Mouse Long-term Bone Marrow Cultures Research Articles

Evolution of malignant plasmacytoma cell lines from K14E7 Fancd2-/- mouse long-term bone marrow cultures.

We tested the effect of expression of the Human Papilloma Virus (HPV E7) oncogene on hematopoiesis in long-term bone marrow cultures (LTBMCs) derived from K14E7 (FVB) Fancd2−/− (129/Sv), K14E7 Fancd2+/+, Fancd2−/−, and control (FVB X 129/Sv) Fl mice. K14E7 Fancd2−/− and Fancd2−/− LTBMCs showed decreased duration of production of total nonadherent hematopoietic cells and progenitors forming day 7 and day 14 multilineage CFU-GEMM colonies in secondary cultures (7 wks and 8 wks respectively) compared to cultures from K14E7 Fancd2+/+ (17 wks) or control mice (18 wks) p < 0.0001. Marrow stromal cell lines derived from both K14E7 Fancd2−/− and Fancd2−/− cultures were radiosensitive, as were IL-3 dependent hematopoietic progenitor cell lines derived from K14E7 Fancd2−/− cultures. In contrast, Fancd2−/− mouse hematopoietic progenitor cell lines and fresh marrow were radioresistant. K14E7 Fancd2−/− mouse freshly explanted bone marrow expressed no detectable K14 or E7; however, LTBMCs produced K14 positive factor-independent (FI) clonal malignant plasmacytoma forming cell lines in which E7 was detected in the nucleus with p53 and Rb. Transfection of an E6/E7 plasmid into Fancd2−/−, but not control Fancd2+/+ IL-3 dependent hematopoietic progenitor cell lines, increased cloning efficiency, cell growth, and induced malignant cell lines. Therefore, the altered radiobiology of hematopoietic progenitor cells and malignant transformation in vitro by K14E7 expression in cells of the Fancd2−/− genotype suggests a potential role of HPV in hematopoietic malignancies in FA patients.

Abstract IA15: Amelioration of ionizing irradiation damage in Fanconi anemia (Fancd2-/- mice) by GS-nitroxide (JP4-039)

Abstract Fancd2-/- mice derived from both the C57BL/6 and FEV/B background demonstrate bone marrow stromal cell radiosensitivity and paradoxical hematopoietic cell radioresistance. Fancd2-/- (B6) mouse long-term bone marrow cultures showed reduced longevity of hematopoiesis consistent with decreased capacity to handle oxidative stress. FA patients demonstrate an increased risk for hematopoietic and epithelial cancers, which are difficult to treat with radiotherapy due to intrinsic normal tissue radiosensitivity. Intraoral administration of GS-nitroxide (JP4-039) in F15 emulsion prior to head and neck irradiation significantly reduces the increased susceptibility of Fancd2-/- mice to oral cavity ulceration. Baseline biomarkers for oxidative stress related proteins in oral cavity and bone marrow of Fancd2-/- mice compared to heterozygote and normal littermates (B6) and FVB/N were exacerbated by oral cavity irradiation. Suppression of femur marrow hematopoiesis by head and neck irradiation, was also increased in Fancd2-/- mice, but was ameliorated by JP4-039/F15 intraoral administration, as were gene transcripts, proteins, and DNA promoter demethylation. Amelioration of normal tissue radiosensitivity by organ specific radioprotective mitochondrial targeted antioxidants is effective in both DNA repair defective and wild type murine epithelial tissues. Citation Format: Joel S. Greenberger. Amelioration of ionizing irradiation damage in Fanconi anemia (Fancd2-/- mice) by GS-nitroxide (JP4-039). [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA15. doi:10.1158/1538-7445.CANSUSC14-IA15

Neuronal/Mitochondrial Nitric Oxide Synthase Homologous Deletion Recombinant Negative Mice (NOS1 −/−) Long-Term Bone Marrow Cultures (LTBMCs) Demonstrate Increased Longevity and Radioresistance of Derived Cell Lines.

Neuronal NOS (NOS1) is localized to mitochondria. Ionizing irradiation results in influx of calcium into mitochondria stimulating production of nitric oxide (NO), and also increases production of superoxide which reacts with NO to produce peroxynitrite. Peroxidation of mitochondrial lipids, release of cytochrome C and apoptosis is directly related to mitochondrial peroxynitrite. We hypothesized that reduction of mitochondrial NO production should provide radioresistance. In addition, since ROS production is associated with aging NOS1−/− mouse LTBMCs should demonstrate greater hematopoietic longevity. LTBMCs established from NOS1 −/− mice demonstrated increased cumulative adherent cobblestone islands (adherent stem cell containing islands), production of total nonadherent cells, and cumulative day 7 and day 14 CFU-GEMM hematopoietic multi-lineage colony forming cells (over 65 weeks) compared to NOS1 +/+ controls (22 weeks) (p < 0.0001). Seven and 14 day CFU-GEMM production in nonadherent cell harvests from NOS1 −/− LTBMCs continued for 65 weeks compared to 15 weeks for NOS1 +/+ LTBMCs (p < 0.0001). Marrow stromal cell lines derived from NOS1 −/− and NOS1 +/+ culture were irradiated to doses from 0 to 800 cGy, plated in 4 well tissue culture plates, stained with crystal violet 7 days later and colonies of greater than 50 cells were counted. NOS1 −/− stromal cell lines had an increased shoulder on the survival curve compared to the NOS1 +/+ cells (n = 32.15 ± 1.21 and 10.47 ± 3.20, respectively, p=0.0026). Cell cycle analysis of NOS1 −/− and NOS1 +/+ cell lines following 10 Gy irradiation demonstrated a G1 arrest 6 hr after irradiation, in both; however, by 24 hr, NOS1 +/+ but not NOS1−/− cells resumed normal cycling. To determine whether the radioresistance of NOS1−/− cells was attributable to expected higher levels of antioxidants, cells were analyzed for glutathione (GSH) and glutathione peroxidase (GPX). NOS1 −/− cells demonstrated increased GSH compared to NOS1 +/+ cells at 0, 30 min and 24 hr following irradiation (p < 0.0001) with no significant difference in GPX before or after irradiation. NOS1 −/− compared to NOS1+/+. IL-3 dependent hematopoietic cells from NOS1 −/− LTBMCs had significantly decreased apoptosis, 24 hrs following 10 Gy irradiation (5.3 ± 2.4 vs. 14.8 ± 3.3 %, respectively, p = 0.049). Therefore, reduction of NOS1 in bone marrow increases hematopoietic longevity in LTBMCs and radioresistance of derived cell lines.

Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin.

The hematopoietic stem cell (HSC) is defined as a cell that can either self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages. Self-replication of the most primitive HSC produces daughter cells that possess a long (possibly unlimited) clonal lifespan, whereas differentiation of HSC produces daughter cells that demonstrate a progressive reduction of their clonal lifespan, a loss of multilineage potential, and lineage commitment. Previous studies indicated that the proliferation of HSC ex vivo favors differentiation at the expense of self-replication, eventually resulting in a complete loss of HSC. In contrast, transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occurs in vivo. Thrombopoietin (TPO) has been shown to function both as a proliferative and differentiative factor for megakaryocytes and as a survival and weakly proliferative factor for HSC. Our studies focused on the effects of exogenous TPO on HSC in mouse long-term bone marrow cultures (LTBMC). Previous results indicate that HSC decline in LTBMC in the absence of TPO. In contrast, the continuous presence of TPO resulted in the generation of both long- and short-term repopulating HSC as detected by an in vivo competitive repopulation assay. HSC were generated over a 4-month period at concentrations similar to normal bone marrow. Our results demonstrate that TPO can mediate the self-replication of HSC in LTBMC, and provide proof that HSC can self-replicate ex vivo.

TNF-alpha inhibits the further development of committed progenitors while stimulating multipotential progenitors in mouse long-term bone marrow cultures.

Abstract We studied simultaneously the effect of TNF on both relatively primitive and more mature hematopoietic progenitors by using a modification of the mouse long-term bone marrow culture system of Dexter. Mycophenolic acid-treated long-term bone marrow cultures were charged with stromally depleted bone marrow cells along with 10 to 500 U/ml recombinant human TNF-alpha, and assayed each week for 5 wk for the presence of primitive (CFU-S and high proliferative potential colony-forming cell) and mature (CFU-C) colony-forming units. The results demonstrate that TNF-alpha has differential effects on hematopoiesis; it virtually eliminates CFU-C, significantly lowers high proliferative potential colony-forming cell (CFC) numbers and increases CFU-S numbers. This effect appears to be independent of CSF production in the cultures as shown by protein assays. Northern blot analyses showed no differences in expression of CSF genes or any expression of those encoding for IL-1, IL-3, or IL-6. These results, obtained in a long-term culture system that closely mimics in vivo bone marrow, demonstrate that many of the apparently contradictory effects of TNF observed in vitro occur concomitantly and are attributable to the presence of cells at various stages of maturity in the cultures. The data suggest a mechanism of this activity in which TNF is able to block the differentiation of primitive progenitors even as they are stimulated to proliferate.

A tumor necrosis factor-responsive long-term-culture-initiating cell is associated with the stromal layer of mouse long-term bone marrow cultures.

Long-term bone marrow cultures provide a model for the study of hematopoiesis. Both an intact, adherent stromal layer and hematopoietic stem cells are necessary components in these cultures. Mycophenolic acid treatment of mouse long-term bone marrow cultures depletes them of all assayable hematopoietic precursors. The residual stromal cells are functional and support hematopoiesis if new progenitor cells are supplied. We now show that these mycophenolic acid-treated stromal cell cultures contain cells capable of hematopoietic differentiation without the addition of new progenitors. When treated with tumor necrosis factor alpha (20-200 units/ml), the apparently pure stromal cultures undergo an intense burst of hematopoietic activity. After 4 days such cultures contain approximately 2 x 10(6) hematopoietic cells and, by 1 week, they are indistinguishable from control long-term cultures that were not treated with mycophenolic acid. These results suggest that the stromal cultures either contain hematopoietic stem cells that are maintained quiescent and mycophenolic acid-resistant, perhaps by intimate contact with the stroma, or contain adherent cells that can be induced to differentiate into hematopoietic stem cells. These stem cells are primitive, in that they are capable of multilineage development in the long-term cultures, but are unable to form spleen colonies or myeloid colonies in semisolid medium. These data demonstrate that the adherent fraction of cultured bone marrow contains very primitive hematopoietic cells and that tumor necrosis factor alpha activates their proliferation and differentiation. They also suggest a strategy for obtaining the earliest progenitors free of other, more mature cell types.

Friend virus-infected long-term bone marrow cultures produce colony stimulating factor dependent and independent granulocyte-macrophage progenitor cells for over four years in vitro

C3H/HeJ mouse long-term bone marrow cultures infected at initiation with a cloned polycythemic strain of Friend spleen focus forming virus in a cloned N-tropic murine leukemia virus helper virus coat, persistently produced: colony-forming unit spleen (CFUs) for 55 weeks that formed macroscopic spleen colonies in syngeneic or allogeneic C57B10.Br/J mice; and L-cell or WEHI-3 cell conditioned medium-dependent granulocyte-macrophage colony forming unit culture (GM-CFUc); and morphologically normal granulocytes for over 245 weeks. Colony stimulating factor (CSF)-independent colony forming progenitor cells were first detectably produced in vitro at 75 weeks, and when subcultured generated karyotypically distinct permanent factor-independent tumorigenic cell lines. Nonadherent cells removed from long-term marrow cultures at 19 but not at 77 weeks reconstituted donor origin hematopoiesis in C57B10.Br/J mice as measured by B-cell lineage surface immunoglobin allotype. Nonadherent cells removed at 77 weeks produced lethal splenomegaly and marrow infiltration with culture origin cells in C57B10.Br/J mice. Despite generation of clonal malignant cell lines, L-cell CSF (CSF-1, MCSF) responsive GM-CFUc that were simultaneously produced over 4 years in the same long-term marrow cultures, grew to 7 day colonies in semisolid medium and terminally differentiated. Thus, adherent stromal cells in Friend virus-infected long-term bone marrow cultures simultaneously support CSF-responsive and malignant CSF-independent hematopoietic progenitor cells.

Effects of bacterial lipopolysaccharide on the production of colony-stimulating activity in C3H/HeJ mouse long-term bone marrow cultures

Bacterial lipopolysaccharide (LPS)-induced colony-stimulating activity (CSA) in murine long-term bone marrow culture system was investigated. Bone marrow culture cells of LPS-nonresponsive C3H/HeJ mice responded to LPS in terms of CSA production as efficiently as bone marrow culture cells of LPS-responsive C3H/slc mice. On the other hand, both peritoneal macrophages and bone marrow macrophages from C3H/HeJ mice did not produce CSA in vitro after treatment with LPS. Percoll density gradient separation of adherent layer cells in bone marrow cultures showed that two cell populations were present. One population was nonspecific esterase positive, productive of high CSA to LPS stimulation and light density cells, the other population was nonspecific esterase negative, productive of low CSA to LPS stimulation and high density cells, and CSA production stimulated by LPS in C3H/HeJ mice bone marrow culture cells was mainly attributed to the latter population of cells. These results suggest that CSA production stimulated by LPS in C3H/HeJ mice is regulated by different cell populations, respectively in vivo and in vitro.

Viral expression in two myelomonocytic cell lines obtained from long-term bone marrow culture infected with the friend polycythemia-inducing virus (FV-P)

Two subclones of monomyelocytic cell lines (317 and G-31), issued from mouse long-term bone marrow cultures infected with Friend polycythemia-inducing virus, were studied for virus release and viral protein expression. The 317 cell line permanently produced biologically active spleen focus-forming virus (SFFV) and expressed the gp52 SFFV which has been described as encoded by the SFFV genome. In contrast, the G-31 cell line was devoid of any SFFV expression although it released Friend helper leukemia virus (FMuLV). Both lines expressed a new gp70-related glycoprotein with an apparent molecular weight of 66,000. This gp66 species was metabolically unstable and was not packaged into viral particles nor released in culture medium. It was recognized by an anti-Rauscher gp70 antiserum but not by anti-MCF gp70 or anti-p15E antisera. gp66 was not found in erythroblastic Friend tumor cell lines, in fibroblasts infected with virus released by lines 317 and G-31, or in nonmyelomonocytic infected hematopoietic cells. However, it was constantly observed in cell lines with a myelomonocytic origin established in vivo and in vitro. Therefore, gp66 is a F-MuLV gp70-related product specifically linked to the myeloid lineage.

Virus and Cell Requirements for Friend Virus Granulocytic Leukemogenesis in Long-Term Bone Marrow Cultures of NIH Swiss [N:NIH(S)] Mice&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

The effect of hematopoietic stem cell age on leukemogenesis in vitro was tested in nonrecharged, corticosterold-supplemented NIH Swiss [N:NIH(S)] mouse long-term bone marrow cultures infected with Friend murine leukemia virus of anemia-inducing strain (F-MuLV-A) or spleen focus-forming virus (SFFV) [Rauscher murine leukemia virus (R-MuLV)], a pseudotype virus derived by rescue of the SFFV genome from SFFV-Balb/3T3 clone A31 nonproducer cells with clonal helper R-MuLV. Cultures at 33 degrees C derived from 10-day-old or adult mouse marrow generated colony-forming unit culture granulocytic macrophage (CFUc) progenitor cells for over 20 weeks and colony-forming unit spleen cells for 14 weeks and generated permanent granulocytic leukemia cell lines after infection with F-MuLV-A at week 1, 2, or 4 but not at week 8. Leukemia lines were of granulocyte phenotype whether induced by F-MuLV-A or SFFV (R-MuLV) and synthesized myeloperoxidase and lysozyme but were restricted in ability to generate superoxide in response to phorbol myristate acetate stimulation. Cultures (31 degrees C) infected with temperature-sensitive (ts) helper virus mutant pseudotypes of SFFV as well as SFFV (R-MuLV) generated granulocytic leukemia lines, whereas only SFFV (R-MuLV) pseudotype virus-infected cultures became leukemic at 37 degrees C. R-MuLV wild type or ts mutant helper virus infection alone increased cell proliferation and numbers of CFUc but did not generate leukemia. These data indicated that gene(s) specific to F-MuLV-A or a virus rescued from SFFV-Balb/3T3 clone A31 nonproducer cells are required for transformation in vitro of a hematopoietic stem cell present in early but absent in late bone marrow cultures.