typical of PML-RARα activity, and exhibit a shift in myelopoiesis toward CFU-G formation. We found that expression of PML-RARα following transient ER-T2-Cre activation leads to myeloid self-renewal ex vivo and clonal expansion in vivo. Bone marrow cells from DH mPML-PRflox/ER-T2- Cre mice exposed to Tamoxifen could be serially replated in methylcellulose. With successive replating, the proportion of cells carrying a floxed PML-RARα allele increased from 30% in bone marrow cells to 95% following the third replating; DH mPML-PRflox/ ER-T2-Cre bone marrow cells that were not exposed to Tamoxifen, and bone marrow cells from wild type mice, could not be serially replated. Cells bearing a floxed PML-RARα allele also expanded in vivo. In DH mPML-PRflox/ER-T2-Cre mice, a single dose of Tamoxifen (4 mg) resulted in 5% of peripheral blood cells carrying a floxed PML-RARα allele on day 7, but this population expanded progressively to 40% on day 80 without further Tamoxifen exposure (n=5). Five doses of Tamoxifen (4 mg) lead to 20% peripheral blood cells caring a floxed PML-RARα allele on day 18, and this increased progressively to 80% on day 98 (n=4). Peripheral blood of DH mPML-PRflox/ER-T2-Cre mice unexposed to Tamoxifen carried undetectable or trace numbers of cells with a floxed PML-RARα allele on day 80 (n=3). In contrast, myeloid progenitors from DH mPMLPRflox/ Lysozyme M-Cre mice did not display self-renewal ex vivo or an expansion of floxed cells with successive methylcellulose replating. DH mPML-PRflox/Lysozyme M-Cre mice did develop promyelocytic leukemia with long latency (14 months) and low penetrance (7%, n=3), which could be transplanted to secondary recipients. These tumors displayed a high percentage of floxed PML-RARα alleles in peripheral blood, spleen and bone marrow cells (range 50 – 95%) and possessed 4.5 fold higher expression levels of PML-RARα mRNA than our previously characterized Cathepsin G PML-RARα knock-in tumors. Importantly, non-leukemic,18 month-old DH mPML-PRflox/Lysozyme M-Cre mice displayed little evidence of PML-RARα dependent clonal expansion. In these mice, peripheral blood cells and spleen cells maintained low levels of the floxed PML-RARα allele (10–20%), equivalent to 6 week-old mice (n=36). These data suggest that PML-RARα expression by the murine PML locus leads directly to a myeloid self-renewal program and clonal expansion. Since Lysozyme M-Cre is expressed at low levels in early myeloid progenitors, the low penetrance of leukemia and rare clonal expansion in DH mPML-PRflox/Lysozyme M-Cre animals suggests that APL leukemogenesis may require PML-RARα expression in an early myeloid progenitor compartment, rather than a late compartment.

Granulocyte Colony-Stimulating Factor Induces High Expression of HLA-G In CD3+CD4+ T Lymphocytes and CD3+CD8+ T Lymphocytes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4870-4870
Author(s):  
Qifa Liu ◽  
Yinkui Chen ◽  
Xiuli Wu ◽  
Xuan Du ◽  
Rui Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Peripheral Blood Cells ◽  
Blocking Antibody ◽  
Cd8 T Lymphocytes ◽  
Ifn Γ ◽  
Marrow Cells

Abstract Abstract 4870 Background and objectives: Human leukocyte antigen –G (HLA–G) is a nonclassic HLA class I molecule and has the immunosuppressive activity. We investigate the effects of Granulocyte colony-stimulating factor (G-CSF) on the expression and secretion level of HLA-G in peripheral blood and bone marrow cells, and attempt to explain the mechanism of the low incidence of graft-versus-host disease (GVHD) in G-CSF-primed bone marrow (G-BM) or peripheral blood stem cells (G-PBSC) transplantation. Methods: Bone marrow and peripheral blood cells of 10 donors were collected pre-mobilization and at day 5 after G-CSF mobilization. Flow cytometry was used to detect the expression of membrane-bound HLA-G (mHLA-G) of CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, CD19+ B lymphocytes, CD56+ NK cell, CD14+ monocytes and CD33+ granulocytes. The levels of soluble HLA-G (sHLA-G) and cytokine were determined by enzyme-linked immunosorbent assay (ELISA). Bone marrow cells and peripheral blood cells of pre-mobilization were incubated for 24h, which are divided into 5 groups: (a) blank group, (b) with G-CSF, (c) with G-CSF and blocking antibody of IL-10, (d) with G-CSF and blocking antibody of IFN-γ, (e) with G-CSF, blocking antibody of IL-10 and blocking antibody of IFN-γ. Results: The levels of mHLA-G of CD3+CD4+ lymphocytes, CD3+CD8+ lymphocytes and the levels of sHLA-G at day 5 after G-CSF mobilization groups were significantly higher than the pre-mobilization groups both in bone marrow and peripheral blood cells, especially in CD3+CD8+ lymphocytes. The levels of mHLA-G of CD19+ B lymphocytes, CD56+ NK cells, CD14+ monocytes and CD33+ granulocytes had no significant difference in the two groups. The levels of IL-10 or IFN-γ in plasma of bone marrow or peripheral blood at day 5 after G-CSF mobilization groups were much higher than the pre-mobilization groups. The levels of mHLA-G of CD3+CD4+ lymphocytes, CD3+CD8+ lymphocytes and the levels of sHLA-G in bone marrow cells or peripheral blood cells incubating with G-CSF for 24h were significantly higher than the groups without incubating with G-CSF. And the blocking antibody of IL-10 or the blocking antibody of IFN-γ could not reduce the expression levels of mHLA-G and sHLA-G in vitro. Conclusion: G-CSF can act on cells directly to increase the expression levels of HLA-G in CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes. It might be assciated with the low incidence of GVHD in G-BM transplantation or G-PBSC transplantation. Supported by National Natural Science Foundation of China (30971300), Science and Technology Planning Project of Guangdong Province of China (2009A030200007) and China Postdoctoral Science Foundation (200902332, 20080440776). Disclosures: No relevant conflicts of interest to declare.

Influence of Different Genetic Alterations On the Engraftment Capacity and Serial Transplantation Efficiency of AML Patient-Derived Bone Marrow and Peripheral Blood Cells in NSG Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3511-3511
Author(s):  
Julia Schüler ◽  
Peter Haas ◽  
Kerstin Klingner ◽  
Björn W. Hackanson ◽  
Heinz-Herbert Fiebig ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Growth Behavior ◽  
Peripheral Blood Cells ◽  
Nsg Mice ◽  
Marrow Cells

Abstract Abstract 3511 Introduction: In order to allow a better understanding of acute myeloid leukemia (AML) and develop more promising therapeutic strategies the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation best. Here, we performed extensive analysis of AML engraftment in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice comparing tail vein versus intratibial injection and growth behavior of AML patient-derived bone marrow versus peripheral blood cells. Furthermore, tumor growth characteristics in the murine host were correlated with the disease stage and the molecular risk factor profile of the individual donors. Methods: Bone marrow and peripheral blood cells from 17 AML patients were injected intratibially into NSG mice (n=4–8/patient, 82 mice in total). As controls, 14 mice received bone marrow from three different donors and 5 mice were mock-injected. Tumor growth was monitored via a) determination of overall survival, b) fluorescence-based in vivo imaging (IVI, Kodak FX, Alexa750 labeled anti-human CD45 or CD33 and c) confirmation of IVI data by histological and immunohistochemical examination of bone marrow and spleen. When highly positive IVI signals and/or the overall condition of individual mice indicated enlarged tumor burden, the respective animals were sacrificed and the human AML cells transferred to another animal. In parallel the engraftment pattern of AML cells 2–4 weeks posttransplant was correlated with clinical disease activity, application route and origin of the particular tumor cells. Results: Patients included in the present study represent multiple different French-American-British (FAB) subtypes, various karyotypes and molecular features in terms of the mutational status of NPM1 and FLT3. All patient-derived specimens were capable of recapitulating the disease in NSG mice at 4–6 weeks after transplantation. Over a period of 13 months 12 out of 17 xenografts could be passaged once and 9 at least twice. Up to six passages were performed for an individual AML xenograft. In contrast, engraftment of healthy donor bone marrow cells could be determined merely until day 56 after implantation. The human bone marrow cells of the healthy donors did not engraft in serial passages. Mean survival time of AML bearing animals ranged between 21 and 82 days for a respective xenograft. No differences could be determined between engraftment capacities of peripheral blood or bone marrow cells of one patient. Neither karyotype, FAB classification nor leucocyte count or the percentage of monomorphic blasts in the bone marrow seemed to have an impact on engraftment capacity in the murine organism. However, mice bearing AML xenografts with mutations in FLT3 as well as in NPM1 showed particular short overall survival times and high tumor cell engraftment determined by IVI. This phenomenon became more obvious along the different passages. The intratibial approach proved to be superior in comparison to the intravenous application as cells of an individual patient engrafted faster when injected directly into the bone marrow microenvironment. Determination or tumor load via IVI permits to closely monitor not only the growth behavior but also the homing characteristics of the human cells over time. The positive IVI signals in bone marrow and spleen could be confirmed by histological examination as well as by immunohistochemistry specific for human CD45 and CD33. Conclusions: Our xenografts show a close resemblance to the AML-disease regarding the level of dissemination and organ involvement. Collection of whole-body IVI data proved to be a time- and animal-saving analysis that allows to closely monitor AML growth. As AML is characterized by an increasing number of molecular subtypes with completely different therapeutic options it seems to be extremely worthwhile to develop patient derived xenograft models representing as many AML subtypes as possible. Our results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of AML. Disclosures: No relevant conflicts of interest to declare.

The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 796-796
Author(s):  
Benjamin Povinelli ◽  
Michael Nemeth
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk+ HSPCs increased at the expense of Ryk-/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin-), Sca-1+, CD48- cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk+ HSPCs (12.9 ± 1.7%) compared to Ryk-/low HSPCs (32.4 ± 5.3%, p < 0.001, n = 3). To test whether this effect was limited to 5-FU, we performed a similar study in which we irradiated C57BL/6 mice with 3 cGy of total body irradiation (TBI) and analyzed bone marrow 72 hours later for apoptotic HSPCs (for this experiment, defined by a Lin-, c-kit+, Sca-1+, CD150+, CD48- immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk+ HSPCs (3.1 ± 0.2%) compared to Ryk-/low HSPCs (9.2 ± 1.5%, p < 0. 001, n = 3) in mice receiving 3 cGy TBI. These results demonstrated an association between Ryk expression and survival of HSPCs following myeloablative injury. To determine whether in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G2/M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p < 0.05, n = 3). This was associated with a decrease in the percentage of LSK, SLAM cells in G1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p < 0.01, n = 3). The percentage of G0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G1. Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p < 0.05, n = 3). To determine if α-Ryk treatment altered HSPC function, we transplanted whole bone marrow cells from C57BL/6 mice treated with two days of α-Ryk or isotype IgG at a 1:1 ratio with whole bone marrow from untreated Ubc-GFP transgenic mice into lethally irradiated B6.SJL mice. Four weeks after transplant, we analyzed peripheral blood cells for the percentage of CD45.2+ GFP- cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x106 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2+ peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p < 0.05, n = 5), suggesting that inhibition of Ryk resulted in increased elimination of host HSPCs by 5-FU. Collectively, these data suggest a model in which inhibition of the Ryk receptor results in increased proliferation of HSPCs, rendering them more sensitive to the effects of myeloablative agents such as chemotherapy or TBI. Disclosures: No relevant conflicts of interest to declare.

Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2476-2482 ◽  
Author(s):  
JM Molina ◽  
DT Scadden ◽  
M Sakaguchi ◽  
B Fuller ◽  
A Woon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Immunodeficiency Virus ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Marrow Cells

The pathogenesis of the hematologic abnormalities commonly observed in patients with acquired immunodeficiency syndrome (AIDS) is incompletely understood. We report here that in vitro growth of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from six patients with AIDS was not significantly different from that of normal human immunodeficiency virus (HIV) seronegative donors: 25.3 +/- 5 CFU-GM per 5 x 10(4) low density marrow cells and 33.5 +/- 5 BFU-E were observed in AIDS patients versus 32.7 +/- 5 CFU-GM and 42.1 +/- 5 BFU-E in controls. Furthermore, no HIV-DNA in individual colonies (CFU-GM and BFU-E) could be detected using the polymerase chain reaction (PCR) technique, although HIV-1 DNA was detected in peripheral blood mononuclear cells from the same patients. Similarly, normal bone marrow cells exposed in vitro to different isolates of HIV or recombinant purified HIV-1 envelope glycoprotein (gp) 120 did not exhibit any difference in growth of CFU-GM or BFU-E as compared with mock exposed bone marrow cells. HIV- 1 DNA could not be detected by the PCR technique in individual colonies derived from HIV exposed marrow. This study suggests that committed myeloid and erythroid progenitors from AIDS patients are responsive to hematopoietic growth factors in vitro and do not appear to contain HIV- 1 DNA. Also, HIV or its envelope gp did not alter the growth of hematopoietic progenitor cells in vitro. No evidence of HIV infection of progenitor cells could be demonstrated. Impaired hematopoiesis in patients with AIDS may not be related to direct effects of HIV on committed progenitor cells.

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