The use of metformin to enhance radiation effects on M1 macrophage subtype polarization in bone marrow-derived macrophage in glioblastoma tumor environment.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13513-e13513
Author(s):  
Fei Wang ◽  
Nan Zhao ◽  
Chi Lin ◽  
Chi Zhang
Keyword(s):  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Radiation Effects ◽  
Culture Conditions ◽  
Mouse Bone Marrow ◽  
M1 Macrophage ◽  
Tumor Environment ◽  
In Trans ◽  
High Concentrations

e13513 Background: Given the clinical relevance of tumor-associated macrophage (TAM) with its pro-tumor role and as a cell type compromising a large portion in glioblastoma (GBM), reversing the imbalance of TAM polarization in the tumor environment has emerged as a promising novel field for GBM treatment. Radiation therapy (RT) is the standard treatment for GBM patients after surgery, which has been shown to transiently induce M1 polarization of macrophage (M1Ø). Recent studies suggested that metformin could also promote M1Ø in tumor microenvironment. We thus postulate that metformin may enhance and sustain the M1-inducing effect of radiation in GBM. Methods: We first examined the polarization effect of metformin (0.1mM, 1mM and 2mM) on mouse bone marrow-derived macrophage (BMDM) cultured in GBM tumor environment, including media conditioned by GBM cells in monolayer culture or tumor spheres as well as in trans-well co-culturing system. We irradiated GBM cells with different doses (2 Gy, 8 Gy, and 20 Gy) after the treatment of Metformin at various time points; then we used conditioned media to treat BMDM either cultured alone or co-cultured with GBM cells in trans-well system for 24 or 48 hours. A separate set of experiment was conducted by first irradiating GBM cells and then co-culturing them with BMDM at 24 or 48 hours after radiation with metformin added at the start of co-culture. Percentage of various subtypes of BMDM was calculated after flow cytometry. Results: High concentrations of metformin (1mM and 2mM) significantly increased M1Ø and inhibited M2Ø in all culture conditions. Co-culture with irradiated GBM cells or treatment with medium conditioned by irradiated GBM cells could temporally induce M1Ø polarization in BMDM, with the effects being RT dose-dependent. Metformin at high concentrations further promoted M1Ø and suppressed M2Ø polarization in those conditions mimicking tumor microenvironment. This enhancing effect was sustained for at least 48 hours. Conclusions: Metformin at mili-molar concentrations significantly enhances the effects of radiation on M1Ø polarization in BMDM in vitro.

Rapid and Irreversible Alteration of the Ability of Hematopoietic Stem Cells To Execute Both Symmetric and Asymmetric Self-Renewal Divisions by Exposure to Reduced Steel Factor Concentrations with No Effect on Their Survival or Mitogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 684-684
Author(s):  
David G. Kent ◽  
Brad Dykstra ◽  
Connie J. Eaves
Keyword(s):  
Bone Marrow ◽  
Adult Mouse ◽  
Single Cells ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
High Concentrations

Abstract Hematopoietic stem cells (HSCs) are present in the marrow of adult mice at a frequency of 1/104, as measured by limiting dilution transplantation assays for individual cells that produce lymphoid (B and T) as well as myeloid (GM) cells for at least 4 months in irradiated recipients. HSCs thus defined can be reproducibly isolated in the CD45midlin−Rho−SP fraction of adult mouse bone marrow at a purity of >30%. In mice, mutations in c-kit, the receptor for Steel factor (SF) lead to substantial reductions in the adult HSC population. In vitro, SF has been identified as a potent regulator of HSC self-renewal divisions. High concentrations of SF in combination with IL-11 allow adult HSCs to divide with a net 2–4 fold expansion in HSC numbers after 10 days and low concentrations of SF result in loss of HSC activity. To investigate the cellular mechanisms underlying these different outcomes, we cultured 114 CD45midlin−Rho−SP adult mouse bone marrow cells in single cell cultures containing serum-free medium + 20 ng/ml IL-11 and either 300 or 10 ng/ml of SF. Each culture was then examined every 4–6 hr. The kinetics of division of these cells under both conditions was identical with completion of the 1st division occurring between 22–68 hr. During that time none of the input cells died (<1%). After 10 days of culture, during which time all input cells divided at least 5 times (>50 cells), the HSC content of pooled clones (as measured by in vivo transplantation assays) was found to be >10-fold higher in the clones generated under high vs. low SF conditions (p<0.05). To characterize the types of self-renewal divisions undertaken, 9 doublets generated under the high SF condition were harvested between 4 and 8 hr after they underwent their 1st division and then each of the daughters was injected into a separate irradiated mouse. Analysis of the 18 mice showed that for one of the input cells both daughters were HSCs (evidence of a symmetric self-renewal division) and for 3 more, only one of the 2 daughters was an HSC (evidence of an asymmetric self-renewal division). In contrast no daughter HSCs were identified when 6 doublets produced under the low SF condition were assayed. To determine whether the loss of HSC activity under low SF conditions was a pre- or post-mitotic event, additional in vivo HSC assays were performed on cells harvested from individual wells after 8, 16 and 96 hours of incubation. The results revealed no change in the proportion of wells with either low or high concentrations of SF that contained HSCs after 8 hr of incubation (10/36 positive mice injected with starting single cells and 5/17 (low SF) vs. 6/17 (high SF) positive mice injected with 8-hr single cells, respectively). However, a significant difference (p<0.01) was seen after 96 hr (5/35 vs. 2/43 positive mice, respectively) and, after only 16 hr, before a first mitosis was seen under either condition, a decline in HSCs was apparent under the low SF condition (4/15 vs. 1/15 positive mice injected with cells from the high vs. low SF condition). Together, these studies indicate that HSC exposure to different SF concentrations can rapidly and irreversibly alter the ability of HSCs to execute symmetric as well asymmetric self-renewal divisions in vitro.

scholarly journals Evidence for early recruitment of granulocyte precursors during high- dose methotrexate infusions in mice

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 301-307 ◽  
Author(s):  
HM Pinedo ◽  
BA Chabner ◽  
DS Zaharko ◽  
JM Bull
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Stem Cell Population ◽  
High Dose ◽  
Mouse Bone Marrow ◽  
Drug Infusion ◽  
High Concentrations

Abstract The effects of constant exposure to high concentrations of methotrexate in vivo on the committed stem cell (CFU-C) were studied by in vitro culture of mouse bone marrow. Bone marrow samples were obstained from animals receiving a continuous infusion, and were cultured in a methotrexate-free semisolid gel system. The effects of methotrexate infusion on the pluripotent stem cell population (CFU-S) were studied as well. Constant exposure to 10(-5) M methotrexate produced a rapid decrease in total nucleated cells per femur, reaching 35% of control at 12 hr and remaining at approximately this level throughout 48 hr of drug infusion. A decrease in the number of both CFU-C and CFU-S per femur was observed, which paralleled the drop in nucleated cells during the first 24 hr. However, in contrast to an additional drop in the number of CFU-S, an increase of CFU-C number per femur was observed from 24 to 48 hr. These data indicated a self-limited cell kill of nucleated bone marrow cells, and suggested recruitment of CFU-C from the CFU-S pool between 24 and 48 hr of infusion despite continued methotrexate infusion.

scholarly journals Cooperative effects of colony-stimulating factor 1 and recombinant interleukin 2 on proliferation and induction of cytotoxicity of macrophage precursors generated from mouse bone marrow cell cultures.

1989 ◽  
Vol 169 (3) ◽  
pp. 973-986 ◽  
Author(s):  
H Li ◽  
R Schwinzer ◽  
M Baccarini ◽  
M L Lohmann-Matthes
Keyword(s):  
Bone Marrow ◽  
Marrow Cell ◽  
Interleukin 2 ◽  
Culture Conditions ◽  
Mouse Bone Marrow ◽  
Nk Activity ◽  
Proliferating Cells ◽  
Mouse Bone Marrow Cell ◽  
Cooperative Effects

Precursor cells for NK activity, present in the light fraction of fresh mouse bone marrow, were cultivated in vitro in the presence of either CSF-1, IL-2, or a combination of both factors. In the presence of only CSF-1, strong proliferation was induced. Cells quickly passed the macrophage precursor stage and matured to typical macrophages. Neither granula formation nor NK activity were induced. Under culture conditions with only IL-2 NK activity had developed after 3 d, however, no significant proliferation occurred. In the presence of both factors strong proliferation was induced, and concomitantly, granula formation and NK activity developed. Apparently, proliferation depended on CSF-1 and granula formation, and NK cytotoxicity was induced by IL-2. When proliferating cells with strong anti-YAC-1 activity from a culture in CSF-1 plus IL-2 were further cultivated in only IL-2, the content of granula further increased, whereas proliferation gradually stopped. In contrast, when these cells from CSF-1 plus IL-2 culture were further cultivated in only CSF-1, granula disappeared and NK activity was lost, whereas sustained proliferation and differentiation to macrophages occurred. Only under culture conditions with both factors were proliferation and NK activity both maintained. More than 90% of cells from a 3-d culture in CSF-1 plus IL-2 expressed the NK 1.1. marker, whereas F4/80 was only marginally detected by FACS analysis. After two further days in culture, 70% of the cells expressed F4/80 and 60% coexpressed NK 1.1. and F4/80. By setting the size scatter in order to gate for large granular cells, a population was obtained with 100% coexpression of NK1.1. and F4/80. The data indicate that early cells of the macrophage lineage can develop into different functional and morphological directions depending on the varying influence of IL-2 and CSF-1.

scholarly journals Evidence for early recruitment of granulocyte precursors during high- dose methotrexate infusions in mice

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 301-307
Author(s):  
HM Pinedo ◽  
BA Chabner ◽  
DS Zaharko ◽  
JM Bull
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Stem Cell Population ◽  
High Dose ◽  
Mouse Bone Marrow ◽  
Drug Infusion ◽  
High Concentrations

The effects of constant exposure to high concentrations of methotrexate in vivo on the committed stem cell (CFU-C) were studied by in vitro culture of mouse bone marrow. Bone marrow samples were obstained from animals receiving a continuous infusion, and were cultured in a methotrexate-free semisolid gel system. The effects of methotrexate infusion on the pluripotent stem cell population (CFU-S) were studied as well. Constant exposure to 10(-5) M methotrexate produced a rapid decrease in total nucleated cells per femur, reaching 35% of control at 12 hr and remaining at approximately this level throughout 48 hr of drug infusion. A decrease in the number of both CFU-C and CFU-S per femur was observed, which paralleled the drop in nucleated cells during the first 24 hr. However, in contrast to an additional drop in the number of CFU-S, an increase of CFU-C number per femur was observed from 24 to 48 hr. These data indicated a self-limited cell kill of nucleated bone marrow cells, and suggested recruitment of CFU-C from the CFU-S pool between 24 and 48 hr of infusion despite continued methotrexate infusion.

scholarly journals Effects of normal mouse serum on the IL-3-induced proliferation of bone marrow cells

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 700-705 ◽  
Author(s):  
CA Dahl ◽  
C Lindqvist
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Normal Mouse ◽  
Bone Marrow Cells ◽  
Normal Serum ◽  
Mouse Serum ◽  
Mouse Bone Marrow ◽  
Normal Mouse Serum ◽  
High Concentrations

Abstract Normal mouse serum (NMS) devoid of colony-stimulating factor (CSF) was found to enhance the interleukin 3 (IL-3)-driven colony formation of bone marrow in vitro. Inclusion of NMS in bone marrow colony-forming assays resulted in greatly increased numbers of colonies and clusters following seven days incubation; however, incubation of bone marrow with NMS before the colony-forming assay had no effect on resultant colony number. The levels of serum-enhancing activity (SEA) did not appear to vary significantly with age and in part was species restricted, in that human and guinea pig serum did not enhance mouse bone marrow colony formation. Conversely, NMS had no effect on human bone marrow colony formation. Levels of SEA were found to vary between strains, as did the degree to which bone marrow from various strains was enhanced by the serum. Serum fractionation studies indicated three active fractions with molecular weights of 800–900 Kd, 60–70 Kd, and 20- 30 Kd. The fraction at 800–900 Kd inhibited colony formation at high concentrations and enhanced colony formation on dilution, whereas the two other active fractions contained enhancing activity at all concentrations tested. These results would indicate that normal serum can play a greater role in colony-forming assays than nutritional supplements. The relationship of the SEA factors to other factors that have been reported to modulate bone marrow colony formation is discussed.

scholarly journals Effects of normal mouse serum on the IL-3-induced proliferation of bone marrow cells

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 700-705 ◽  
Author(s):  
CA Dahl ◽  
C Lindqvist
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Normal Mouse ◽  
Bone Marrow Cells ◽  
Normal Serum ◽  
Mouse Serum ◽  
Mouse Bone Marrow ◽  
Normal Mouse Serum ◽  
High Concentrations

Normal mouse serum (NMS) devoid of colony-stimulating factor (CSF) was found to enhance the interleukin 3 (IL-3)-driven colony formation of bone marrow in vitro. Inclusion of NMS in bone marrow colony-forming assays resulted in greatly increased numbers of colonies and clusters following seven days incubation; however, incubation of bone marrow with NMS before the colony-forming assay had no effect on resultant colony number. The levels of serum-enhancing activity (SEA) did not appear to vary significantly with age and in part was species restricted, in that human and guinea pig serum did not enhance mouse bone marrow colony formation. Conversely, NMS had no effect on human bone marrow colony formation. Levels of SEA were found to vary between strains, as did the degree to which bone marrow from various strains was enhanced by the serum. Serum fractionation studies indicated three active fractions with molecular weights of 800–900 Kd, 60–70 Kd, and 20- 30 Kd. The fraction at 800–900 Kd inhibited colony formation at high concentrations and enhanced colony formation on dilution, whereas the two other active fractions contained enhancing activity at all concentrations tested. These results would indicate that normal serum can play a greater role in colony-forming assays than nutritional supplements. The relationship of the SEA factors to other factors that have been reported to modulate bone marrow colony formation is discussed.

scholarly journals Comparative Study on In Vitro Culture of Mouse Bone Marrow Mesenchymal Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-14 ◽  
Author(s):  
Yuxin Hu ◽  
Bin Lou ◽  
Xiafang Wu ◽  
Ruirui Wu ◽  
Huihui Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Adipogenic Differentiation ◽  
Culture Method ◽  
Culture Conditions ◽  
Mouse Bone Marrow ◽  
Differentiation Potential ◽  
Initial Medium

In vitro culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) has been hampered because of the low yield of MSCs during isolation and the contamination of hematopoietic cells during expansion. The lack of specific mouse BM-MSC markers increases the difficulty. Several techniques have been reported to improve the purity and in vitro growth of mouse BM-MSCs. However, systematic report on comparison of characteristics in primary BM-MSCs between different culture conditions is rare. Here, we studied the effects of oxygen concentrations and initial medium replacement intervals, along with cell passages, on mouse BM-MSCs isolated with differential adhesion method. BM-MSCs exhibited elevated proliferative and clonogenic abilities in 5% oxygen compared with 10% and 21% oxygen, as well as a better expression of the MSC marker Sca-1. Adipogenic and osteogenetic differentiation of BM-MSCs can be observed in both 21% and 5% oxygen. Adipogenic differentiation appeared stronger under normoxia conditions. BM-MSCs showed increased proliferative capacity and adipogenic/osteogenetic differentiation potential when initial medium replacement interval was 4 days compared with 1 day. As passage number increased, cells were more MSC-like in morphology and in expression of surface markers (positive for CD29, CD44, and Sca-1 and negative for CD11b, CD19, and CD45). These data provide new insight into optimizing the culture method and understanding the biological characteristics of mouse BM-MSCs during in vitro expansion.

In Vitro, In Silico and Ex Vivo Studies of Dihydroartemisinin Derivatives as Antitubercular Agents

2019 ◽  
Vol 19 (8) ◽  
pp. 633-644 ◽  
Author(s):  
Komal Kalani ◽  
Sarfaraz Alam ◽  
Vinita Chaturvedi ◽  
Shyam Singh ◽  
Feroz Khan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Silico ◽  
Ex Vivo ◽  
Virulent Strain ◽  
Infection Model ◽  
Mouse Bone Marrow ◽  
Significant Activity ◽  
Macrophage Infection ◽  
In Silico Studies

Introduction: As a part of our drug discovery program for anti-tubercular agents, dihydroartemisinin (DHA-1) was screened against Mtb H37Rv, which showed moderate anti-tubercular activity (>25.0 µg/mL). These results prompted us to carry out the chemical transformation of DHA-1 into various derivatives and study their antitubercular potential. Materials and Methods: DHA-1 was semi-synthetically converted into four new acyl derivatives (DHA-1A – DHA-1D) and in-vitro evaluated for their anti-tubercular potential against Mycobacterium tuberculosis H37Rv virulent strain. The derivatives, DHA-1C (12-O-(4-nitro) benzoyl; MIC 12.5 µg/mL) and DHA-1D (12-O-chloro acetyl; MIC 3.12µg/mL) showed significant activity against the pathogen. Results: In silico studies of the most active derivative (DHA-1D) showed interaction with ARG448 inhibiting the mycobacterium enzymes. Additionally, it showed no cytotoxicity towards the Vero C1008 cells and Mouse bone marrow derived macrophages. Conclusion: DHA-1D killed 62% intracellular M. tuberculosis in Mouse bone marrow macrophage infection model. To the best of our knowledge, this is the first-ever report on the antitubercular potential of dihydroartemisinin and its derivatives. Since dihydroartemisinin is widely used as an antimalarial drug; these results may be of great help in anti-tubercular drug development from a very common, inexpensive, and non-toxic natural product.

Differentiation of Mouse Bone-Marrow Mesenchymal Stem Cells into Motor Neuron Cells in vitro

2016 ◽  
Vol 19 (2) ◽  
pp. 111-116
Author(s):  
Rafal Hussamildeen Abdullah ◽  
◽  
Shahlla Mahdi Salih ◽  
Nahi Yosef Yaseen ◽  
Ahmed Majeed Al-Shammari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Motor Neuron ◽  
Mouse Bone Marrow ◽  
Marrow Mesenchymal Stem Cells

scholarly journals INDUCTION OF A HEMOLYSIN RESPONSE IN VITRO

1971 ◽  
Vol 133 (6) ◽  
pp. 1325-1333 ◽  
Author(s):  
Klaus-Ulrich Hartmann
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Close Contact ◽  
Precursor Cells ◽  
Spleen Cells ◽  
High Concentrations ◽  
Thymus Cells ◽  
Bone Marrow Chimeras

Spleen cells of bone marrow chimeras (B cells) and of irradiated mice injected with thymus cells and heterologous erythrocytes (educated T cells) were mixed and cultured together (17). The number of PFC developing in these cultures was dependent both on the concentration of the B cells and of the educated T cells. In excess of T cells the number of developing PFC is linearly dependent on the number of B cells. At high concentrations of T cells more PFC developed; the increase in the number of PFC was greatest between the 3rd and 4th day of culture. Increased numbers of educated T cells also assisted the development of PFC directed against the erythrocytes. It is concluded that the T cells not only play a role during the triggering of the precursor cells but also during the time of proliferation of the B cells; close contact between B and T cells seems to be needed to allow the positive activity of the T cells.

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