Mutant U2AF1(S34F) Expression Alters Hematopoiesis in Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 553-553
Author(s):  
Cara L Lunn ◽  
Justin Tibbitts ◽  
James N Ley ◽  
Jin Shao ◽  
Timothy Graubert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Annexin V ◽  
Single Copy ◽  
Empty Vector ◽  
Transgenic Cells ◽  
Marrow Cells

Abstract Abstract 553 Myelodysplastic syndromes (MDS) are stem cell disorders characterized by ineffective hematopoiesis with increased levels of hematopoietic cell apoptosis. Recent discoveries by our group and others suggest that perturbations in pre-mRNA splicing may play a role in MDS pathogenesis. Indeed, more than half of all MDS patients have a mutation in one of eight splicing factors. U2AF1 (U2AF35), a gene encoding a splicing factor involved in intronic 3'-splice site recognition, is mutated in 8.7% of MDS patients. The consequence of the highly recurrent serine to phenylalanine mutation at position 34 (S34F) of U2AF1 in hematopoiesis is unknown. Therefore, to examine the effects of mutant U2AF1(S34F) on hematopoiesis, we utilized the MSCV-IRES-GFP retroviral system to introduce mutant U2AF1(S34F) or wild type U2AF1(WT), or an empty vector control, into mouse bone marrow cells for in vitro and in vivo studies. Expression of U2AF1(S34F) results in reduced expansion of transduced bone marrow cells (marked by GFP) compared to both U2AF1(WT) or empty vector-transduced cells grown in suspension culture (2 vs 4-fold change, respectively; p<0.001, n=3). Additionally, U2AF1(S34F)-transduced cells have increased levels of apoptosis (Annexin V+/7AAD+) in culture compared to U2AF1(WT) (p=0.03) and empty vector-transduced cells (p=0.02) (n=3). We also examined the effects of the U2AF1(S34F) mutation in vivo using bone marrow transplantation. The percentage of GFP+ cells in the peripheral blood of recipient mice transplanted with MSCV-transduced bone marrow was significantly reduced at 6 months post-transplant with U2AF1(S34F) expression (average=4%) compared to U2AF1(WT) (average=44%) and empty vector (average=65%) (p<0.02, n= 6–9 mice each). Transduction efficiencies were similar within experiments. There was no consistent alteration in lineage distribution of GFP+ cells in the peripheral blood of these mice. To overcome some of the limitations of retroviral models, we created a single-copy, doxycycline-inducible U2AF1(S34F) transgenic mouse to model the effect of U2AF1(S34F) expression on hematopoiesis. As a control for U2AF1 protein overexpression, we created an additional single-copy, doxycycline-inducible U2AF1(WT) transgenic mouse with transgene integration into the same locus as the U2AF1(S34F) mouse. Induction of U2AF1(S34F) transgene expression in bone marrow cells in culture with doxycycline treatment (200 ng/ml for 5 days) resulted in reduced cell numbers when compared to uninduced U2AF1(S34F) transgenic cells (ratio of growth of induced/uninduced cells = 0.38), while cell proliferation was not altered for U2AF1(WT) transgenic cells (ratio of growth of induced/uninduced cells = 1.13) (p<0.001, n=3). In addition, doxycycline-induced U2AF1(S34F) expression results in increased apoptosis (Annexin V+) compared to uninduced U2AF1(S34F) transgenic cells (21% vs 11%, p=0.01) and induced U2AF1(WT) transgenic cells in culture (21% vs 9.3%, p=0.008) (n=4). To examine the effects of mutant U2AF1(S34F) induction in vivo, we transplanted mutant U2AF1(S34F) or U2AF1(WT) transgenic bone marrow cells into congenic wild type recipient mice and induced transgene expression 6 weeks post-transplant using 2 mg/ml doxycycline in the drinking water for 5 days. Induction of U2AF1(S34F) expression in vivo results in reduced number of WBCs in the peripheral blood of recipient mice compared to mice with uninduced U2AF1(S34F) transgenic bone marrow (3.4k vs 5.6k, p=0.01, n=3). In addition, recipient mice with induced U2AF1(S34F) bone marrow had reduced number of bone marrow cells per femur when compared to uninduced U2AF1(S34F) recipient mice (3.9M vs 13.1M, p=0.04) and induced U2AF1(WT) recipient mice (3.9M vs 12.4M, p=0.03) (n=3). The number of neutrophils in peripheral blood (p<0.001), bone marrow (p=0.04), and spleen (p=0.04) of induced U2AF1(S34F) recipient mice were all significantly lower compared to uninduced U2AF1(S34F) mice (n=3). The total numbers of c-Kit+/lineage-/Sca+ hematopoietic progenitor cells were not affected in induced U2AF1(S34F) recipient mice compared to uninduced U2AF1(S34F) (p=0.75) or induced U2AF1(WT) recipient mice (p=0.46, n=3) after 5 days of treatment. Collectively, these results suggest that the U2AF1(S34F) mutation may contribute to abnormal hematopoiesis in vivo. Longer periods of doxycycline-induction in vivo are ongoing and will be presented. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 829-836 ◽  
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.

Loss of Ezh2 Promotes the Development of Mutant-RUNX1 Induced MDS

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 402-402
Author(s):  
Goro Sashida ◽  
Satomi Tanaka ◽  
Makiko Mochizuki-Kashio ◽  
Atsunori Saraya ◽  
Tomoya Muto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
The Self ◽  
Post Transplantation ◽  
Self Renewal ◽  
Myeloid Malignancies ◽  
Empty Vector ◽  
Marrow Cells

Abstract Abstract 402 Polycomb group proteins are transcriptional repressors that epigenetically regulate transcription via histone modifications. There are two major polycomb-complexes, the Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2). PRC2 contains SUZ12, EED, and EZH1/EZH2, and catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3), silencing target-genes. We have shown that the self-renewal of Ezh2-deficient HSCs is not compromised and H3K27me3 marks are not completely depleted in the absence of Ezh2, possibly as a result of Ezh1 complementation. EZH2 is generally thought to act as an oncogene in lymphoma and solid tumors by silencing tumor suppressor genes. Recently however, loss-of-function mutations of EZH2 have been found in myeloid malignancies such as AML, MDS and MPN, suggesting that EZH2 also functions as a tumor suppressor, although it remains unclear how EZH2 prevents the transformation of myeloid malignancies. RUNX1 is a critical transcription factor in the regulation of the self-renewal and differentiation of HSCs. RUNX1 mutations are frequently found in MDS, AML following MDS (MDS/AML) and de novo AML patients. One of the most frequent mutations, RUNX1S291fs, lacks the transactivation domain in C-terminus, but retains the RUNT DNA biding domain, resulting in a dominant negative phenotype. RUNX1S291fs-transduced bone marrow cells have been shown to generate MDS/AML in vivo. Given that RUNX1 and EZH2 mutations coexist in MDS and AML patients as reported recently, we generated a novel mouse model of MDS utilizing RUNX1S291fs retrovirus and Ezh2 conditional knockout mice in order to understand how EZH2 loss contributes to the pathogenesis of MDS upon genetic mutation of RUNX1. We first harvested CD34-Lin-Sca1+c-Kit+(LSK) HSCs from tamoxifen-inducible Cre-ERT;Ezh2wild/wild (EW) and Cre-ERT;Ezh2flox/flox (EF) mice (CD45.2) and transduced these cells with RUNX1S291fs retrovirus or an empty vector, which contains IRES-GFP. Then, we transplanted RUNX1S291fs-transduced Cre-ERT;Ezh2wild/wild (S291EW) or Cre-ERT;Ezh2flox/flox (S291EF) HSCs into lethally irradiated recipient mice (CD45.1) together with life saving dose 1×105 CD45.1 bone marrow cells. At 6 weeks post transplantation, we deleted Ezh2 via administration of tamoxifen, and observed disease progression until 12 months post transplantation. The empty vector transduced control mice with or without Ezh2 (EW and EF) did not develop myeloid malignancies. Two out of 16 S291EW mice died due to MDS progression, while 12 out of 16 and 1 out of 17 S291EF mice developed MDS and MDS/AML, respectively. S291EF mice showed significantly shorter median survival than S291EW mice (314 days versus undefined, p=0.037). In the peripheral blood, we observed significantly lower CD45.2+GFP+ chimerism in S291EF mice; however S291EF mice eventually showed macrocytic anemia and variable white blood cell counts accompanied with dysplastic features of MDS. Despite low CD45.2+GFP+ chimerism in peripheral blood, S291EF mice showed a higher chimerism of CD45.2+GFP+ cells in the bone marrow and had a significantly increased number of LSK and CD34-LSK cells compared to EW, EF, and S291EW mice, indicating that Ezh2 loss promoted HSCs/progenitors expansion, but impaired myeloid differentiation in the presence of RUNX1S291fs. We also saw enhanced apoptosis of CD71+Ter119+ erythroblasts in S291EF MDS mice, which may account for the anemia we observed. Since S291EF MDS bone marrow cells were transplantable in secondary experiments, we performed limiting-dilution assays to evaluate the frequency of MDS initiating cells and found that the frequency of MDS initiating cells was much higher in S291EF pre-MDS Lin-Mac1-Kit+ cells compared to S291EW pre-MDS Lin-Mac1-Kit+ cells. To understand this molecular mechanism, we performed gene expression analysis during MDS progression. S291EF MDS LSKs showed 1979 and 1875 dysregulated (>5-fold) genes, compared to EW LSK and S291EF pre-MDS LSK, respectively. We are now working to understand how these dysregulated genes are involved in the development of RUNX1S291fs-induced MDS after deletion of Ezh2. In summary, we have successfully recapitulated the clinical feature of MDS in mice reconstituted with Ezh2 null HSCs expressing a RUNX1 mutant, and demonstrated that Ezh2 functions as a tumor suppressor in this context. 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.

High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1758-1758
Author(s):  
Axel Schambach ◽  
Bernhard Schiedlmeier ◽  
Jens Bohne ◽  
Dorothee von Laer ◽  
Geoff Margison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

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.

Engraftment Capacity of Intratibialy Injected Multiple Myeloma Patient-Derived Bone Marrow-Cells In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3010-3010
Author(s):  
Julia Schüler ◽  
Dagmar Wider ◽  
Kerstin Klingner ◽  
Heinz-Herbert Fiebig ◽  
Monika Engelhardt
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Expression Pattern ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Nsg Mice ◽  
Cell Engraftment ◽  
Healthy Donors ◽  
Marrow Cells

Abstract Abstract 3010 Introduction: In order to allow a better understanding of multiple myeloma (MM), the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation advantageously. Here, the engraftment capacity of MM patient-derived bone marrow cells implanted into NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice was meticulously investigated. Material and Methods: Bone marrow cells from 7 MM patients were injected intratibialy into NSG mice (n=5/patient). As controls, 5 mice received healthy donor bone marrow and 5 mice were mock-injected. Tumor growth was monitored via a) daily MM-symptom acquisition, such as hind limb paresis, apathy and consistent foot dragging, b) FACS (human HLA-A,B,C; CD138; CD45; CD38) and c) fluorescence-based in vivo imaging (FI, Kodak FX, Alexa750 labeled anti-human CD138, CD38, CD45 and HLA-ABC) in bone marrow, peripheral blood, spleen and lymph node sites of the respective animals. Results: There were significant differences in engraftment capacity, persistence of human cells and expression of selected markers between bone marrow of MM patients and healthy donors: 1.) infiltration of the spleen and lymph nodes was exclusively detected in NSG-mice bearing patient-derived MM cells, whereas cells of healthy donors were - if detected - exclusively found within the murine bone marrow; 2.) mean FI-areas in the bone marrow of MM-patient-derived injected mice were significantly increased as compared to mice bearing bone marrow cells of healthy donors (p=0.006); 3.) patient-derived MM cells expressed CD138, CD38 and HLA-ABC. In contrast, bone marrow cells of healthy donors expressed exclusively CD45 and CD138. The CD138 cell population determined by FACS in patients' bone marrow cells (before NSG-injection) decreased from a median of 11.3% to 0.8% 56 days after implantation (in NSG mice), either due to preferably CD138-negative plasma cell engraftment or the CD138 loss within the murine environment as previously described. Fifty-six days after implantation, patient-derived MM cells could be detected in all animals via FACS-analysis. Follow-up analyses by FI confirmed, that bone marrow engraftment was prominent and observed in all (35/35) NSG mice, albeit also in others organs. Patient-derived MM cells within the bone marrow could be detected in parallel via FACS- and FI-analyses in 10 NSG mice and within the peripheral blood in 12 NSG mice (total of 35 mice being examined). Maximal bone marrow-, peripheral blood- and spleen-engraftment numbers in NSG mice were as high as 4%, 25% and 52%, respectively, suggesting that in peripheral blood- and spleen-sites, MM-cell engraftment could even surmount that of bone marrow-sites. Spleen and other organ involvement observed in our xenografts have been confirmed in previous murine MM-models (Murillo et al. Clin Cancer Res, 2008), postulating that similarly to spleen-colony-forming-cells in hematopoiesis, spleen and other sites serve as fertile tumor engraftment locations.Differences in engraftment capacity and expression pattern between respective patient-derived MM specimen were evident, but did not strikingly correlate with MM-patients' characteristics, such as MM-subtypes, disease stage or expression pattern of the primary material; this observation also well correlating with previous reports (e.g. (Pilarski et al. Blood, 2000). Conclusions: Murine MM-models have shown to be exceedingly challenging in their ability to induce valid and trustworthy MM-patient-derived cell engraftment; here our NSG model suggest to harbor MM-cells. Our data demonstrates that intratibially-injected NSG mice mimic the clinical MM disease with respect to the disseminated nature of the disease and the indispensable engraftment of clonogenic plasma cells into the bone marrow. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
In Utero ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Show Evidence ◽  
Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

Distribution of sister chromatid exchanges on the mouse chromosomes in vivo with reference to the replication properties of the X chromosome

1984 ◽  
Vol 26 (2) ◽  
pp. 152-157
Author(s):  
S. M. Singh ◽  
D. L. Reimer
Keyword(s):  
Bone Marrow ◽  
X Chromosome ◽  
Sister Chromatid ◽  
Bone Marrow Cells ◽  
Relative Length ◽  
Chromosome Length ◽  
Sister Chromatid Exchanges ◽  
Chromatid Exchanges ◽  
Marrow Cells

Frequency of sister chromatid exchanges (SCE) were recorded separately for different chromosomes from bone marrow cells of female mice of the two genetic strains (C3H/S and C57BL/6J). SCEs were evaluated following different doses of 5-bromo-2′deoxyuridine (BrdU) as nine hourly i.p. injections. The SCE per cell increased with increasing BrdU doses which was slightly higher in C3H/S than in the C57BL/6J. SCEs per cell were variable at every treatment – strain combination, possibly reflecting the heterogeneous nature of the bone marrow cells. In general, there is a positive correlation between SCE per chromosome and the relative chromosome length. Total SCEs on one of the large chromosomes (most likely the X chromosome), however, are significantly higher than expected on the basis of relative length alone. Most of this increase is attributable to one of the homologues of this chromosome, which is not in synchrony with the rest of the chromosomes and may represent the late-replicating X. These results when viewed in the light of replication properties of the heterochromatinized X, suggest a direct involvement of DNA replication in SCE formation and may argue against the replication point as the sole site for the SCEs.Key words: sister chromatid exchange, BrdU, recombination, replication, X chromosome.

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