Research Article Effect of Metformin on Viability...

Research ArticleEffect of Metformin on Viability Morphology andUltrastructure of Mouse Bone Marrow-Derived MultipotentMesenchymal Stromal Cells and Balb3T3 EmbryonicFibroblast Cell Line

Agnieszka Umieszek12 Aleksandra Czyrek2 Katarzyna Basinska1 Justyna Trynda3

Aneta SkaradziNska4 Anna SiudziNska1 Monika Marwdziak12 and Krzysztof Marycz12

1Electron Microscopy Laboratory The Faculty of Biology and Animal Science University of Environmental and Life SciencesKozuchowska 5b Street 50-631 Wroclaw Poland2Wrocławskie Centrum Badan EIT+ Stablowicka 147 Street 54-066 Wroclaw Poland3Department of Experimental Oncology Ludwik Hirszfeld Institute of Immunology and Experimental TherapyPolish Academy of Sciences 53-114 Wroclaw Poland4Department of Biotechnology and Food Microbiology Faculty of Food Science University of Environmental and Life SciencesChelmonskiego 3741 51-630 Wroclaw Poland

Correspondence should be addressed to Agnieszka Smieszek smieszekagnieszkagmailcom

Received 8 January 2015 Revised 27 March 2015 Accepted 15 April 2015

Academic Editor Wiep Scheper

Copyright copy 2015 Agnieszka Smieszek et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Metformin a popular drug used to treat diabetes has recently gained attention as a potentially useful therapeutic agent for treatingcancer In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromalcells (BMSCs) and Balb3T3 fibroblast at concentration of 1mM 5mM and 10mM Obtained results indicated that metforminnegatively affected proliferation activity of investigated cells The drug triggered the formation of autophagosomes and apoptoticbodies in all tested cultures Additionally we focused on determination of expression of genes involved in insulin-like growthfactor 2 (IGF2) signaling pathway The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs andBalb3T3 Further the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and10mM metformin treatments In case of Balb3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5mMand increased after 10mM of metformin Our results suggest that metformin influences the cytophysiology of somatic cells in adose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure

1 Introduction

Metformin is a common drug used worldwide in the treat-ment of diabetes mellitus It belongs to the group of biguani-dine drugs among which it has the best safety profile [1] Thegeneral systemic effect of metformin involves the reductionof glucose concentration and increased insulin sensitivityHowever mounting evidence indicates that the range ofmetformin action may be significantly wider and thus theapplication of metformin may open new perspectives in thetreatment of various medical conditions [2 3] In cell culture

metformin inhibits the proliferation of a range of cancercells including breast [4ndash6] oral cavity [7] pancreas [8] andovarian cells [9] Effectiveness of this agent as an anticancerdrug is associated not only with its cytostatic properties butalso with proapoptotic action in tumor cells [7 10 11]

Metformin is also assigned to the conceptual group ofdrugs known as calorie restriction mimetics (CRM) Ithas been demonstrated that calorie restriction is a veryeffective way of increasing the lifespan by reducingmorbidityand mortality in mice with tumors [12] The key signalingpathways underlying the antiaging effects of metformin or

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 769402 14 pageshttpdxdoiorg1011552015769402

2 BioMed Research International

other CRM drugs have not been fully explored It seems thatmetformin affects endocrine regulatory systems and insulin-like growth factors [13] Signaling pathway of insulin-likegrowth factors (IGF) regulates cell proliferation differenti-ation aging and life span thus its role is principal for thedevelopment of the organism and has remained unchangedduring evolution [14] IGF2 together with theH19 gene forman imprinted tandem both in humans and in mice that playsan important role not only during embryonic developmentbut also during the proliferation of stem cells residing in adulttissues [14 15]

Bone marrow provides a niche for various populationsof stem cells the interplay of which is essential for bodyhomeostasis Biology of the bone marrow-derived multipo-tent mesenchymal stromal cells (BMSCs) is continuouslybeing studied Their potential for self-renewal as well as highphenotypic plasticitymanifested by the ability to differentiateinto bone cartilage or adipose tissue is extremely importantin terms of regenerative medicine [16] Mesenchymal stromalstem cells (MSCs) due to a high phenotypic and cellularplasticity are a suitable model for in vitro assessment ofvarious biological and chemical agents [17] Additionallyevaluation of alterations in MSC morphology provides valu-able information that reflects complex biological processescontrolled by the interactions between the cytoskeleton andthe extracellular environment [18]

The properties of self-renewal and differentiation of stemcells might be regulated by octamer-binding protein 4 (Oct-4) a transcription factor crucial for embryonic development[19] The expression of Oct-4 was reported in bone marrow-derived stromal cells which confirms high phenotypic plas-ticity of these cells [20] Impairment of the proliferationpotential of mesenchymal stem cells may account for regen-erative potential deficiency of the organism Mesenchymalstem cells seem to participate in the process of bioactivestroma formation [21] and affect the biological properties ofsurrounding tissues Due to the fact that metformin increasesglucose uptake in connective and embryonic tissues [22]their effect on proliferative activity of BMSCs and othercells of connective tissue such as fibroblasts should beconsidered In the present work we have evaluated the effectof metformin in vitro using murine primary cultures of bonemarrow-derived multipotent mesenchymal stromal cells andBalb3T3 fibroblast cell line

We have investigated the effect of metformin in cellcultures at doses cytotoxic for cancer cells [4 5 7ndash9] Ourobjective was to determine how different concentrations ofmetformin affect the physiology of stromal cellsThe analysisincluded BMSC and Balb3T3 proliferation activity assaysand evaluation of the morphology and ultrastructure of cellsinvestigatedWe have also aimed to determine the expressionof IGF signaling components (IGF2 IGF2R and H19) as wellas the expression of Oct-4

2 Materials and Methods

All reagents used in this experiment were purchased fromSigma-Aldrich (Poland) unless indicated otherwise

21 Ethical Approval The study was conducted with theapproval of the Bioethics Committee as stated by the SecondLocal Bioethics Committee at the Department of Biology andAnimal Breeding Wroclaw University of Environmental andLife Sciences Wroclaw Chelmonskiego 38C Poland (Decnumber 1772010 of 11152010)

22 Cell Population Two types of mouse cells were used inthe experiments multipotent stromal cells (BMSCs) derivedfrom bone marrow (primary cultures) and Balb3T3 embry-onic fibroblasts (cell line obtained from the Institute ofImmunology and Experimental Therapy Polish Academy ofSciences)

23 Isolation of BMSCs Bone marrow-derived multipotentmesenchymal stromal cells (BMSCs) were isolated fromtwelve 4-week-old C57BL6 mice (Animal Vivarium Wro-clawMedical School Poland) Femurs were collected directlyafter euthanasia of the animal and placed in a sterile Hanksrsquobalanced salt solution (HBSS) Cells were isolated from thebone marrow by flushing with an insulin syringe U-40(29G X 1210158401015840 needle) filled with HBSS Cell suspension wastransferred into falcon tube and centrifuged at 300timesg for 4minutes For FACS analysis bone marrow cells were lysedin BD lysing buffer (BD Biosciences San Jose CA USA) for15min at room temperature and washed twice in phosphate-buffered saline (PBS) For cell culture pellets were resus-pended in Dulbeccorsquos Modified Eaglersquos Medium (DMEM)containing Hamrsquos F-12 nutrient mixture supplemented with10 of foetal bovine serum (FBS) and transferred to cultureflasks

24 Characterization of BMSCs Phenotype Flow CytometryBone marrow cell suspensions isolated by flushing femursand tibia were lysed in BD lysing buffer (BD Biosciences SanJose CA USA) for 15min at room temperature and washedtwice in phosphate-buffered saline (PBS) The cells were sub-sequently stained for Sca-1 antigen and hematopoietic lineagemarkers (Lin) for 30min in medium containing 2 foetalbovine serum The following anti-mouse antibodies (BDPharmingen) were used for staining Sca-1 (FITC clone D7)B220 (PE clone RA3-6B2) T-cell receptor-120573 (PE clone H57-597) T-cell receptor-120574120575 (PE clone GL3) CD11b (PE cloneM170) Ter119 (PE clone TER-119) and Gr-1 (PE clone RB6-8 C5) Sca-1+Linminus cells were isolated by a multiparameterlive-cell sorting (INFLUX BD)

To phenotype BMSC cell surface antigens Sca-1+Linminuscells were stained using the following CD31 (APC clone390) CD45 (APC-Cy7 clone 30-F11) CD51 (biotin cloneRMV-7 with streptavidin conjugated to PE-Cy5) CD73(FITC clone B5) CD90 (PB clone 53-21) and CD105 (PEMJ718) All monoclonal antibodies (mAbs) were added atsaturating concentrations and the cells were incubated for30 minutes on ice washed twice resuspended in stainingbuffer at a concentration of 5 times 106 cells per millilitre andanalyzed using an LSR II (BD Biosciences Mountain ViewCA httpwwwbdbiosciencescom) Anti-mouse mAbswere purchased fromBDPharmingen (SanDiego CA httpwwwbdbiosciencescom)

BioMed Research International 3

25 Determination of Multipotent Character of BMSCs Adi-pogenic and osteogenic differentiation of BMSCs was in-duced using commercial kits (StemPro Life Technologies)Stimulation toward adipocytes lasted 14 days while osteoge-nesis was induced during a 21-day period To evaluate adi-pogenic and osteogenic differentiation two specific stainingmethods were used that is Oil-Red O for the detection ofneutral lipid deposits and Alizarin red for calcium depositsPreparations were analyzed using an Axio Observer A1inverted microscope (Carl Zeiss Jena Germany) Documen-tation was made using Canon PowerShot camera

26 Propagation of Cells Cultures were maintained at 37∘Cin a humidified atmosphere of 5 CO

2and 95 air Primary

and subsequent cultures of BMSCs were propagated inDulbeccorsquos Modified Eaglersquos Medium (DMEM) with HamrsquosF-12 nutrient mixture while Balb3T3 were maintained inDMEM containing 4500mgL of glucose All culture mediawere supplemented with 10 of FBS and 1 of antibiotics(penicillin and streptomycin) Medium was changed everytwo days The passage of cells was performed at 80ndash90confluence Prior to the experiment the cells were passagedthree times using trypsin solution (TrypLE Life Technolo-gies) according to the manufacturersrsquo instruction

27 Cultures with Metformin Metformin (Metformax 850Teva Pharmaceuticals Poland) was grinded with a mor-tar and dissolved in the culture medium at the followingconcentrations 1mM 5mM and 10mM Nontreated cellsserved as a control for comparison with the test cultures Forthe analysis of proliferation morphology ultrastructure andgene expression both BMSCs and Balb3T3 were inoculatedinto 24-well plates while measurements of DNA synthesiswere performed in cultures propagated in 96-well platesInitial concentration of cells in 24-well dishes was 3 times 104per well The cells were inoculated in a 05mL volume ofculture medium per well Seeding density of cells in 96-wellplates was 5times103 Cells were inoculated in 01mL of completegrowth medium per well The first dose of the drug testedwas added to the medium after 24 hours when adhesionand spreading of cells were observed on the plate surfaceDuring cell propagationmediumwas changed every dayTheexperiment was performed in three independent replicates

28 Proliferation of Cells

281 Analysis Using Resazurin Assay Cell viability wasevaluated after 24 48 and 72 hours using resazurin-resorufinsystem To perform the assay medium was removed andreplaced with a medium containing 10 of the dye Cellswere incubated in a CO

2incubator for 2 hours and then the

supernatants were collected and transferred into the 96-wellmicroplate reader (Spectrostar Nano BMG Labtech) Super-natants after BMSCs and Balb3T3 cultures were derivedfrom three independent experiments The absorbance ofthe supernatants was measured spectrophotometrically at awavelength of 600 nm for resazurin and 690 nmas a referencewavelength Each test included a blank containing completemedium without cells

282 Measurement of DNA Synthesis BrdU Assay DNAsynthesis was assessed by measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into cellular DNA Prolifera-tion of cells was analyzed three times independently after 2448 and 72 hours of the experiment The assay was carriedout using BrdU Cell Proliferation ELISA Kit based on theprotocol provided by the manufacturer (Abcam) Brieflycultures were treated with BrdU and incubated overnightat 37∘C in a humidified atmosphere After incubation withBrdU cells were fixed and DNA was denatured using aFixing Solution provided by the manufacturer BrdU incor-porationwas detected using anti-BrdUmonoclonal antibodyIncubation of cells with specific antibody was performed atroom temperature and lasted for 1 hour Goat anti-mouseIgG conjugated with horseradish peroxidase (HRP) was usedas secondary antibody Incubation with secondary antibodywas performed at room temperature for 30 minutes Colorreaction was developed using 331015840551015840-tetramethylbenzidine(TMB) as substrate and stopped after 30 minutes Incubationwith substrate was performed at room temperature avoidingexposition to excessive light Signal intensity was measuredwith a spectrophotometer microplate reader (SpectrostarNano BMG Labtech) at a wavelength of 450550 nm

283 Morphology of Cells The morphology of the studiedcells was evaluated with an epifluorescent microscope (ZeissAxio Observer A1) and scanning electronmicroscope (SEMZeiss Evo LS 15) The analysis of morphology was performedafter 48 h of the experimental culture in 24-well platesPreparation of cells for fluorescence microscopy was asfollows cells were (i) washed three times using HBSS 1minute each wash (ii) fixed in 4 ice cold paraformaldehydeovernight at 4∘C (iii) washed (as described above) (iv)permeabilized for 15minutes with 01 Triton X-100 at roomtemperature (v) washed (as described above) (vi) stainedwith atto-488-labeled phalloidin (1 800) for 30 minutes inthe dark at room temperature and (vii) counterstained usingdiamidino-2-phenylindole (DAPI 1 1000) for 5 minutes atroom temperature as described previously [23] In additionpropidium iodide staining was performed to detect deadcells in the population Propidium iodide was diluted in PBS(1 1000) Cultures were incubated with a dye for 15 minutesat 37∘C Images of stained cultures were captured using aPowerShotCamera (Canon) SEManalysis was as follows cellcultures were (i) fixed in 25 glutaraldehyde in DMEM (ii)rinsed with HBSS (iii) dehydrated in a graded ethanol series(from50 to 100 increasing 10 at each step) (iv) air-driedfor 30 minutes at room temperature and (v) coated with goldparticles using 300-second program (Edwards Scancoat six)Prepared samples were imaged using SE1 detector at 10 kVfilament tension (SEM Zeiss Evo LS 15) and 500x and 5000xmagnification as described previously [24 25]

284 Ultrastructure of Cells Ultrastructure analysis of cellswas performed by using a scanning transmission electronmicroscope (TEM Zeiss Evo LS 15) as described previously[26]The cells were fixed overnight at 4∘C in 25 glutaralde-hyde in DMEM After fixing the cells were centrifuged at2000timesg for 10minutes and rinsed with PBS (01M pH = 70)


Table 1 Sequences of primers used in qPCR

Gene Abbreviation Sequence 51015840-31015840 Loci Amplicon length [bp] Accession number

Beta-2 microglobulin b2m F CATACGCCTGCAGAGTTAAGCA 341ndash362 73 NM 0097353R GATCACATGTCTCGATCCCAGTAG 413ndash390

Insulin-like growthfactor 2 IGF2 F TCAGTTTGTCTGTTCGGACCG 223ndash243 223 NM 0011227371

R TTGGAAGAACTTGCCCACG 445ndash427Insulin-like growthfactor 2 receptor IGF2R F GGCTGCGATCGATATGCATCT 2616ndash2636 106 NM 0105152

R GGCCTATCTTTGCAACTCCCA 2721ndash2701

H19 H19 F AGGTGAAGCTGAAAG 2031ndash2045 97 NR 0015921R GCAGAGTTGGCCATGAAGATG 2127ndash2107

Octamer bindingtranscription factor 4 Oct-4 F TTCTGCGGAGGGATGGCATA 258ndash277 232 NT 0396498

R GTTCTAGCTCCTTCTGCAGGG 489ndash469

for 30 minutes at room temperature as described previouslyThen the cells were centrifuged once again using settingsprovided aboveThe resulting pellets were incubated with 1osmium tetroxide in PBS for 2 hours Next cells were washedusing 01M PBS and centrifuged After this procedure cellswere dehydrated in a graded acetone series (30ndash100) andembedded in Agar Low Viscosity Resin Kit (Agar ScientificLtd Stansted Essex UK) Ultrathin sections (80 nm) ofthe specimens were collected on copper grids Cells werecontrasted with uranyl acetate (30 minutes incubation) andlead citrate (15 minutes incubation) The cells were observedwith the TEM detector at 10 kV filament tension

285 Analysis of Gene Expression Real-Time Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) Cellswere rinsed twice using HBSS after 48 h culture and thenhomogenized using 08mL of TRI Reagent Total RNA wasisolated according to a single-step method described byChomczynski and Sacchi [27] The resulting samples werediluted in DEPC-treated water Quantity and quality oftotal RNA was determined using nanospectrometer (WPABiowave II) Traces of genomic DNA (gDNA) were digestedwith DNase I RNase-free kit (Thermo Scientific) Eachreaction contained 200 ng of total RNA ComplementaryDNA (cDNA) was obtained in the reaction with MoloneyMurine Leukemia Virus Reverse Transcriptase (M-MLV RT)and oligo(dT)15 primers (Verte KIT oligo(dT)15 Novazym)Both RNA purification and cDNA synthesis were performedin accordance with the manufacturersrsquo instructions using aT100 Thermo Cycler (Bio-Rad) Sequences of the primersused in the amplification are listed in Table 1 QuantitativeRT-PCR was carried out in a total volume of 20120583L usingSensiFast SYBR amp Fluorescein Kit (Bioline) PCR mixturecontained 20 of cDNA Concentration of primers in eachreaction was 500 nM The following cycling conditions wereapplied 95∘C for 2 minutes followed by 45 cycles of 95∘Cfor 5 s annealing temperature gradient for 10 s and 72∘C for5 s with a single fluorescence measurement To determinethe specificity of the PCR products analysis of the dissoci-ation curve of amplicons was performed Melting curve wasdetermined with a program ramped up from 65 to 95∘C ata heating rate of 02∘Cs and continuous measurement of

the fluorescence The value of the threshold cycle (Ct) wasused to calculate the fold change in relation to the expressionof housekeeping gene beta-2 microglobulin (1205732m) Real-timePCRwas performedusingCFXConnect Real-TimePCRDetection System (BioRad)

286 Enzyme-Linked Immunosorbent Assay (ELISA) Thesupernatants of the cultures were analyzed by ELISA todetermine the concentration of secreted IGF2 Supernatantswere collected after 48 hours of cell propagation from controland experimental cultures Aliquots were kept at minus20∘C untilELISA analysis Marker protein was assayed using specificmouse IGF-II ELISA kit (DuoSet ELISA Development kitRampD Systems Poland) Before each assay all samples werebriefly centrifuged and twofold diluted The substrate forperoxidase used in ELISA was 331015840551015840-tetramethylbenzidine(TMB) and the reaction was stopped with 2N sulfuricacid (H

2SO4) Readouts were conducted at a wavelength of

450 nm using a spectrophotometer (BMG Labtech)

287 Statistical Analysis Normality of the population datawas determined using the Shapiro-Wilk test while equality ofvariances was assessed by Levenersquos test Differences betweengroups were determined using one- or two-way analysisof variance (ANOVA) Statistical analysis was performedwith STATISTICA 100 software (StatSoft Inc Statistica forWindows Tulsa OK USA) Differences with a probability of119901 lt 005 were considered significant

3 Results

31 Phenotypic Characterization of BMSCs and Their Mul-tipotent Properties The analysis showed that BMSCs iso-lated according to the method described expressed markersspecific for multipotent mesenchymal stromal cells that isCD51 CD73 CD90 and CD105 Cells were negative forhematopoietic marker that is CD45 and endothelial markerCD31 (Figure 1(a)) Additionally multipotent nature of cellswas confirmed by their ability to differentiate into bone andadipocyte precursors In contrast to the cells cultured instandard conditions (Figure 1(b)) specific staining showedthat stimulation of BMSCs with the adipogenic medium


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Figure 1 Characterization of BMSC phenotype and determination their multipotency Phenotype of BMSCs was determined by flowcytometry The analysis revealed that BMSCs isolated by the described method were negative for CD45 and CD31 In addition the cellswere strongly positive for CD51 CD73 CD90 and CD105 (panel (a)) Morphology of BMSC culture in standard (b) adipogenic (c) andosteogenic conditions (d) Specific stainings were carried out to determine the BMSC differentiation to adipocytes and osteocytes Oil-RedO staining was used to detect lipid droplet formation during adipogenic differentiation (c) Alizarin red staining was used to detect calciumdeposition during osteogenic differentiation (d) Images of differentiated cultures were captured at 100x magnification (scale bar = 200 120583m)


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(b)Figure 2 Influence of metformin on the proliferative activity of BMSCs (a) and mouse embryonic fibroblast cell line Balb3T3 (b)Proliferation of control cultures was compared to the cultures propagated with metformin (a) No difference in the proliferation rate ofBMSCs was recorded after 24 and 48 hours (119901 gt 005) when cells were cultured with the addition of 1mM and 5mMmetformin A significantdecrease in the proliferative activity of BMSCs treated with 1mM and 5mM concentrations was noticed after 72 hoursThe addition of 10mMmetformin to the BMSC culture significantly reduced the proliferation of BMSCs from the second day of culture (b) Proliferative activity ofBalb3T3 in experimental cultures decreased significantly after 48 hours Statistically significant differences in the experimental and controlculture of Balb3T3 were also observed after 72 hours of propagation An asterisk () indicates a statistically significant difference (119901 lt 001)Significance was determined by two-way ANOVA test Each test included a blank containing complete medium without cells The 119909-axisrefers to the time of cell propagation while ΔΔ119860 mark on 119910-axis refers to the difference between absorbance read at 600 nm and 690 nmincluding blank sample

promotes the formation of lipid droplets while inductionof osteogenesis resulted in the formation of calcium-richdeposits (Figures 1(c) and 1(d))

32 Proliferation of Cells Cellular proliferative activity ofboth of BMSCs as well as mouse embryonic fibroblast cellline Balb3T3 was evaluated after 24 48 and 72 hours ofculture (Figures 2 and 3) The results of resazurin-basedassay revealed that exponential pattern of cell growth wasobserved only in the control cultures of BMSCs andBalb3T3The proliferation of BMSCs treated with metformin at aconcentration of 1mM and 5mM was comparable with thecontrol culture until 48 hours of propagation A slighthowever not statistically significant increase in the rate ofBMSCs proliferation was recorded when cells were treatedwith 1mM metformin The proliferation of BMSCs in cul-tures exposed to 1mM and 5mM metformin decreasedsignificantly after 72 hours The significant inhibition ofBMSCs proliferation treated with 10mM metformin wasobserved after 48 h of culture (Figure 2(a)) The analysis ofcellular activity measured by BrdU incorporation into DNAof actively proliferating cells showed a decrease of DNAsynthesis after 72 h Significant changes of BMSCproliferativeactivity were observed in cultures treated with 5mM and

10mM metformin in comparison to the control cultureBMSCs treated with 10mM metformin showed reducedDNA synthesis from the beginning of the experiment(Figure 3(a))

After 24 hours of propagation no significant changeswere observed in the proliferation of Balb3T3 in control andexperimental cultureHowever 48-hourmetformin exposureof Balb3T3 cultures negatively influenced metabolic activityof cells Interestingly in Balb3T3 culture metformin at 1mMand 5mM concentrations exerted comparable effects onthe cell proliferation Growth curves of these cultures hadsimilar patterns indicating culture restoration and increaseof cell proliferative activity after 72 hours Similarly asin BMSC cultures the activity of cells was significantlyreduced after exposure to 10mM metformin (Figure 2(b))BrdU incorporation assay showed that synthesis of DNA inBalb3T3 was not altered by metformin after 24 hours ofpropagation Interestingly after 48 hours of culture 1mMmetformin induced DNA synthesis in Balb3T3 Cytotoxiceffect of 10mM metformin on Balb3T3 was observed after48 hours of culture Reduced DNA synthesis was found in allexperimental cultures after 72 hours of propagation howeverstatistical significance was reached in the case of culturestreated with 5mM and 10mMmetformin (Figure 3(b))


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(b)Figure 3 Results of BrdU incorporation assay (a) Cytotoxic effect of 10mM metformin was recorded starting from 24th hour of BMSCculture Metformin at 5mM concentration significantly reduced DNA synthesis in BMSC cultures after 72 hours of treatment Metformin atthe lowest investigated concentration did not affect DNA synthesis (b) Increase of DNA synthesis was observed in Balb3T3 after 48 hours ofculture with 1mMmetformin Cytotoxic effect of metformin was prominent after 72 hours of culture especially when Balb3T3 were treatedwith metformin at 5 and 10mM doses Fold change in DNA synthesis was calculated by comparing BrdU signals of metformin-treated cellsto that of the control culture to which a value of 1 was assigned An asterisk (lowast) indicates a statistically significant difference (119901 lt 005)Significance was determined by two-way ANOVA test (119901 lt 005)

33 Morphology of Cells in Cultures with Metformin Evalua-tion of morphological changes was performed after 48-hourpropagation based on the results of cytotoxic assay (Figure 4)

Control culture of murine BMSCs was characterizedby heterogeneous morphology Three distinct cell typeswere present population of fibroblast-like cells with thepredominance of bi- or multipolar cells the most apparentlarge flat cells of irregular shape and small cells adhering tothe surface of large cells Cytoskeleton of smaller fibroblastcells and large flat cells was well developed and nuclei werecentrally localized while the cytoskeleton of small cells wasless developed forming a thin rim around the oval nucleiMoreover small cells had characteristic actin projections onthe edge of the cell bodymdashlamellipodia Small cells weremore numerous in BMSC culture treated with metformin ata concentration of 1mM even though both fibroblast-likeand large cells were still prevalent No signs of cytoskeletondeformation or nuclei degradation were observed Moreoverthe confluence of culture treated with 1mM metforminwas similar to the control culture The amount of deadcells visualized with propidium iodide was increasing withthe metformin dose as shown by quantitative analysis(Figure 5)

Additionally SEM analysis showed that the cells fromcultures propagated in the presence of 1mM metformindeveloped numerous thin cytoskeletal projections (filopodia)and secreted many microvesicles similarly to the cells of thecontrol culture (Figure 6) Metformin added at a concentra-tion of 5mM caused the enlargement of flat cells Isolatedsmall cells attached to the large cells were observed In addi-tion cells with peripherally located nuclei were detectedTheanalysis of cell surface demonstrated that the projectionswerereduced and shortened Significantmorphological changes ofBMSCs were observed when 10mM metformin was addedto the culture The number of cells decreased resulting in

the partial loss of intracellular connections The remainingcells in the culture with 10mM metformin had an irregularshape and the cellular projections were underdeveloped

Although small oval or spindle-shaped cells were char-acteristic for the control culture of Balb3T3 fibroblastsenlarged and multinuclear cells were also present Thecytoskeleton of Balb3T3 cells was well developed in thecase of cells with a typical fibroblast morphotype or largecell body Cytoplasmic organelles of oval-shaped cells werelimited to the rim around nuclei No signs of apoptosis wereobserved The pattern of growth of 3T3control culture wasrandom cells formed aggregates next to densely and evenlyarranged areas of culture The introduction of metformin to3T3Balb culture at a concentration of 1mMand 5mMhadnosignificant effect on the pattern of growth andmorphology ofcells nevertheless a decrease in the number of largemultinu-clear cells was visible Furthermore evaluation of cell surfaceshowed that the cells from cultures with 1mM and 5mMmetformin had well developed filopodia and lamellipodiaCytotoxic effect of metformin at 10mM concentration wasapparent when cell morphology was evaluated Cell bodieswere significantly reduced and cellular debris was dominantin the image SEManalysis showed lack of cellular projectionsand shrunken cell bodies

34 Ultrastructure of Cells Transmission electron micros-copy revealed changes in the structure and arrangement oforganelles after treatment withmetformin (Figure 7) In bothcell types the most evident changes were related to the shapeof the nucleus The nuclei of BMSCs were malformed evenwhen treated with metformin at the lowest concentrationwhile in the case of Balb3T3 changes in the shape of nucleiwere visible after treatment with 5mMmetformin

BMSCs treated with 1mMmetformin had a better devel-oped rough reticulum when compared to the control culture


BMSCs Balb3T3 Balb3T3 M

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Results of propidium iodide stainingResults of phalloidin and DAPI staining

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Figure 4 Morphology of murine BMSCs (left column) and Balb 3T3 (right column) in the control and experimental cultures Three distinctcell types of BMSCs are indicated with white arrows large flat cells (thick arrows) smaller fibroblast-like cells (arrows with dotted shaft) andsmall round cells (thin arrows) Morphotypes of Balb3T3 cells are indicated with yellow arrows large multinucleated cells (thick arrows)fibroblast-shaped cells (thin arrows) and small round cells (arrows with dotted shafts) Cytoskeleton was stained using atto-488 phalloidintherefore cellular bodies are stained in green Nuclei stained with DAPI are visible as white dots while dead cells visualized in the reactionwith propidium iodide are stained in red Magnification 100x scale bar = 200120583m

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(b)Figure 5 The percentage of dead cells quantified after propidium iodide staining Calculation was performed based on the images obtainedfrom three independent experiments Evaluation of cell viability in cultures after 48 h of treatment with 1mM 5mM and 10mMmetforminThe number of dead cells in BMSC cultures (a) and Balb3T3 cultures (b) increased after treatment with metformin at 5 and 10mMconcentration Statistical analysiswas performed in relation to the results obtained for control culture (nometformin) An asterisk () indicatesa statistically significant difference (119901 lt 00001) Significance was determined by one-way ANOVA test


BMSC Balb 3T3

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Figure 6 SEM analysis of cellular membrane projections Imageswere captured at magnification 5000-fold scale bar = 2 120583m

Golgi apparatus was also more apparent The number ofendosomes and peroxisomes in BMSCs treated with 1mMconcentration was comparable to the control culture butmicrovesicles were more abundant The ultrastructural anal-ysis of BMSCs cultured with 5mM concentration of the drugshowed an increase in the number of late endosomes andlysosomes Long cellular projections were also characteristicof BMSCs cultured in 5mM metformin but microvesicleswere observed sporadically BMSCs treated with 10mMmet-formin had an irregular shape and the cytoplasm was filledwith vacuoles The initial stage of apoptotic body formationwas also recorded

A distinctive feature of Balb3T3 cells cultured with1mM metformin was a ridged cellular membrane releasingmicrovesicles and exosomes The addition of metforminat a concentration of 5mM resulted in enhanced produc-tion of endosomes and peroxisomes while the release ofmicrovesicles was reduced Complete damage of Balb3T3cells was recorded in the culture with 10mM metforminMicroscope imaging showed only fragmented nuclei andsmall apoptotic bodies

35 Analysis of Gene Expression The next stage of the studywas to determine the expression of genes associated withproliferative potential of cells Consequently the analysiswas performed on cells derived from cultures propagatedfor 48 hours Quantitative analysis of transcripts revealed

BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m

Figure 7Ultrastructure of BMSCs andBalb3T3 cells of control andexperimental cultures Scale bar = 2120583m nu nucleus en endoso-mes le late endosomes ex exosomes er endoplasmic reticulummv mesenchymal microvesicles ga Golgi apparatus cp cellularprojections ly lysosomes p peroxisomes v vacuoles ab apoptoticbodies

that the expression of H19 and IGF2 in BMSCs was notaltered in the experimental cultures The level of IGF2Rtranscript in BMSCs was constant in cultures with 1mMand 5mM metformin but decreased in the cultures with10mM concentration In turn the expression of Oct-4 genewas increased in BMSC cultures propagated with 10mMmetformin while the transcript level in cultures with 1mMand 5mMmetforminwas not changedwhen compared to thecontrol culture (Figure 8(a))

The expression of H19 gene in Balb3T3 cells was reducedin cultures treated with metformin however significantchanges in transcript level compared to the control culturewere observed only when cells were treated with 5 and 10mMmetformin The investigated concentrations of metformindid not influence the amount of IGF2 transcript in Balb3T3cells in turn lower level of IGF2RmRNAwas observedwhencompared to the control cultureThe level of Oct-4 transcriptwas significantly decreased in cultures treated with 5 and10mMmetformin (Figure 8(b))

36 Concentration of IGF2 Protein in Culture SupernatantsQuantitative analysis of IGF2 concentration showed that thepropagation of BMSCs with 5mM and 10mM metformindecreased the level of this protein Different patterns wereobserved for Balb3T3 where an increase of metformin levelwas positively correlated with the concentration of IGF2protein (Figure 9)


00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)

Figure 8 mRNA expression of H19 insulin-like growth factor 2 (IGF2) its receptor (IGF2R) and Oct-4 in BMSCs (a) and Balb3T3 cellline (b) in control and experimental cultures The level of expression of all genes was calculated in relation to the housekeeping gene beta2 microglobulin (1205732m) Results are presented as the mean of three independent experiments plusmn standard deviation (plusmnSD) Significance wasdetermined by one-way ANOVA test (119901 lt 005) Description of observed relationships was included in the main body of the paper


BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L) lowastlowastlowast

(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast

(b)Figure 9 Quantitative analysis of IGF2 protein level in the supernatants after BMSC (a) and Balb3T3 (b) cultures Statistically significantdifferences were observed at 119901 lt 005 () and 119901 lt 001 ()

4 Discussion

Currentlymetformin is perceived not only as a hypoglycemicagent but also as a comprehensive medication The admin-istration among many others is recommended in medicalconditions associated with metabolic disorders Howeverthe greatest expectations are held with the application ofmetformin in the treatment of cancer and activation ofendogenous adult stem cells [28] Antiaging activity of met-formin combined with the potential of mesenchymal stromalstem cells would provide a perfect solution for the needs ofregenerative medicine mainly due to the fact that metforminmay modulate proliferation of MSCs and promote theirdifferentiation towards osteoblast cells Nevertheless a reviewof the literature indicates that biological effects of metforminare predominantly studied using immortalized cell lineswhile studies that focus on the influence of metformin onmesenchymal multipotent stem cells are limited

In this study we decided to analyze the effect of met-formin on the proliferative activity morphology and ultra-structure of two populations of cells (i) primary culturesof bone marrow-derived multipotent mesenchymal stromalstem cells and (ii) well established fibroblast Balb3T3 cellline As opposed to the committed cells (here fibroblasts)mesenchymal stem cells possess the unique ability to self-renew and to differentiate into other cells of mesodermallineages [29ndash32]

Antitumor effect of metformin was established for dosesfrom 5 to 30mM [4 5 7 8 33] The concentrations ofmetformin added to cultures are well above the concentrationrange in which metformin can be safely used in vivo [3435] However it was also proven that the concentration ofmetformin accumulating in the tissuesmight be several timeshigher than in blood and thus metformin is present at asignificantly higher levels in the target organs [36]

The effect of metformin on BMSCs has been investigatedby Gao et al [37] and Molinuevo et al [38] The study

of Gao et al [37] demonstrated that BMSCs cultured inosteogenic conditions and treated with 100120583M metforminresponded with an increase in proliferative activity andosteogenesis Molinuevo et al [38] reported that metformininduced osteogenesis of BMSCs both in vitro and in vivoand enhanced the process of bone repair in diabetic andnondiabetic rats However both experiments were disputedby the Jeyabalan et al [39] Both Gao and Molinuevo usedlow doses of metformin in their in vitro models while wewere interested in the effect of higher concentrations ofmetformin that is 1mM 5mM and 10mM which are thedoses exerting antitumor effect Our results demonstratedsignificant inhibition of BMSC proliferation rate at 10mMconcentration ofmetforminThe addition of 1mM and 5mMdoses decreased proliferative activity of mouse BMSCs butonly after 72 hours of propagation Recently Abu-Zaiton [40]showed that the proliferation of dermal fibroblasts is affectedby metformin in a dose-dependent manner Our resultsshowed that metformin at a concentration of 1mM and5mM exerted comparable effect on the proliferative activityof Balb3T3 fibroblasts Interestingly fibroblast proliferativepotential after application of 1mM and 5mMmetformin wasrestored after 72 hours of cultureNevertheless the effect of allconcentrations investigated significantly reduced fibroblastproliferative activity and this result is consistent with Abu-Zaitonrsquos findings

The influence of metformin on physiology of BMSCs andfibroblasts has been thus far predominantly studied in thecontext of their proliferation [38 40] However these studiesused lower concentration of metformin and experimentswere performed independently In our model due to theuse of two cell populations of stromal origin but differingin the context of proliferative potential (nonimmortalizedBMSCs and immortalized Balb3T3) we could perform com-parative analysis of their cytophysiological properties Ourexperiment shows that proliferation activity of BMSCs andBalb3T3 fibroblasts is significantly inhibited after exposition


to 10mM metformin Growth of both BMSCs and Balb3T3was significantly influenced by metformin at 1mM and5mM concentration after 72 hThe difference in proliferativepotential of BMSCs and Balb3T3 is significant which isconfirmed by the growth curves Balb3T3 cells attemptedto restore the population despite the inhibition of metabolicactivity after exposition to 1mM and 5mMmetformin whileBMSC growth was declined

Decrease in cell proliferation can be associated withcytotoxic effect of metformin which has been previouslyobserved in various cancer cell lines [4 5 7 8 33]The resultsof the BrdU assay confirmed that metformin has great impacton cellular activity of both BMSCs and Balb3T3 in a dose-dependent manner Significant inhibition of the number ofactively proliferating cells expressed by the reduction ofDNA synthesis was noted after treatment with 5mM and10mM metformin This finding was also consistent withthe results of propidium iodide staining The percentage ofdead cells increased in cultures treated with 5 and 10mMmetformin Metformin affected physiology of the cells notonly by reducing proliferating activity but also by causingmorphological and ultrastructural alterations

Undeniably significant part of our experimental modelwas focused on the evaluation of morphological and ultra-structural changes of cells after metformin treatment Weargue that the analysis of cellular organization should be a keyparameter when determining the effects of active agents suchasmetformin even though the evaluation of cell proliferationand gene expression becomes an important complement tosuch analysis Detailed investigation of cell morphology isoften neglected due to the difficult and time-consumingtechniques or is sometimes reduced to the analysis of cellshapes and growth patterns with an inverted light or flu-orescence microscope as in the papers discussed [38 40]To the best of our knowledge the present study is the firstreport showing morphological and ultrastructural changesof BMSCs and Balb3T3 fibroblasts under the influence ofmetformin

Morphology is an important large-scale manifestation ofthe global organizational and physiological state of the cells[41] therefore we believe that the presented approach willhelp understand how structure and function of cells exam-ined are interrelated The analysis of cell morphology in thecontext of anticancer compounds may provide meaningfuldata of clinical significance because traditional diagnosisof pathology is established on the basis of morphologicalfindings [42]

Epifluorescence microscopy in BMSC control culturerevealed the occurrence of three distinct morphotypes Ourobservation corresponds with findings presented by Ren etal [20] and Wieczorek et al [43] who have also describedthree different types of BMSCs (i) large flat cells (ii)smaller fibroblast-like cells and (iii) small round cells Ourresults demonstrated that with increasing concentrations ofmetformin the number of small round cells is reduced Thestudy of Ren et al [20] indicated that cultures enriched insmall cells had a greater multipotent differentiation potentialthan the cultures with a higher ratio of large cells Thereforewe concluded that high doses of metformin may negatively

affect the ldquostemnessrdquo of BMSCs Similarly SEM analysisshowed that the number of cellular projections decreasedwith increasing concentrations of metformin Ultrastructuralevaluation showed signs of BMSC autophagy after met-formin treatment at a concentration of 5mM reflected inthe fusion of late endosomes with lysosomes [43] Nucleardisorganization was also characteristic of cells treated withmetformin Three types of cells could also be distinguishedin the Balb3T3 fibroblast cultures that were typical forthis cell line that is (i) small round cells (ii) fibroblast-shaped cells and (iii) multinucleated large cells Holt andGrainger [44] argued that immortalized fibroblasts may formmultinucleated cells via fusionwith other fibroblasts which istypical of various pathologies such as fibrosis cancer agingand foreign body response (FBR) Our results showed thatthe number of multinucleated giant cells is reduced in theBalb3T3 cultures treated with metformin at a concentrationof 1mM and 5mM Cytotoxic effect of the highest dosageof metformin in Balb3T3 was confirmed by means offluorescent and scanning electron microscopy Evaluationof cellular projections with SEM showed that the culturestreated with 1mM and 5mM metformin had well developedfilopodia and lamellipodia Ultrastructural analysis revealedsignificant alterations in cellular organization after treatmentwith 5mMmetformin

Analysis of gene expression of the components of IGF2signaling pathway showed that the expression of IGF2 genewas not affected by metformin treatment neither in BMSCnor Balb3T3 cell line The expression of H19 in BMSCsdemonstrated that metformin did not influence the levelof mRNA What is more the level of H19 transcript wascomparable with IGF2 mRNA level which contributes tomaintaining the proliferation balance [15] Expression ofIGF2 receptor mRNA was decreased in all experimentalcultures of Balb3T3 while in BMSCs IGF2R expressionwas reduced in cultures treated with 10mM metforminThe expression of H19 gene in the case of Balb3T3 wassignificantly reduced after treatment with 5mM and 10mMconcentrations when compared to control

Synthesis of IGF2 mRNA seems to be functioning ina constitutive manner IGF2 is strongly involved in cellproliferation survival and migration [15 45] A decreasein the IGF2 protein contents in the supernatants collectedafter BMSC cultures with metformin correlated with the lossof proliferation potential of the cells However an increaseof IGF level was positively correlated with higher dosesof metformin in Balb3T3 cell line We believe that thisphenomenon might be associated with prevention of celldeath particularly in view of a significant decrease in theexpression of Oct-4 gene in Balb3T3 cells treated with 5 and10mM metformin It was shown that fibroblasts show basalexpression of certain pluripotency related genes includingOct-4 and culture conditions may affect the expression pat-tern of these genes [46] Our results indicate that metforminat 5 and 10mM concentration may decrease the transcriptlevel ofOct-4 in Balb3T3 cultures In turn the differentiationmarkers of BMSC linage are well established whereas onlyfew literature reports concern the expression of pluripotentgenes including Oct-4 which is a critical transcription factor


regulating self-renewal and differentiation of stem cells [2047] As previously reported [48] Oct-4 expression may beinduced in somatic cells in response to various stress stimuliThis thesis could be true in the case of BMSC culture asthe expression of Oct-4 was significantly enhanced aftertreatment with 10mMmetformin

5 Conclusions

In summary metformin introduced to BMSC and Balb3T3cultures at a concentration equal to 5mM and 10mM exertedcytotoxic effect which was reflected in (i) a decrease of cellproliferation (ii) increase in the incidence of cell death and(iii) disintegration of cultures manifested with morphologi-cal and ultrastructural changes Undoubtedly it is necessaryto elucidate the molecular mechanisms determining theunderlying effects of metformin both in vitro and in vivonevertheless our results demonstrate that the analysis ofcell morphology and ultrastructure may provide additionalinformation that may help to understand the complexityof metformin action We believe that the analysis of cellmorphology in the context of anticancer compounds mayprovide meaningful information of clinical significance

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The research was supported by Wroclaw Research CentreEIT+ under the Project ldquoBiotechnologies and AdvancedMedical Technologiesrdquo BioMed (POIG010102-02-00308)founded from the European Regional Development Fund(Operational Program Innovative Economy 112) Publica-tion supported by Wroclaw Centre of Biotechnology pro-gramme the Leading National Research Centre (KNOW) foryears 2014ndash2018

References

[1] L B A Rojas and M B Gomes ldquoMetformin an old but stillthe best treatment for type 2 diabetesrdquoDiabetology ampMetabolicSyndrome vol 5 no 1 article 6 2013

[2] T V Kourelis and R D Siegel ldquoMetformin and cancer newapplications for an old drugrdquo Medical Oncology vol 29 no 2pp 1314ndash1327 2012

[3] B Viollet B Guigas N Sanz Garcia J Leclerc M Foretz and FAndreelli ldquoCellular and molecular mechanisms of metforminan overviewrdquo Clinical Science vol 122 no 6 pp 253ndash270 2012

[4] I N Alimova B Liu Z Fan et al ldquoMetformin inhibits breastcancer cell growth colony formation and induces cell cyclearrest in vitrordquo Cell Cycle vol 8 no 6 pp 909ndash915 2009

[5] B Liu Z Fan S M Edgerton et al ldquoMetformin induces uniquebiological and molecular responses in triple negative breastcancer cellsrdquo Cell Cycle vol 8 no 13 pp 2031ndash2040 2009

[6] E Giovannucci D M Harlan M C Archer et al ldquoDiabetesand cancer a consensus reportrdquoDiabetes Care vol 33 no 7 pp1674ndash1685 2010

[7] Q Luo D Hu S Hu M Yan Z Sun and F Chen ldquoIn vitro andin vivo anti-tumor effect of metformin as a novel therapeuticagent in human oral squamous cell carcinomardquo BMC Cancervol 12 article 517 2012

[8] E Karnevi K Said R Andersson and A H RosendahlldquoMetformin-mediated growth inhibition involves suppressionof the IGF-I receptor signalling pathway in human pancreaticcancer cellsrdquo BMC Cancer vol 13 article 235 2013

[9] W H Gotlieb J Saumet M-C Beauchamp et al ldquoIn vitrometformin anti-neoplastic activity in epithelial ovarian cancerrdquoGynecologic Oncology vol 110 no 2 pp 246ndash250 2008

[10] N Wu C Gu H Gu H Hu Y Han and Q Li ldquoMetformininduces apoptosis of lung cancer cells through activatingJNKp38 MAPK pathway and GADD153rdquo Neoplasma vol 58no 6 pp 482ndash490 2011

[11] H-P Chen J-J Shieh C-C Chang et al ldquoMetformin decreaseshepatocellular carcinoma risk in a dose-dependent mannerpopulation-based and in vitro studiesrdquo Gut vol 62 no 4 pp606ndash615 2013

[12] V N Anisimov L M Berstein I G Popovich et al ldquoIfstarted early in life metformin treatment increases life span andpostpones tumors in female SHR micerdquo Aging vol 3 no 2 pp148ndash157 2011

[13] T Chiba T Tsuchiya T Komatsu R Mori H Hayashi andI Shimokawa ldquoDevelopment of calorie restriction mimetics astherapeutics for obesity diabetes inflammatory and neurode-generative diseasesrdquo Current Genomics vol 11 no 8 pp 562ndash567 2010

[14] S N Hardouin R Guo P-H Romeo A Nagy and J EAubin ldquoImpaired mesenchymal stem cell differentiation andosteoclastogenesis in mice deficient for Igf2-P2 transcriptsrdquoDevelopment vol 138 no 2 pp 203ndash213 2011

[15] M Z Ratajczak ldquoIgf2-H19 an imprinted tandem gene is animportant regulator of embryonic development a guardianof proliferation of adult pluripotent stem cells a regulator oflongevity and a lsquopasskeyrsquo to cancerogenesisrdquo Folia Histochemicaet Cytobiologica vol 50 no 2 pp 171ndash179 2012

[16] C D Porada and G Almeida-Porada ldquoMesenchymal stemcells as therapeutics and vehicles for gene and drug deliveryrdquoAdvanced Drug Delivery Reviews vol 62 no 12 pp 1156ndash11662010

[17] M Scanu L Mancuso and G Cao ldquoEvaluation of the useof human mesenchymal stem cells for acute toxicity testsrdquoToxicology in Vitro vol 25 no 8 pp 1989ndash1995 2011

[18] K Marycz J Krzak-Ros A Donesz-Sikorska and A SmieszekldquoThe morphology proliferation rate and population doublingtime factor of adipose-derivedmesenchymal stem cells culturedon to non-aqueous SiO

2 TiO2 and hybrid sol-gel-derived oxide

coatingsrdquo Journal of Biomedical Materials Research A vol 102no 11 pp 4017ndash4026 2014

[19] G Wu and H R Scholer ldquoRole of Oct4 in the early embryodevelopmentrdquo Cell Regeneration vol 3 no 1 article 7 2014

[20] H Ren Y Cao Q Zhao et al ldquoProliferation and differentiationof bone marrow stromal cells under hypoxic conditionsrdquoBiochemical and Biophysical Research Communications vol 347no 1 pp 12ndash21 2006

[21] P J Mishra J W Glod and D Banerjee ldquoMesenchymal stemcells flip side of the coinrdquo Cancer Research vol 69 no 4 pp1255ndash1258 2009

[22] M Fischer K Timper T Radimerski et al ldquoMetformin inducesglucose uptake in human preadipocyte-derived adipocytes


from various fat depotsrdquoDiabetes Obesity and Metabolism vol12 no 4 pp 356ndash359 2010

[23] K Marycz A Smieszek J Grzesiak A Donesz-Sikorska and JKrzak-Ros ldquoApplication of bone marrow and adipose-derivedmesenchymal stem cells for testing the biocompatibility ofmetal-based biomaterials functionalized with ascorbic acidrdquoBiomedical Materials vol 8 no 6 Article ID 065004 2013

[24] M Maredziak K Marycz A Smieszek D Lewandowski andN Y Toker ldquoThe influence of static magnetic fields on canineand equine mesenchymal stem cells derived from adiposetissuerdquo In Vitro Cellular and Developmental BiologymdashAnimalvol 50 no 6 pp 562ndash571 2014

[25] A Witek-Krowiak D Podstawczyk K Chojnacka A Dawiecand K Marycz ldquoModelling and optimization of chromiumIII biosorption on soybean mealrdquo Central European Journal ofChemistry vol 11 no 9 pp 1505ndash1517 2013

[26] K Kalinski K Marycz J Czogała E Serwa and W JaneczekldquoAn application of scanning electron microscopy combinedwith roentgen microanalysis (SEMndashEDS) in canine urolithia-sisrdquo Journal of ElectronMicroscopy (Tokyo) vol 61 no 1 pp 47ndash55 2012

[27] P Chomczynski and N Sacchi ldquoSingle-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextractionrdquo Analytical Biochemistry vol 162 no 1 pp 156ndash1591987

[28] J A Menendez and A Vazquez-Martin ldquoRejuvenating regen-eration metformin activates endogenous adult stem cellsrdquo CellCycle vol 11 no 19 pp 3521ndash3522 2012

[29] P J Marie and O Fromigue ldquoOsteogenic differentiation ofhuman marrow-derived mesenchymal stem cellsrdquo RegenerativeMedicine vol 1 no 4 pp 539ndash548 2006

[30] E Luzi F Marini S C Sala I Tognarini G Galli and M LBrandi ldquoOsteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting ofthe SMAD1 transcription factorrdquo Journal of Bone and MineralResearch vol 23 no 2 pp 287ndash295 2008

[31] K Tamama C K Sen and A Wells ldquoDifferentiation ofbone marrow mesenchymal stem cells into the smooth musclelineage by blocking ERKMAPK signaling pathwayrdquo Stem Cellsand Development vol 17 no 5 pp 897ndash908 2008

[32] L A Solchaga K J Penick and J F Welter ldquoChondrogenicdifferentiation of bone marrow-derived mesenchymal stemcells tips and tricksrdquo Methods in Molecular Biology vol 698pp 253ndash278 2011

[33] M Zakikhani R Dowling I G Fantus N Sonenberg andM Pollak ldquoMetformin is an AMP kinase-dependent growthinhibitor for breast cancer cellsrdquo Cancer Research vol 66 no21 pp 10269ndash10273 2006

[34] C-F Sum J M Webster A B Johnson C Catalano B GCooper and R Taylor ldquoThe effect of intravenous metformin onglucose metabolism during hyperglycaemia in type 2 diabetesrdquoDiabetic Medicine vol 9 no 1 pp 61ndash65 1992

[35] M Foretz S Hebrard J Leclerc et al ldquoMetformin inhibits hep-atic gluconeogenesis inmice independently of the LKB1AMPKpathway via a decrease in hepatic energy staterdquo Journal ofClinical Investigation vol 120 no 7 pp 2355ndash2369 2010

[36] C Wilcock and C J Bailey ldquoAccumulation of metformin bytissues of the normal and diabetic mouserdquo Xenobiotica vol 24no 1 pp 49ndash57 1994

[37] Y Gao J Xue X Li Y Jia and J Hu ldquoMetformin regulatesosteoblast and adipocyte differentiation of rat mesenchymal

stem cellsrdquo Journal of Pharmacy and Pharmacology vol 60 no12 pp 1695ndash1700 2008

[38] M S Molinuevo L Schurman A D McCarthy et al ldquoEffect ofmetformin on bone marrow progenitor cell differentiation invivo and in vitro studiesrdquo Journal of Bone andMineral Researchvol 25 no 2 pp 211ndash221 2010

[39] J Jeyabalan B Viollet P Smitham et al ldquoThe anti-diabeticdrug metformin does not affect bone mass in vivo or fracturehealingrdquo Osteoporosis International vol 24 no 10 pp 2659ndash2670 2013

[40] A S Abu-Zaiton ldquoEffect of metformin on proliferation of skinderived stem cells in the case of normoxia and hypoxiardquo Journalof Biological Sciences vol 14 no 2 pp 149ndash153 2014

[41] Z Pincus and J A Theriot ldquoComparison of quantitativemethods for cell-shape analysisrdquo Journal of Microscopy vol 227no 2 pp 140ndash156 2007

[42] M Nakamura and K KakudoMolecular Mechanism and Mor-phology in Cancer Foreword Bentham eBooks Chiba Japan2009

[43] G Wieczorek C Steinhoff R Schulz et al ldquoGene expressionprofile of mouse bone marrow stromal cells determined bycDNA microarray analysisrdquo Cell and Tissue Research vol 311no 2 pp 227ndash237 2003

[44] D J Holt and D W Grainger ldquoMultinucleated giant cells fromfibroblast culturesrdquo Biomaterials vol 32 no 16 pp 3977ndash39872011

[45] T Schilling C Schonefeldt S Zeck et al ldquoLocal expression ofinsulin-like growth factor (IGF) signaling components duringin vitro aging of mesenchymal stem cellsrdquo Journal of Stem Cellsamp Regenerative Medicine vol 2 no 1 pp 41ndash42 2007

[46] R L Page S Ambady W F Holmes et al ldquoInduction ofstem cell gene expression in adult human fibroblasts withouttransgenesrdquo Cloning and Stem Cells vol 11 no 3 pp 417ndash4262009

[47] Y X Fan C H Gu Y L Zhang et al ldquoOct4 and Sox2 overex-pression improves the proliferation and differentiation of bonemesenchymal stem cells in Xiaomeishan porcinerdquo Genetics andMolecular Research vol 12 no 4 pp 6067ndash6079 2013

[48] H Obokata T Wakayama Y Sasai et al ldquoStimulus-triggeredfate conversion of somatic cells into pluripotencyrdquo Nature vol505 no 7485 pp 641ndash647 2014

Submit your manuscripts athttpwwwhindawicom

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Anesthesiology Research and Practice

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Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


other CRM drugs have not been fully explored It seems thatmetformin affects endocrine regulatory systems and insulin-like growth factors [13] Signaling pathway of insulin-likegrowth factors (IGF) regulates cell proliferation differenti-ation aging and life span thus its role is principal for thedevelopment of the organism and has remained unchangedduring evolution [14] IGF2 together with theH19 gene forman imprinted tandem both in humans and in mice that playsan important role not only during embryonic developmentbut also during the proliferation of stem cells residing in adulttissues [14 15]

Bone marrow provides a niche for various populationsof stem cells the interplay of which is essential for bodyhomeostasis Biology of the bone marrow-derived multipo-tent mesenchymal stromal cells (BMSCs) is continuouslybeing studied Their potential for self-renewal as well as highphenotypic plasticitymanifested by the ability to differentiateinto bone cartilage or adipose tissue is extremely importantin terms of regenerative medicine [16] Mesenchymal stromalstem cells (MSCs) due to a high phenotypic and cellularplasticity are a suitable model for in vitro assessment ofvarious biological and chemical agents [17] Additionallyevaluation of alterations in MSC morphology provides valu-able information that reflects complex biological processescontrolled by the interactions between the cytoskeleton andthe extracellular environment [18]

The properties of self-renewal and differentiation of stemcells might be regulated by octamer-binding protein 4 (Oct-4) a transcription factor crucial for embryonic development[19] The expression of Oct-4 was reported in bone marrow-derived stromal cells which confirms high phenotypic plas-ticity of these cells [20] Impairment of the proliferationpotential of mesenchymal stem cells may account for regen-erative potential deficiency of the organism Mesenchymalstem cells seem to participate in the process of bioactivestroma formation [21] and affect the biological properties ofsurrounding tissues Due to the fact that metformin increasesglucose uptake in connective and embryonic tissues [22]their effect on proliferative activity of BMSCs and othercells of connective tissue such as fibroblasts should beconsidered In the present work we have evaluated the effectof metformin in vitro using murine primary cultures of bonemarrow-derived multipotent mesenchymal stromal cells andBalb3T3 fibroblast cell line

We have investigated the effect of metformin in cellcultures at doses cytotoxic for cancer cells [4 5 7ndash9] Ourobjective was to determine how different concentrations ofmetformin affect the physiology of stromal cellsThe analysisincluded BMSC and Balb3T3 proliferation activity assaysand evaluation of the morphology and ultrastructure of cellsinvestigatedWe have also aimed to determine the expressionof IGF signaling components (IGF2 IGF2R and H19) as wellas the expression of Oct-4

2 Materials and Methods

All reagents used in this experiment were purchased fromSigma-Aldrich (Poland) unless indicated otherwise

21 Ethical Approval The study was conducted with theapproval of the Bioethics Committee as stated by the SecondLocal Bioethics Committee at the Department of Biology andAnimal Breeding Wroclaw University of Environmental andLife Sciences Wroclaw Chelmonskiego 38C Poland (Decnumber 1772010 of 11152010)

22 Cell Population Two types of mouse cells were used inthe experiments multipotent stromal cells (BMSCs) derivedfrom bone marrow (primary cultures) and Balb3T3 embry-onic fibroblasts (cell line obtained from the Institute ofImmunology and Experimental Therapy Polish Academy ofSciences)

23 Isolation of BMSCs Bone marrow-derived multipotentmesenchymal stromal cells (BMSCs) were isolated fromtwelve 4-week-old C57BL6 mice (Animal Vivarium Wro-clawMedical School Poland) Femurs were collected directlyafter euthanasia of the animal and placed in a sterile Hanksrsquobalanced salt solution (HBSS) Cells were isolated from thebone marrow by flushing with an insulin syringe U-40(29G X 1210158401015840 needle) filled with HBSS Cell suspension wastransferred into falcon tube and centrifuged at 300timesg for 4minutes For FACS analysis bone marrow cells were lysedin BD lysing buffer (BD Biosciences San Jose CA USA) for15min at room temperature and washed twice in phosphate-buffered saline (PBS) For cell culture pellets were resus-pended in Dulbeccorsquos Modified Eaglersquos Medium (DMEM)containing Hamrsquos F-12 nutrient mixture supplemented with10 of foetal bovine serum (FBS) and transferred to cultureflasks

24 Characterization of BMSCs Phenotype Flow CytometryBone marrow cell suspensions isolated by flushing femursand tibia were lysed in BD lysing buffer (BD Biosciences SanJose CA USA) for 15min at room temperature and washedtwice in phosphate-buffered saline (PBS) The cells were sub-sequently stained for Sca-1 antigen and hematopoietic lineagemarkers (Lin) for 30min in medium containing 2 foetalbovine serum The following anti-mouse antibodies (BDPharmingen) were used for staining Sca-1 (FITC clone D7)B220 (PE clone RA3-6B2) T-cell receptor-120573 (PE clone H57-597) T-cell receptor-120574120575 (PE clone GL3) CD11b (PE cloneM170) Ter119 (PE clone TER-119) and Gr-1 (PE clone RB6-8 C5) Sca-1+Linminus cells were isolated by a multiparameterlive-cell sorting (INFLUX BD)

To phenotype BMSC cell surface antigens Sca-1+Linminuscells were stained using the following CD31 (APC clone390) CD45 (APC-Cy7 clone 30-F11) CD51 (biotin cloneRMV-7 with streptavidin conjugated to PE-Cy5) CD73(FITC clone B5) CD90 (PB clone 53-21) and CD105 (PEMJ718) All monoclonal antibodies (mAbs) were added atsaturating concentrations and the cells were incubated for30 minutes on ice washed twice resuspended in stainingbuffer at a concentration of 5 times 106 cells per millilitre andanalyzed using an LSR II (BD Biosciences Mountain ViewCA httpwwwbdbiosciencescom) Anti-mouse mAbswere purchased fromBDPharmingen (SanDiego CA httpwwwbdbiosciencescom)




2and 95 air Primary



























3 Results



0

500

15 k

10 k

717838

844

556

128106

Cou

nt

0

500

15 k

10 k

Cou

nt

0

500

15 k

10 k

Cou

nt

0

500

20 k

15 k

25 k

10 kCou

nt

CD 45

0

500

15 k

10 k

Cou

nt

CD 51

CD 31

0minus103 103 104 105

0minus103 103 104 105

0minus103 103 104 105 0minus103 103 104 105

CD 45 APC Cy70minus103 103 104 105

0minus103 103 104 105

CD 31 APC

CD 51 PE Cy5

0

200

400

600

Cou

nt

CD 73

675823

CD 73 FITC

656

894

CD 90 PB

CD 90

971

761

CD 105 PE

CD 105

(a)

(b) (c) (d)



000

005

010

015

020

025

030

035

24 48 72

lowast

lowast

lowast

lowast


ΔΔA

CTRL1mM

5mM10mM

(h)

(a)

lowast

lowast

lowast lowast000

005

010

015

020

025

030

035

ΔΔA


24 48 72

CTRL

(h)

1mM5mM10mM







00

05

10

15

20Re

lativ

e DN

A sy

nthe


lowastlowast

lowastlowast

24 48 72

(h)

(a)

00

05

10

15

20

Relat

ive D

NA

synt

hesis

lowast

lowast

lowast

lowast

24 48 72

(h)












etfo

rmin

conc

entr

atio

nBMSCs


0mM(CTRL)

1mM

5mM

10mM


0

20

40

60

CTRL 1mM 5mM 10mM

lowast

lowast

Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f BM

SCS

()


(a)

lowast

lowast

0

20

40

60


Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f Bal

b3

T3(

)



BMSC Balb 3T3

1mM

Met

form

in co

ncen

trat

ion

0mM(CTRL)

1mM

5mM

10mM





BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m






00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




2and 95 air Primary



























3 Results



0

500

15 k

10 k

717838

844

556

128106

Cou

nt

0

500

15 k

10 k

Cou

nt

0

500

15 k

10 k

Cou

nt

0

500

20 k

15 k

25 k

10 kCou

nt

CD 45

0

500

15 k

10 k

Cou

nt

CD 51

CD 31

0minus103 103 104 105

0minus103 103 104 105

0minus103 103 104 105 0minus103 103 104 105

CD 45 APC Cy70minus103 103 104 105

0minus103 103 104 105

CD 31 APC

CD 51 PE Cy5

0

200

400

600

Cou

nt

CD 73

675823

CD 73 FITC

656

894

CD 90 PB

CD 90

971

761

CD 105 PE

CD 105

(a)

(b) (c) (d)



000

005

010

015

020

025

030

035

24 48 72

lowast

lowast

lowast

lowast


ΔΔA

CTRL1mM

5mM10mM

(h)

(a)

lowast

lowast

lowast lowast000

005

010

015

020

025

030

035

ΔΔA


24 48 72

CTRL

(h)

1mM5mM10mM







00

05

10

15

20Re

lativ

e DN

A sy

nthe


lowastlowast

lowastlowast

24 48 72

(h)

(a)

00

05

10

15

20

Relat

ive D

NA

synt

hesis

lowast

lowast

lowast

lowast

24 48 72

(h)












etfo

rmin

conc

entr

atio

nBMSCs


0mM(CTRL)

1mM

5mM

10mM


0

20

40

60

CTRL 1mM 5mM 10mM

lowast

lowast

Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f BM

SCS

()


(a)

lowast

lowast

0

20

40

60


Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f Bal

b3

T3(

)



BMSC Balb 3T3

1mM

Met

form

in co

ncen

trat

ion

0mM(CTRL)

1mM

5mM

10mM





BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m






00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


















3 Results



0

500

15 k

10 k

717838

844

556

128106

Cou

nt

0

500

15 k

10 k

Cou

nt

0

500

15 k

10 k

Cou

nt

0

500

20 k

15 k

25 k

10 kCou

nt

CD 45

0

500

15 k

10 k

Cou

nt

CD 51

CD 31

0minus103 103 104 105

0minus103 103 104 105

0minus103 103 104 105 0minus103 103 104 105

CD 45 APC Cy70minus103 103 104 105

0minus103 103 104 105

CD 31 APC

CD 51 PE Cy5

0

200

400

600

Cou

nt

CD 73

675823

CD 73 FITC

656

894

CD 90 PB

CD 90

971

761

CD 105 PE

CD 105

(a)

(b) (c) (d)



000

005

010

015

020

025

030

035

24 48 72

lowast

lowast

lowast

lowast


ΔΔA

CTRL1mM

5mM10mM

(h)

(a)

lowast

lowast

lowast lowast000

005

010

015

020

025

030

035

ΔΔA


24 48 72

CTRL

(h)

1mM5mM10mM







00

05

10

15

20Re

lativ

e DN

A sy

nthe


lowastlowast

lowastlowast

24 48 72

(h)

(a)

00

05

10

15

20

Relat

ive D

NA

synt

hesis

lowast

lowast

lowast

lowast

24 48 72

(h)












etfo

rmin

conc

entr

atio

nBMSCs


0mM(CTRL)

1mM

5mM

10mM


0

20

40

60

CTRL 1mM 5mM 10mM

lowast

lowast

Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f BM

SCS

()


(a)

lowast

lowast

0

20

40

60


Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f Bal

b3

T3(

)



BMSC Balb 3T3

1mM

Met

form

in co

ncen

trat

ion

0mM(CTRL)

1mM

5mM

10mM





BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m






00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

500

15 k

10 k

717838

844

556

128106

Cou

nt

0

500

15 k

10 k

Cou

nt

0

500

15 k

10 k

Cou

nt

0

500

20 k

15 k

25 k

10 kCou

nt

CD 45

0

500

15 k

10 k

Cou

nt

CD 51

CD 31

0minus103 103 104 105

0minus103 103 104 105

0minus103 103 104 105 0minus103 103 104 105

CD 45 APC Cy70minus103 103 104 105

0minus103 103 104 105

CD 31 APC

CD 51 PE Cy5

0

200

400

600

Cou

nt

CD 73

675823

CD 73 FITC

656

894

CD 90 PB

CD 90

971

761

CD 105 PE

CD 105

(a)

(b) (c) (d)



000

005

010

015

020

025

030

035

24 48 72

lowast

lowast

lowast

lowast


ΔΔA

CTRL1mM

5mM10mM

(h)

(a)

lowast

lowast

lowast lowast000

005

010

015

020

025

030

035

ΔΔA


24 48 72

CTRL

(h)

1mM5mM10mM







00

05

10

15

20Re

lativ

e DN

A sy

nthe


lowastlowast

lowastlowast

24 48 72

(h)

(a)

00

05

10

15

20

Relat

ive D

NA

synt

hesis

lowast

lowast

lowast

lowast

24 48 72

(h)












etfo

rmin

conc

entr

atio

nBMSCs


0mM(CTRL)

1mM

5mM

10mM


0

20

40

60

CTRL 1mM 5mM 10mM

lowast

lowast

Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f BM

SCS

()


(a)

lowast

lowast

0

20

40

60


Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f Bal

b3

T3(

)



BMSC Balb 3T3

1mM

Met

form

in co

ncen

trat

ion

0mM(CTRL)

1mM

5mM

10mM





BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m






00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


000

005

010

015

020

025

030

035

24 48 72

lowast

lowast

lowast

lowast


ΔΔA

CTRL1mM

5mM10mM

(h)

(a)

lowast

lowast

lowast lowast000

005

010

015

020

025

030

035

ΔΔA


24 48 72

CTRL

(h)

1mM5mM10mM







00

05

10

15

20Re

lativ

e DN

A sy

nthe


lowastlowast

lowastlowast

24 48 72

(h)

(a)

00

05

10

15

20

Relat

ive D

NA

synt

hesis

lowast

lowast

lowast

lowast

24 48 72

(h)












etfo

rmin

conc

entr

atio

nBMSCs


0mM(CTRL)

1mM

5mM

10mM


0

20

40

60

CTRL 1mM 5mM 10mM

lowast

lowast

Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f BM

SCS

()


(a)

lowast

lowast

0

20

40

60


Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f Bal

b3

T3(

)



BMSC Balb 3T3

1mM

Met

form

in co

ncen

trat

ion

0mM(CTRL)

1mM

5mM

10mM





BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m






00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00

05

10

15

20Re

lativ

e DN

A sy

nthe


lowastlowast

lowastlowast

24 48 72

(h)

(a)

00

05

10

15

20

Relat

ive D

NA

synt

hesis

lowast

lowast

lowast

lowast

24 48 72

(h)












etfo

rmin

conc

entr

atio

nBMSCs


0mM(CTRL)

1mM

5mM

10mM


0

20

40

60

CTRL 1mM 5mM 10mM

lowast

lowast

Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f BM

SCS

()


(a)

lowast

lowast

0

20

40

60


Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f Bal

b3

T3(

)



BMSC Balb 3T3

1mM

Met

form

in co

ncen

trat

ion

0mM(CTRL)

1mM

5mM

10mM





BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m






00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



etfo

rmin

conc

entr

atio

nBMSCs


0mM(CTRL)

1mM

5mM

10mM


0

20

40

60

CTRL 1mM 5mM 10mM

lowast

lowast

Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f BM

SCS

()


(a)

lowast

lowast

0

20

40

60


Dea

d ce

lls in

expe

rimen

tal c

ultu

res o

f Bal

b3

T3(

)



BMSC Balb 3T3

1mM

Met

form

in co

ncen

trat

ion

0mM(CTRL)

1mM

5mM

10mM





BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m






00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


BMSC Balb 3T3

1mM

Met

form

in co

ncen

trat

ion

0mM(CTRL)

1mM

5mM

10mM





BMSC Balb 3T3

Met

form

in co

ncen

trat

ion

nu

nu

nu

nu

nu

nu

nu

er

ga

er

en

en

p

p

mvmv

er

lyle

mv

cp

le

en v

nu

ab

mvex

p

en en

0mM(CTRL)

1mM

5mM

10mM

mv

2120583m

2120583m 2120583m

2120583m

2120583m2120583m

2120583m 2120583m






00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

00

02

04

06

08

10

H19

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

lowast lowast

H19

1205732

m m

RNA

(AU

)

IGF2

1205732

m m

RNA

(AU

)

IGF2

R1205732

m m

RNA

(AU

)

Oct

-41205732

m m

RNA

(AU

)

Oct-4

IGF2

IGF2R

(a)

lowast lowast

lowast

lowast lowast

lowastlowast

00

02

04

06

08

10 H19

H19

1205732

m m

RNA

(AU

)

00

02

04

06

08

10

IGF2

1205732

m m

RNA

(AU

)

IGF2

00

02

04

06

08

10

IGF2

R1205732

m m

RNA

(AU

)

00

02

04

06

08

10

Oct

-41205732

m m

RNA

(AU

)

Oct-4IGF2R

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

(b)



BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


BMSC

s CTR

L

BMSC

s1m

M

BMSC

s5m

M

BMSC

s10

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm


(a)

Balb3

T3CT

RL

Balb3

T31

mM

Balb3

T35

mM

Balb3

T310

mM

0

500

1000

1500

2000

2500

IGF2

conc

entr

atio

n (p

gm

L)

lowast lowast

lowastlowast


4 Discussion


















5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












5 Conclusions




Acknowledgments


References


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Effect of Metformin on Viability...

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Transcript of Research Article Effect of Metformin on Viability...