Bone Marrow Of Irradiated Mice Research Articles

The miRNA Content of Bone Marrow-Derived Extracellular Vesicles Contributes to Protein Pathway Alterations Involved in Ionising Radiation-Induced Bystander Responses.

Extracellular vesicles (EVs), through their cargo, are important mediators of bystander responses in the irradiated bone marrow (BM). MiRNAs carried by EVs can potentially alter cellular pathways in EV-recipient cells by regulating their protein content. Using the CBA/Ca mouse model, we characterised the miRNA content of BM-derived EVs from mice irradiated with 0.1 Gy or 3 Gy using an nCounter analysis system. We also analysed proteomic changes in BM cells either directly irradiated or treated with EVs derived from the BM of irradiated mice. Our aim was to identify key cellular processes in the EV-acceptor cells regulated by miRNAs. The irradiation of BM cells with 0.1 Gy led to protein alterations involved in oxidative stress and immune and inflammatory processes. Oxidative stress-related pathways were also present in BM cells treated with EVs isolated from 0.1 Gy-irradiated mice, indicating the propagation of oxidative stress in a bystander manner. The irradiation of BM cells with 3 Gy led to protein pathway alterations involved in the DNA damage response, metabolism, cell death and immune and inflammatory processes. The majority of these pathways were also altered in BM cells treated with EVs from mice irradiated with 3 Gy. Certain pathways (cell cycle, acute and chronic myeloid leukaemia) regulated by miRNAs differentially expressed in EVs isolated from mice irradiated with 3 Gy overlapped with protein pathway alterations in BM cells treated with 3 Gy EVs. Six miRNAs were involved in these common pathways interacting with 11 proteins, suggesting the involvement of miRNAs in the EV-mediated bystander processes. In conclusion, we characterised proteomic changes in directly irradiated and EV-treated BM cells, identified processes transmitted in a bystander manner and suggested miRNA and protein candidates potentially involved in the regulation of these bystander processes.

RNA Profiling Reveals a Common Mechanism of Histone Gene Downregulation and Complementary Effects for Radioprotectants in Response to Ionizing Radiation.

High-dose ionizing radiation (IR) alters the expression levels of non-coding RNAs (ncRNAs). However, the roles of ncRNAs and mRNAs in mediating radiation protection by radioprotectants remain unknown. Microarrays were used to determine microRNA (miRNA), long ncRNA (lncRNA), and mRNA expression profiles in the bone marrow of irradiated mice pretreated with amifostine, CBLB502, and nilestriol. Differentially expressed mRNAs were functionally annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Some histone cluster genes were validated by real-time PCR, and the effects of radioprotectant combinations were monitored by survival analysis. We found that these radioprotectants increased the induction of lncRNAs and mRNAs. miRNA, lncRNA, and mRNA expression patterns were similar with amifostine and CBLB502, but not nilestriol. The radioprotectants exhibited mostly opposite effects against IR-induced miRNAs, lncRNAs, and mRNAs while inducing a common histone gene downregulation following IR, mainly via nucleosome assembly and related signaling pathways. Notably, the effects of nilestriol significantly complemented those of amisfostine or CBLB502; low-dose drug combinations resulted in better radioprotective effects in pretreated mice. Thus, we present histone gene downregulation by radioprotectants, together with the biological functions of miRNA, lncRNA, and mRNA, to explain the mechanism underlying radioprotection.

Changes in the Activity of Genes Involved in the Regulation of Hematopoiesis during Tumorigenesis in Irradiated Mice.

We used 183 F1 CBA×C57Bl hybrid mice to study the delayed effects of low-power long-term γ-irradiation at a dose of 12.6 Gy (10 mGy/min) 8 and 10 months after the treatment. Eight months after the treatment we found the increased expression of the transcription factor NFκB and its target genes iNOS and G-SCF in the bone marrow (BM). Ten months after the treatment malignant lymphomas were revealed in 14 of 94 mice in the liver, abdominal cavity, and subcutaneously. In the BM of these mice, the transcription of the PTEN, NFκB, and iNOS genes was inhibited and the contents of long non-coding RNAs (lncRNAs) lnc p21, NEAT1, and microRNA miR-125b were decreased. The expression of the NFκB(p65) gene and miR-125b was inhibited in the BM of irradiated mice without tumors ten months after the treatment. These data show the deregulation of the P53 system supporting the genome stability in the BM of irradiated mice. These indices will be studied as potential markers of risk of development of irradiation-induced tumors.

Antioxidant and genoprotective properties of the antidiabetic drug metformin under X-ray irradiation

The antioxidant and genoprotective properties of the antidiabetic drug metformin have been investigated. The research was performed both in vitro and in vivo under X-ray irradiation. It was shown that metformin reduced the formation of hydroxyl radicals and hydrogen peroxide in aqueous solutions irradiated with X-rays. The particular features of its influence on the formation of H2O2 were revealed. It has been established that metformin reduced the formation of 8-oxoguanine, a key biomarker of oxidative damage to DNA, in vitro. The effect of metformin on the frequency of micronucleus formation in polychromatic erythrocytes of bone marrow of irradiated mice was investigated. Its genoprotective and radiomimitatory properties at different concentrations and times of application are shown.

The early response cells to bone marrow injury

We have previous studied the bone marrow response in submyeloablatively (6 Gy) irradiated mice. This allowed a detailed analysis of immature Lin-c-Kit+ (LK) cells only after approximately two weeks. These studies revealed significant expansion of Sca-1+ (S+) LK CD150+CD48+ cells with decreased c Kit expression level (Blood 2014 124:5112). Furthermore, they showed that not only LS+K cells responded to the injury but also Sca 1- (S-) late myeloid progenitors were activated and some of them were induced to re-expressed Sca 1 antigen. In present study, we focused on the response of LK cells occurring shortly after bone marrow damage by analyzing bone marrow 1-5 days after irradiation of mice with 2-4 Gy. The number of bone marrow cells significantly decreased after 2-4 Gy irradiation during one day. In LK cells, the LS-K/LS+K ratio decreased (below 2) due to Sca-1 induction. However, it recovered to normal level in a range of 5-7 after 3-5 days. We used CD150 and CD48 SLAM markers to distinguish between various subtypes of LSK cells, and CD34 and CD16/32 markers to characterize various types of LS-K myeloid progenitor cells. This revealed an early activation of megakaryocyte-erythroid and granulocyte macrophage myeloid progenitors. The activation was characterized by re-expression of Sca 1 in a part of these cells and by significantly decreased c-Kit expression level. During 3-5 days, c Kit recovered fully in 2 Gy- and partly in 4 Gy-irradiated mice. The transcription factor SCL/TAL1 with other co-factors is known to upregulate c-Kit gene and c-Kit expression. Therefore, we examined SCL/TAL1 mRNA level in LK cells in bone marrow of irradiated mice. Necas E, Szikszai Forgacova K, Faltusova K, et al: Lin-Sca-1+c-KitlowCD48+CD71+ Cells Are the Engine of Bone Marrow Regeneration. Blood 2014 124:5112 (abstract). Research was supported by the Grant Agency of the Czech Republic (GACR 17-01897S).

Neuropeptide Y Is Critical for Bone Marrow Stromal Cells and Hematopoietic Recovery Following Injury

Bone marrow suppression is the most common limiting side-effect of conventional cancer chemo/radio therapy and is the primary cause of morbidity/mortality after accidental exposure to a high dose of ionizing radiation. The mechanisms mediating radiation-induced hematopoietic stem and stromal cell dysfunction however are not well understood. Radiation therapy causes substantial sensory neuropathy in patients. Recent studies reveal that bone marrow cells are highly innervated by sympathetic nerve fibers and that chemotherapy induced nerve-damage can impair hematopoietic regeneration, suggesting a contribution of nerve fibers in the regulation of hematopoietic stem cell and stromal cell activities. Whether irradiation- mediated nerve injury is a crucial lesion that causes deficits in hematopoietic recovery is not known. We recently discovered that differential signaling from the neuropeptide Y (NPY) receptors on bone marrow endothelial cells regulates vascular permeability and stem cell egress. NPY is an important neurotransmitter of the sympathetic nervous system and the principal adreno-medullary hormone. In this study, we found that NPY is important for reconstitution of the bone marrow niche and hematopoietic regeneration following sublethal irradiation (650 cGy). The levels of NPY were significantly reduced in bone marrow of irradiated mice suggesting damage to nerve fibers. Treatment of wild-type mice with full length NPY (1µg/mouse/day) for 3 consecutive days after irradiation markedly reduced the loss of mesenchymal stem cells (CD45-Ter119-CD31-Nestin+PDGFR+CD51+), endothelial cells (CD45-Ter119-CD31+VE-cadherin+) and hematopoietic stem and progenitor cells (SLAM LSK and LSK) in the bone marrow and promoted faster hematopoietic recovery. In addition, pharmacological NPY treatment prevented irradiation mediated nerve fiber damage. In contrast, in NPY knockout mice, regeneration of CD45neg stromal cells, SLAM LSK and LSK cells after irradiation was significantly reduced compared to wild-type controls. This reduced hematopoietic recovery in NPY deficient mice following irradiation was associated with increased apoptosis/necrosis of stromal cells and hematopoietic stem and progenitor cells. We also examined whether NPY played an intrinsic or extrinsic role in stem cell homing. Wild-type or NPY deficient BM cells were transplanted into wild-type or NPY knockout recipients. Strikingly, the homing of wild-type donor cells into NPY deficient recipients or NPY knockout donor cells into wild-type recipients were both reduced. To explore whether NPY regulates human stem cells, we treated human cord blood CD34+ cells ex vivo with NPY for 3 days and evaluated cell expansion. Long-term culture assays demonstrated that NPY treatment enhanced the clonal expansion of CD34+ cells. In conclusion, our studies suggests that NPY plays both an intrinsic and extrinsic role in hematopoiesis and that NPY-mediated protection of the sympathetic nervous system within the bone marrow can facilitate stem cell niche regeneration and enhance regenerative hematopoiesis following irradiation/injury. DisclosuresNo relevant conflicts of interest to declare.

Gradual Rarefaction of Hematopoietic Precursors and Atrophy in a Depleted microRNA 29a, b and c Environment.

BackgroundThe self-renewing ability of HSCs is fundamental for the maintenance of a pool of bone marrow precursors throughout the life of an individual. The genetic mechanisms underlying such a complex process are still poorly understood.Results and SignificanceHere, we show that constitutive in vivo deletion of miR29ab1 leads to reduced number of HSCs and that miR29ab1 deficient bone marrow cannot repopulate the bone marrow of irradiated mice. An Affymetrix analysis of the miR29ab1 knockout mice identifies key proteins that could be responsible for this phenotype, as DNMT3a and b. Moreover, our findings reveal that whereas miR29b2c knockout mice do not exhibit any spontaneous abnormality, the double knock out – miR29ab1b2c – has marked generalized atrophy, raising the possibility that the two bi-cistrons might cooperate in order to maintain the stem cell number in general, not only limited to the bone marrow.

Uterine-Derived Stem Cells Reconstitute the Bone Marrow of Irradiated Mice

Hematopoietic stem cells (HSCs) can be found in several tissues of mesodermal origin. Uterine tissue contains stem cells and can regenerate during each menstrual cycle with robust new tissue formation. Stem cells may play a role in this regenerative potential. Here, we report that transplantation of cells isolated from murine uterine tissue can rescue lethally irradiated mice and reconstitute the major hematopoietic lineages. Donor cells can be detected in the blood and hematopoietic tissues such as spleen and bone marrow (BM) of recipient mice. Uterine tissue contains a significant percentage of cells that are Sca-1(+), Thy 1.2(+), or CD45(+) cells, and uterine cells (UCs) were able to give rise to hematopoietic colonies in methylcellulose. Using secondary reconstitution, a key test for hematopoietic potential, we found that the UCs exhibited HSC-like reconstitution of BM and formation of splenic nodules. In a sensitive assay for cell fusion, we used a mixture of cells from Cre and loxP mice for reconstitution and demonstrated that hematopoietic reconstitution by UCs is not a function of fusion with donor BM cells. We also showed that the hematopoietic potential of the uterine tissue was not a result of BM stem cells residing in the uterine tissue. In conclusion, our data provide novel evidence that cells isolated from mesodermal tissues such as the uterus can engraft into the hematopoietic system of irradiated recipients and give rise to multiple hematopoietic lineages. Thus, uterine tissue could be considered an important source of stem cells able to support hematopoiesis.

Induced Hematopoietic Differentiation Of Common Marmoset Embryonic Stem Cells By LYL1 Can Give Rise To Hematopoietic Stem Cells Having The Enhanced Bone Marrow Reconstitution Capability

Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host's hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1.We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro.The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point.Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures:No relevant conflicts of interest to declare.

Long-lived Inflammatory Signaling in Irradiated Bone Marrow Is Genome Dependent

Ionizing radiation is carcinogenic, but genotype is a key determinant of susceptibility. Mutational DNA damage is generally attributed to cause disease, but irradiation also affects multicellular interactions as a result of poorly understood bystander effects that may influence carcinogenic susceptibility. In this study, we show that the bone marrow of irradiated mice will retain the ability to kill hemopoietic clonogenic stem cells and to induce chromosomal instability for up to 3 months after irradiation. Chromosomal instability was induced in bone marrow cells derived from CBA/Ca mice, a strain that is susceptible to radiation-induced acute myeloid leukemia (r-AML), but not in C57BL6 mice that are resistant to r-AML. Similarly, clonogenic cell lethality was exhibited in C57BL/6 mice but not CBA/Ca mice. Mechanistic investigations revealed that these genotype-dependent effects involved cytokine-mediated signaling and were mediated by a cyclooxygenase-2-dependent mechanism. Thus, our results suggested that inflammatory processes were responsible for mediating and sustaining the durable effects of ionizing radiation observed on bone marrow cells. Because most exposures to ionizing radiation are directed to only part of the body, our findings imply that genotype-directed tissue responses may be important determinants of understanding the specific consequence of radiation exposure in different individuals.

-tocotrienol protects mouse and human hematopoietic progenitors from -irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling

Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments. CD2F1 mice and human hematopoietic progenitor CD34(+) cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay. δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage(-)/Sca-1(+)/ckit(+) stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34(+) cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34(+) cells. Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34(+) cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.

Vascular Homeostasis Regulators, Edn1 and Agpt2, are Upregulated as a Protective Effect of Heat-treated Zinc Yeast in Irradiated Murine Bone Marrow

To elucidate the mechanism underlying the in vivo radioprotection activity by Zn-containing, heat-treated Saccharomyces cerevisiae yeast (Zn-yeast). Zn-yeast suspension was administered into C3H/He mice immediately after whole body irradiation (WBI) at 7.5 Gy. Bone marrow was extracted from the mice 6 hours after irradiation and analyzed on a microarray. Expression changes in the candidate responsive genes differentially expressed in treated mice were re-examined by qRT-PCR. The bone marrow was also examined pathologically at 6 h, 3, 7, and 14 days postirradiation. Thirty-six genes, including Edn1 and Agpt2, were identified as candidate responsive genes in irradiated mouse bone marrow treated with Zn-yeast by showing a greater than three-fold change compared with control (no irradiation and no Zn-yeast) mice. The expressions of Cdkn1a, Bax, and Ccng, which are well known as radioresponsive genes, were upregulated in WBI mice and Zn-yeast treated WBI mice. Pathological examination showed the newly formed microvessels lined with endothelial cells, and small round hematopoietic cells around vessels in bone marrow matrix of mice administered with Zn-yeast after WBI, while whole-body irradiated mice developed fatty bone marrow within 2 weeks after irradiation. This study identified a possible mechanism for the postirradiation protection conferred by Zn-yeast. The protective effect of Zn-yeast against WBI is related to maintaining the bone marrow microenvironment, including targeting endothelial cells and cytokine release.

Differentiation of reprogrammed somatic cells into functional hematopoietic cells

Recent advances have demonstrated that the differentiated somatic cells could be reprogrammed into pluripotent state. Consequently, the reprogrammed somatic cells recapitulate the capacity to differentiate into specific cell lineages under appropriate culture conditions, which provides unlimited cell sources for cell transplantation-based therapy. In the present study, testicular Sertoli cells were successfully reprogrammed into pluripotent stem cells through somatic cell nuclear transfer (SCNT). Hematopoietic differentiation potential of the reprogrammed somatic cells was investigated in parallel to fertilization-derived ES (F-ES) cells. Our results demonstrated that the reprogrammed Sertoli cells (NT-ES) could efficiently differentiate into hematopoietic embryoid bodies (EBs). The hematopoietic-related genes including FLK-1, Bmp4, Runx1, etc. were dynamically expressed during the differentiation of the reprogrammed somatic cells in vitro. Transplantation of these differentiated reprogrammed cells into the bone marrow of irradiated mice could allow differentiation into different functional hematopoietic lineages in vivo. Moreover, blast-colony-forming cells (BL-CFCs) could be generated from both NT-ES and F-ES cells with similar efficiency in vitro. Our study indicates that the reprogrammed somatic cells possess the equivalent potency as F-ES cells in differentiating into functional hematopoietic cells.

Protective effect of Bixa orellana L. against radiation induced chromosomal aberration in Swiss Albino Mice.

Radioprotective effect of hydroalcoholic extract of seeds of Bixa orellana have been studied by examining chromosome aberration in cells of bone marrow in irradiated mice. Healthy adult Swiss mice were injected intraperitoneally (ip) with 500 mg kg–1 body weight, 1000 mg kg–1 of or double distilled water (DDW). They were exposed to whole body irradiation of 2.0 Gy gamma radiation 30 min later. After 24 h, chromosomal aberrations were studied in the bone marrow of the femur by routine metaphase preparation after colchicine treatment. Radiation (4.0 Gy) increased the number of aberrant cells from less than 1% in controls to almost 20%. Pre-treatment with the extract compounds resulted in a significant reduction in the percentage of aberrant metaphases as well as in the different types of aberration scored. The extract was not toxic at 1500 mg kg–1 body weight. Being non toxic and easily available natural source Bixa orellana extract may be use for as radioprotective for human beings. Keywords: – radioprotective, Bixa orellana

Recombinant PDGF-BB Enhances Platelet Recovery in Mice with Radiation Induced Thromobocytopenia.

Platelet-derived growth factor (PDGF) involves in the regulation of hematopoietic stem cells. Imatinib mesylate (Gleevec), a PDGF receptor inhibitor, induces thrombocytopenia in 50% of patients with chronic myeloid leukaemia (CML). Our previous studies showed that PDGF enhances megakaryocytopoiesis in vitro via its receptors directly. However, the in vivo effect of recombinant PDGF in thrombocytopenic models has not been reported. In this study, we investigated the in vivo effect of PDGF as well as thrombopoietin (TPO) on hematopoietic stem cells and platelet production in a radiation treated mouse model. We also explored its potential molecular mechanisms on thrombopoiesis. A radiation induced myelosuppression with thrombocytopenic model was established using 4-Gy-irradiated mice. PDGF-BB (1 mg/kg/day) and TPO (1 mg/kg/day) were injected separately after radiotherapy into the mice. Peripheral blood platelets, WBC and RBC from PDGF-BB, TPO and control groups were collected and analyzed on day 0, 7, 14 and 21 respectively. The mice were sacrificed on day 21 and their bone marrows were harvested for CFU assays and histology analysis. PDGF-BB, like TPO, showed a significant promoting effect in platelet production (n=5 p= 0.0078) as well as CFU-MK formation (n=5 p=0.0019) compared with the untreated control group. Histology images also indicated that PDGF-BB increased the number of the megakaryocytic cells and its progenitors in bone marrow of irradiated mice. In order to find out the underlying mechanism, we further studied the in vitro effect of PDGF using megakaryocytic cell line M-07e. Our results demonstrated that PDGF alone (100–200ng/ml) significantly activated the PI-3K/AKT signaling pathway (n=9 p=0.014). PDGF plus PI-3K inhibitor (wortmannin, 100nM) was shown to attenuate the expression of AKT (n=9, p=0.0112). Consistently, imatinib mesylate (Gleevec, 1uM) negatively interfered the expression of AKT when combined with PDGF-BB ((n=4, p= 0.0288). PDGF-BB was found to have a similar anti-apoptotic effect as TPO on M07e cells as showed by Annexin V (n=8, p=0.0281), Mitochondrial Membrane Potential (JC-1) (n=7, p=0.0042), and Caspase-3 (n=5, p=0.0080) assays respectively. In summary, our findings showed that PDGF-BB had an in vivo thromobopoietic effect on the radiation induced myelosuppressive mice models. This effect was likely mediated via PDGF receptor with subsequent activation of PI-3K/AKT pathway, which leads to anti-apoptotic effect on megakaryocytes.

Resveratrol Reduces Radiation-Induced Chromosome Aberration Frequencies in Mouse Bone Marrow Cells

Resveratrol, a polyphenol compound with reported antioxidant and anticarcinogenic effects, a wide range of molecular targets, and toxicity only at extreme doses, has received considerable attention. We evaluated the radioprotective effect of orally administered resveratrol on the frequencies of chromosome aberrations in irradiated mouse bone marrow cells. CBA/CaJ mice were divided into four groups: (1) no treatment, (2) resveratrol only, (3) radiation only, and (4) resveratrol and radiation. Resveratrol treatment (100 mg/kg daily) was initiated 2 days prior to irradiation. Bone marrow was then harvested at 1 and 30 days after a single dose of 3 Gy whole-body gamma radiation. A statistically significant (P < 0.05) reduction in the mean total chromosome aberration frequency per metaphase at both times postirradiation in the resveratrol and radiation group compared to the radiation-only group was observed. This study is the first to demonstrate that resveratrol has radioprotective effects in vivo. These results support the use of resveratrol as a radioprotector with the potential for widespread application.

Bone-marrow-derived cell differentiation into microglia: A study in a progressive mouse model of Parkinson's disease

The migration of peripheral bone-marrow-derived cells (BMDCs) to the brain was studied in a chronic mouse model of Parkinson's disease (PD). BMDCs expressing the enhanced green fluorescent protein (GFP) were aseptically obtained from C57 BL/6-EGFP-Tg mice and intravenously injected into C57 BL/6j mice which had received a total body irradiation of 8 Gy to induce bone marrow ablation. Implanted GFP-BMDCs replenished the bone marrow of irradiated mice, and progressively crossed the blood-brain barrier (BBB), penetrating different mesencephalic and telencephalic brain regions in the following months. The progressive degeneration of dopamine (DA) cells with a small daily dose (4 mg/kg/day for 20 days) of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) increased the penetration of GFP-BMDCs into the brain, particularly into those regions with marked DA innervation and which showed the clearest DA cell loss. BMDC penetration increased before the DA cell loss was evident and persisted for a long time after MPTP withdrawal. Under these conditions, most BMDCs differentiated into microglia (CD68 expression was observed in 50% of GFP cells 60 days after MPTP administration). BMDC-derived microglia showed morphological characteristics of cell activation, with the glial cell line-derived neurotrophic factor only being expressed in 3% of the cells. No differentiation into neurons (NeuN expression), astrocites (GFAP), cytotoxic lymphocytes (CD8) and T-helper lymphocytes (CD4) was observed. Taken together, the present data suggest that a significant portion of microglial cells is of a peripheral origin. Bearing in mind that microglial reaction is a significant part of the degenerative process in PD, the increase of BMDC penetration into DA-rich areas during DA cell degeneration and their differentiation into microglia suggest that cells coming across the BBB may participate in the neurodegeneration process. The precise role of such a cell inflow into the brain requires further study. Nevertheless, this may represent an opportunity to develop neuroprotective therapeutic strategies for PD.

Influence of glutathione on the induction of chromosome aberrations, delay in cell cycle kinetics and cell cycle regulator proteins in irradiated mouse bone marrow cells

Purpose: Reduced glutathione (GSH) is an endogenous thiol and has long been thought to affect the sensitivity of cells to radiation. The aim was to see the influence of GSH on: (i) the production of all types of radiation-induced chromosome aberrations (CA), and (ii) the radiation-induced delay in cell cycle and the levels of cell cycle regulator proteins.Materials and Methods: Cell cycle kinetics were determined by scoring the mitotic index (MI). CA and MI were scored in γ-irradiated buthionine sulfoximine (BSO) (10 h) or GSH (1 h) pretreated and untreated mouse bone marrow cells (BMC). The expression of p53 and p21 proteins after 2 and 6 h of irradiation and for the B-cell lymphoma 2 (Bcl-2) associated X-protein (Bax) after 24 h of irradiation with or without BSO or GSH treatment was analyzed by immunoblotting.Results: Radiation delays mouse BMC in their passage through the cell cycle and induces CA. Exogenous addition of GSH protected CA uniformly at lower doses of radiation but differentially at higher doses, whereas GSH-depletion by BSO increased the frequency of radiation-induced CA. Both GSH and BSO-pretreated cells reduced the delay in cell kinetics after irradiation. Levels of both p53 and p21 were enhanced after irradiation to BSO-pretreated cells. However, in GSH-pretreated cells the level of these proteins was reduced.Conclusion: Data indicate that the induction of CA and delay in cell kinetics by radiation may not always be interlinked and that the level of endogenous GSH exerts its influence on these parameters. Both GSH and BSO pretreatment reduce delays in cell kinetics of irradiated cells which may die apoptotically, since they have either a higher frequency of exchange aberrations or CA, respectively.

A novel technique of spermatogonial stem cell isolation

Objective: Adult stem cells offer the possibility of tissue regeneration following injury. However, for most stem cells, markers are not available for cell identification and purification. Accordingly, alternative approaches for stem cell identification have been developed. Hematopoetic stem cells can be enriched by exploiting their ability to home to the bone marrow of irradiated mice. In this study, we describe the development of a similar technique to isolate and enrich a population of adult spermatogonial stem cells for germ cell transplantation. Our ultimate goal is to develop a novel technique for the purification of adult spermatogonial stem cells to be used for transplantation as a therapy for primary or secondary male infertility

Accelerated recovery from irradiation injury by angiotensin peptides.

Angiotensin peptides have been shown to affect the proliferation and chemotaxis of multiple cell types. More recent studies in this laboratory have shown that angiotensin II (AII) can increase colony formation and proliferation by hematopoietic progenitors and mesenchymal cells in vitro. As white blood cell (WBC) recovery after bone marrow injury requires progenitor proliferation, the effect of AII and angiotensin (1-7) [A(1-7)], a non-hypertensive fragment of AII, on recovery from total body irradiation was evaluated in C57Bl/6 mice. The effect of angiotensin peptides on hematopoietic recovery and the number of progenitors in the bone marrow of irradiated C57Bl/6 mice was evaluated. Treatment of animals with angiotensin peptides accelerated hematopoietic recovery and increased the number of hematopoietic progenitors in bone marrow and in the blood. The increase in WBC concentration continued for a longer time after cessation of AII therapy than after treatment with filgrastim. Specifically, the number of WBCs continued to increase 21 days after irradiation with 7 days of angiotensin peptide administration. In contrast, the number of WBCs increased through day 13 with 7 days of filgrastim administration. On day 35 after irradiation (28 days after the last treatment), AII was shown to have increased the number of CFU-GM in the bone marrow of irradiated mice, whereas filgrastim administration had not. Angiotensin peptides also reduced the drop in platelet concentration after irradiation and increased the number of megakaryocyte precursors and megakaryocytes in the bone marrow. Receptor blocking studies indicated that losartan, an antagonist of the angiotensin type 1 receptor, blocked recovery of WBC levels in response to treatment with AII. In contrast, the increase in WBC levels in response to treatment with A(1-7), a ligand for other angiotensin receptors, was not affected by losartan. These findings suggest that these peptides utilize distinct receptors in the stimulation of hematopoietic recovery. In summary, systemic administration of angiotensin peptides led to an acceleration in hematopoietic recovery after irradiation. These peptides act to stimulate the formation of bone marrow progenitors, thereby facilitating recovery after myelosuppressive irradiation.