Conditioned Media from Human Adipose Tissue-Derived … C Biomed Res Int. 2014.pdf · 2014. 8. 8. · ResearchArticle Conditioned Media from Human Adipose Tissue-Derived Mesenchymal

Research ArticleConditioned Media from Human Adipose Tissue-DerivedMesenchymal Stem Cells and Umbilical Cord-DerivedMesenchymal Stem Cells Efficiently Induced the Apoptosisand Differentiation in Human Glioma Cell Lines In Vitro

Chao Yang1 Deqiang Lei2 Weixiang Ouyang3 Jinghua Ren4

Huiyu Li1 Jingqiong Hu1 and Shiang Huang1

1 Stem Cell Center Wuhan Union Hospital Tongji Medical College Huazhong University of Science and TechnologyWuhan Hubei 430022 China

2Neurosurgery Department Wuhan Union Hospital Tongji Medical College Huazhong University of Science and TechnologyWuhan Hubei 430022 China

3Department of Gynaecology and Obstetrics Wuhan Union Hospital Tongji Medical College Huazhong University ofScience and Technology Wuhan Hubei 430022 China

4Cancer Center Wuhan Union Hospital Tongji Medical College Huazhong University of Science and TechnologyWuhan Hubei 430022 China

Correspondence should be addressed to Jingqiong Hu jingqionghu2006sinacom andShiang Huang shianghuang2000yahoocom

Received 6 December 2013 Revised 25 March 2014 Accepted 19 April 2014 Published 27 May 2014

Academic Editor Mouldy Sioud

Copyright copy 2014 Chao Yang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Human mesenchymal stem cells (MSCs) have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy But very limited studies investigated the antitumor properties ofMSCs themselves In this studyweinvestigated the antiglioma properties of two easily accessibleMSCs namely human adipose tissue-derivedmesenchymal stem cells(ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) We found (1) MSC conditioned media can significantlyinhibit the growth of human U251 glioma cell line (2) MSC conditioned media can significantly induce apoptosis in human U251cell line (3) real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 andsignificant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251cells (4) furthermore MSCs conditioned media culture induced rapid and complete differentiation in U251 cells These resultsindicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line Whereas UC-MSCs aremore efficient for apoptosis induction thanASCs their capability of differentiation induction is not distinguishable from each otherOur findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future gliomatherapy

1 Introduction

Gliomas are the most common malignant cancer affectingthe central nervous system with a very poor prognosisin particular with high-grade tumors such as glioblastomamultiforme [1ndash3] Current therapy includes surgery radi-ation and chemotherapy But these treatments are rarelycurative Gliomas usually grow in a highly invasive mannerand always infiltrate neighboring tissues and therefore

surgical resection rarely removes the tumor completelyAfter a certain period gliomas inevitably recur and finallycause the death of the patient Radiation therapy usuallyirradiates normal brain tissues surrounding the tumor aswell therefore causing multiple side effects Chemotherapy isalso with limited effects since most chemicals have difficultyin crossing the blood brain barrier [4] Temozolomide isthe most effective chemotherapeutic reagent but can usuallyprolong the lifespan for only 3ndash6 months in some patients

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 109389 13 pageshttpdxdoiorg1011552014109389

2 BioMed Research International

[5 6] with side effects Novel therapy approaches includecombinations of molecular targeted agents such as epidermalgrowth factor receptor vascular endothelial growth factorreceptor and mammalian target of rapamycin But theseagain have only very limited effects [7] Overall gliomas havea poor prognosis and developing novel modalities to increaseantiglioma effects and decrease side effects is necessary

Mesenchymal stem cells (MSCs) are a population of stemcells with self-renewal andmultipotentiality which hold greatpromise for regenerative medicine [8 9] In recent yearsMSCs have gained increased attention because it has beenshown that these cells have an intrinsic property for homingtowards tumor sites and can be used as tumor-tropic vectorsfor tumor therapy [10ndash17] Tumor cell-derived substancesand factors associated with tumor-induced inflammationand tumor neovascularization can specifically attract stemcells to invasive gliomas Injected mesenchymal stem cellsengineered to produce antitumor substances have shownstrong therapeutic effects in experimental gliomamodels Butvery limited studies investigated the antitumor properties ofMSCs themselves There are some preliminary experimentsshowing that MSCs can suppress human glioma growth [18]but the exact mechanisms remain poorly studied

To further explore this phenomenon we examined theinteraction of two different types of mesenchymal stem cellsnamely human adipose tissue-derived mesenchymal stemcells (ASCs) and umbilical cord-derived mesenchymal stemcells (UC-MSCs) with the U251 human glioma cell lineBoth types of MSCs are easily accessible and have com-parative characteristics We found that conditioned mediafrom both types of MSCs can significantly induce apoptosisin U251 cells Interestingly we found a strong effect ofMSC conditioned medium on induction of differentiationof U251 glioma cells Induced U251 cells are evidenced bya morphology similar to normal glial cells and dramaticallydecreased tumor infiltration ability These results indicatehuman mesenchymal stem cells can efficiently induce bothapoptosis and differentiation in the U251 human gliomacell line Our findings suggest that MSCs themselves havefavorable antitumor characteristics and should be furtherexplored in future glioma therapy

2 Material and Methods

21 Isolation Culture and Phenotyping of ASCs and UC-MSCs Human subcutaneous adipose tissues and umbilicalcords were obtained frommothers (18ndash30 years old) planningon cesarean sections after obtaining written informed con-sent and approval by the Ethics Committee of Wuhan UnionHospital ASCs and UC-MSCs were isolated and amplified asdescribed elsewhere [19] ASCs and UC-MSCs were analyzedby multichannel flow cytometry using a standard Becton-Dickinson FACS Aria instrument and the CellQuest Prosoftware (BD Biosciences)

22 Multidifferentiation Capabilities of ASCs and UC-MSCsAdipogenic osteogenic and neurogenic differentiationexperiments were performed as described elsewhere [19]

Briefly for adipogenic differentiation ASCs and UC-MSCswere cultured in DMEMF12 supplemented with 10 FBS1mM dexamethasone 05mM methylisobutylxanthine10mgmL insulin and 100mM indomethacin (all fromSigma) for 3 weeks and analyzed by Oil Red O (Sigma)staining

For osteogenic differentiation ASCs and UC-MSCs werecultured in DMEMF12 supplemented with 10 FBS 01mMdexamethasone 10mM b-glycerophosphate and 50mMascorbic acid (all from Sigma) for about 3 weeks and assayedby alizarin red (Sigma) staining

For neurogenic differentiation ASCs andUC-MSCswerecultured in DMEMF12 supplemented with 5120583molL retinoicacids (RA) for 6ndash10 days and analyzed by immunofluores-cence microscopy [19]

23 U251 Cell Line Cultivation The U251 cell line waspurchased from the cell bank of the Chinese Academyof Sciences Shanghai China The cells were maintainedin DMEMF12 supplemented with 10 fetal bovine serum(FBS) (Hyclone Logan UT) and 1 penicillin-streptomycin(Invitrogen Carlsbad CA)

24 Harvest of ASC-Conditioned Medium and UC-MSC-ConditionedMedium ASCs andUC-MSCs at passage 3 werecultured in mesenchymal stem cell growth medium (whichis comprised of DMEMF12 supplemented with 10 fetalbovine serum) until cells were approximately 80 confluentat that time the medium was replaced with serum-freeDMEMF12 Following incubation in serum-free mediumfor 48 h the conditioned medium was collected as ASC-conditioned medium (ASC-CM) and UC-MSC-conditionedmedium (UC-MSC-CM) The conditioned medium was cen-trifuged at 1000 rpm for 5min filtered through a 022120583msyringe filter and conserved at 4∘C until use

25 CCK-8 Cell Proliferation Assay U251 cells weretrypsinized and adjusted to a concentration of 2 times 104mLand then 100 120583L of cell suspension (5000 cells per well) wasseeded in 96-well plates (Costar) 6 hours later the mediumwas replaced with either ASC-conditioned medium or UC-MSC-conditionedmedium 24 hours later CCK-8 (10 120583Lwell)was added to experimental wells and control wells (U251cells cultured alone) and after 4 hours of incubation theirabsorbance values at 450 nm were measured by enzymeimmunoassay analyzer and the mean values were calculatedThe experiment was performed three times (119899 = 3) with 3wells per condition each time

26 Flow Cytometric Assay for Apoptosis Using AnnexinV and Propidium Iodide U251 cells were plated in 6-wellplates and grown until confluencyThemediumwas replacedwith serum-free ASC-conditioned medium or UC-MSC-conditioned medium and the induction was carried out fortwo consecutive days The experiment was performed threetimes (119899 = 3) with 3 wells per condition each time 48hours after induction U251 cells were digested with 02trypsin for 10 minutes at 37∘C Dissociated cells were washed

BioMed Research International 3

Table 1 Primers used for real-time PCR

Primer sequencesForward Reverse

Caspase 3 CAGTGGAGGCCGACTTCTTG TGGCACAAAGCGACTGGATCaspase 9 TGTCCTACTCTACTTTCCCAGGTTTT GTGAGCCCACTGCTCAAAGATSurvivin ACCACCGCATCTCTAC TCCTCTATGGGGTCGTXIAP GGCCAGACTATGCCCATTTA CGAAGAAGCAGTTGGGAAAActin F GTCGTACCACTGGCATTGT CAGCTGTGGTGGTGAAGCT

with phosphate-buffered saline (PBS) two times resuspendedin 500120583L binding buffer and then incubated for 1 hourat room temperature in the presence of 05 120583gmL annexinV-FITC (RampD) and 5 120583L propidium iodide for 5 minutesin bindingbuffer as described by the manufacturer Afterincubation the cells were analyzed by flow cytometry

27 Real-Time PCR Assay for Apoptotic Induction of U251RNA was isolated from U251 human glioma cells before andafter the induction usingTrizol reagent (Invitrogen CarlsbadCD USA) cDNA was transcribed using superscript IIIfirst strand cDNA synthesis kit following the manufacturerrsquosinstructions (Invitrogen Carlsbad CD USA) Quantita-tive real-time PCR was performed with SYBR Green PCRreagents on an ABI Prism 7300 detection system (AppliedBiosystems Foster City CA) Beta-actin was used as an inter-nal control The normalized fold expression was obtainedusing the 2minusΔΔCT method Primers used for real-time PCR aresummarized in Table 1

28 Immunostaining and Cellomics High Content Screening(HCS) System Assay for Glial Cell Differentiation U251 cellswere plated in 6-well plates and grown until confluencyThemediumwas replaced with serum-free ASC-conditionedmedium and UC-MSC-conditioned medium and the induc-tion was carried out for two consecutive days U251 cells werethen fixed with 4 paraformaldehyde for 15min followed bythree washes with phosphate-buffered saline (PBS) After a1-hour blocking with 02 Triton X-100 and 3 goat serumin PBS the cells were incubated with primary antibodiesovernight at 4∘C The primary antibodies used in this studyinclude rabbit polyclonal GFAP antibody (1 200 dilutionSanta cruz) and mouse monoclonal Tuj1 (1 200 dilutionMillipore) subsequently the cells were washed with PBSthree times and then incubated with Dylight 549 goatanti-rabbit secondary antibody (Jackson ImmunoresearchLaboratories) and Dylight 488 goat anti-mouse secondaryantibody (Jackson) and finally stained with 4101584061015840-diamidino-2-phenylindole hydrochloride (DAPI) for 5 minutes

For quantitative imaging fluorescent images of the cellswere collected automatically by use of the Cellomics highcontent screening (HCS) system using a 10x objective lensFor axon outgrowth assays images of GFAP and DAPI stain-ing were analyzed using Neuronal Profiling BioApplicationsoftware (Cellomics)

29 Tumor Cell Migration and Infiltration Assay For tumorcell migration assay U251 cells and U87 cells were platedin 6-well plates and grown until confluency A scratch wasmade in the confluent monolayer by using a 200120583L pipet tipThemediumwas replaced with serum-free ASC-conditionedmedium and UC-MSC-conditioned medium and the induc-tion was carried out for two consecutive days

For the Matrigel tumor cell invasion assay 25 times 104U251cells or 104U87 cells (normally cultured or induced in serum-free ASC-conditioned mediumUC-MSC-conditionedmedium for two days) were placed in transwells with 8 120583mpore size polycarbonate filters (Corning IncorporatedCorning NY) precoated with 30 uL of 1 6 diluted Matrigel(BD Biosciences) The lower wells were filled with 500 120583Lof DMEM with 10 fetal bovine serum The cells wereincubated at 37∘C for 24 hours Nonmigratory cells onthe upper surface of the transwells were removed with acotton swab and the migratory cells from the lower surfaceof the transwell were fixed in 70 methanol and stainedwith 1 crystal violet Cells were photographed and cellnumbers in four different fields were counted under 100xmagnification for each condition Average cells per field werecalculated Experiments were done in triplicates on threetimes independently

210 Preparation of ASC-CM and UC-MSC-CM and Pro-tein Microarray Analysis of U251 Cells for Apoptosis RelatedProteins ASCs and UC-MSCs at passage 3 were culturedin mesenchymal stem cell growth medium (which is com-prised of DMEMF12 supplemented with 10 fetal bovineserum) until cells were approximately 80 confluent themediumwas replacedwith serum-freeDMEMF12 Two dayslater the conditioned medium was harvested as ASC-CMand UC-MSC-CM U251 cells were cultured in DMEMF12supplemented with 10 fetal bovine serum (FBS) (HycloneLogan UT) and 1 penicillin-streptomycin (InvitrogenCarlsbad CA) until confluency The medium was replacedwith DMEMF12 for two days Then U251 cells were inducedusing either ASC-CM or UC-MSC-CM 48 hours followinginduction U251 cells were collected and lysed using RayBiolysis buffer The cell lysates were assayed by RayBio AAH-APO-1 (RayBiotech Inc Norcross GA) which can simul-taneously detect the expression levels of 43 human apoptoticrelated proteins in cell lysates

211 Statistical Analysis All values are expressed as mean plusmnSD Comparisons between two groups were analyzed by


Studentrsquos 119905-test and comparisons between more than twogroups were analyzed by ANOVA A value of 119875 lt 005was considered statistically significant All analyses wereperformed with SPSS 160

3 Results

31 Morphologies of ASCs and UC-MSCs ASCs morphologyand UC-MSCs morphology are basically indistinguishablefrom each other Both displayed spindle or whirlpool-likemorphology Primary ASCs and UC-MSCs usually adheredwithin 24ndash48 hours after plating These cells proliferatedrapidly within 5ndash7 days and gradually fused into a singlelayer (see Supplementary Figure 1 in SupplementaryMaterialavailable online at httpdxdoiorg1011552014109389) andhave a very uniform fibroblast-like morphology by passage 3

32 Flow Cytometry Multichannel flow cytometric assays forsurface receptor molecule expression were performed ASCsand UC-MSCs both are usually positive for CD13 CD44CD90 and CD105 expression and negative for CD14 CD45and CD34 expression Representative flow cytometry resultsare shown in Supplementary Figure 2 Statistical analysisshowed the phenotypes of ASCs and UC-MSCs are basicallyindistinguishable from each other (data not shown)

33 Multidifferentiation Capabilities of ASCs and UC-MSCsASCs andUC-MSCswere both able to efficiently differentiateinto adipocytes osteocytes and neurons and representativepictures are shown in Supplementary Figure 3

34 Both ASC-Conditioned Medium and UC-MSC-Conditioned Medium Significantly Inhibited the Proliferationof U251 Cells In order to compare the growth curves ofU251 in different conditioned medium CCK-8 assay wasperformed As is shown in Figure 1 both ASC-conditionedmedium and UC-MSC-conditioned medium significantlyinhibited the proliferation of U251 cells The growthinhibition effect weakened with increased cell number perwell More significant growth inhibition is seen in UC-MSCstreated cultures with inhibition being more than 50

Interestingly we also found that the growth of severalother tumor cell lines including lung cancer cell line A549rectal cancer cell line HT29 and breast cancer cell line MCF-7 was inhibited by MSC conditioned media (Figure 2)

35 Apoptosis Assay Using Both Flow Cytometry and Quanti-tative Real-Time PCR Confirmed Increased Apoptosis of U251Human Glioma Cells in MSC Conditioned Medium CultureIn order to find out whether the significant inhibitionof growth in U251 cells is due to growth retardation orelevated apoptosis we did apoptosis assay using both flowcytometry and quantitative real-time PCR Annexin and PIflow cytometric assays showed that UC-MSCs conditionedmedium induced more prominent apoptosis in U251 cellsthan ASCs conditioned medium 5052 plusmn 1123 apoptosisin U251 cells for UC-MSCs conditioned medium culture and

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Figure 1 Growth inhibition of U251 glioma cells in MSCs con-ditioned media The proliferation of U251 cells was inhibited byconditioned media from both ASC-CM and UC-MSC-CM Thenumber of cells in control cultures of U251 glioma cells was takenas 100 The effect on proliferation of U251 under experimentalconditions was calculated in relation to single cultures of U251 cellsNumbers on the119883-axis indicated plated cell number per well

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3428 plusmn 1445 apoptosis in U251 cells for ASCs conditionedmedium culture as is shown in Figure 3

Caspases play a key role during the execution phase invarious forms of apoptosis Survivin and XIAP are identifiedinU251 cells andwere assumed to act as antiapoptosis factorsTo explore the mechanism of MSC-induced cell death ofglioma cells we did quantitative real-time PCR for caspases 3and 9 survivin and XIAP

Quantitative real-timePCR showedmuchmore increasedcaspase 3 and caspase 9 expression and much lower survivinand XIAP expression after conditioned medium treatmentin U251 cells After addition of MSCs conditioned mediumsurvivin RNA expression decreased to only 20 of the levelof that before treatment whereas the increase of caspase 9 andcaspase 3 expressions is around 5-fold to 10-fold respectively

XIAP (X-linked inhibitor of apoptosis protein) is usuallyoverexpressed inU251 cells After coculture XIAP expressionis greatly decreased as is shown in Figure 4


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Figure 3 Annexin and PI flow cytometric assay (a) Representative annexin and PI flow cytometric picture of U251 cells under ASC- andUC-MSC-conditioned media coculture conditions (b) Statistical results of three independent flow experiments

36 MSCs Conditioned Medium Coculture Induced Cell CycleArrest inU251Cells Cell cycle deregulation and apoptosis areclosely related events and disruption of cell cycle progressionmay ultimately lead to apoptotic death The effects of MSCsconditioned medium on U251 proliferation were determinedusing cell cycle assay

MSCs conditioned medium increased the fraction ofG0G1 from 5521plusmn 352 in control to 6123plusmn 574 (forUC-MSC) and 7026 plusmn 312 (for ASC) at the 48-hour time points(Figure 5) The G2-M and S phases were slightly decreasedafter treatment indicating that MSC conditioned mediummay induce G0G1 growth arrest in U251 cells

37 MSCs Conditioned Medium Coculture Leads to SignificantDifferentiation of U251 Cells towards a Normal Glial CellPhenotype and Decrease in Tumor Infiltration CapabilityRather unexpectedly MSCs conditioned medium also ledto significant differentiation of U251 cells towards a normalglial cell phenotype As early as 8 hours after conditionedmedium treatment U251 glioma cells experienced significantmorphological change U251 glioma cells usually have shortprocesses Many more processes were seen after MSCs con-ditioned medium induction and these processes intercon-nected with neighboring cells as is shown in Figure 6 GFAPstaining was performed on U251 cells before and after MSCmedium exposure Although U251 is a human glioma cellline control U251 cells expressed very weak GFAP Howeverafter two days of growth in MSCs conditioned mediumGFAP expression became much more prominent and theoutgrowing processes can be clearly stained as is shown

in Figure 8 These morphological changes led to a relativelynormal glial cell phenotype Quantitative real-time PCR forGFAP expression was performed MSC conditioned mediumtreatment led to significant upregulation of GFAP mRNAwhich is more than 35-fold as is shown in Figure 4

We are interested to see whether this relative normalmor-phology change also leads to a decreased tumor migrationand infiltration capability

For tumormigration assay a scratch assay was performedusing U251 cells After scratching U251 cells soon grow overthe scratch line as if the scratch line never existed But in theMSC conditioned medium group only a few cells grew overthe line as is shown in Figure 7

Next we used in vitro Matrigel invasion assay to deter-mine whether the increased migration corresponded to anincrease in invasive properties We did this side by side withU87 cell line another commonly used human glioma cellline Noninduced control U251 and U87 cells can infiltratetheMatrigel But after 2 days of conditionedmedia inductiononly very few U251 cells can migrate through Matrigel asis shown in Figure 8 There was a significantly decreasedcell invasion capability of U251 cells after conditioned mediaculture and this difference is statistically significant U87 cellsalso displayed decreased tumor infiltration capability but to alesser extent

Cellomics high content screening (HCS) system analyseswere performed to analyze the change in length of cellprocesses Statistical analysis showed much more prominentcell processes outgrowth after MSCs conditioned mediainduction in U251 cells as is shown in Figure 9


Caspase 3

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Figure 4 Quantitative real-time PCR of caspase 3 caspase 9 survivin XIAP and GFAP after MSCs conditioned medium induction MSCsconditioned medium treatment leads to significant overexpression of caspase 3 and caspase 9 and significant downregulation of survivin andXIAP GFAP expression is also significantly increased after induction

38 Apoptotic Protein Microarray Analysis of ASC-CM andUC-MSC-CM To find out what apoptotic related proteinsare responsible for the increased apoptosis in U 251 cells ahuman apoptosis antibody array serial analysis were carriedout Lysates of U251 cells before and after MSC conditionedmedium induction were examined by high-throughput pro-tein array in which 43 human apoptosis related proteins wereexamined in one array Representative Apoptotic Proteinmicroarray analyses are shown in Table 2 As expectedUC-MSCs and ASCs conditioned medium induction led tosignificant overexpression of pro-apoptotic proteins such asBad Bax Bim Bid etc and significant downregulation ofanti-apoptotic proteins such as Bcl-2 Survivin and XIAP

4 Discussion

Human malignant gliomas are the most commonly diag-nosed malignant adult primary brain tumors Resistance to

induction of cell death by apoptosis in response to radio-therapy or chemotherapy is one of its cardinal characteristics[20] Most glioma patients have very unfavorable expectationupon diagnosis whereas for GBM (glioblastomamultiforme)patients a period of only 3ndash6 months is expected Withsuch a miserable prognosis searching for new treatment isnecessary

Mesenchymal stem cells (MSCs) are a population of stemcells with great self-renewal and multipotentiality MSCs arepotential seed cells in regenerative medicine and have beenused for treatment of various neurological diseases such asspinal cord injury and cerebral palsy [21] Current researchfor exploration of MSCs in cancer therapy mainly uses MSCsas a vehicle for targeting tumor cells because these cellshave been found to have tumor chemotactic capabilities andcan migrate towards tumor sites [22ndash24] When transducedwith therapeutic molecules such as IFN-a IFN-b TRAILand thymidine kinase (TK) anticancer activity was often


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Figure 5 Cell cycle analysis of U251 cells after ASCs and UC-MSCs conditioned medium induction MSCs conditioned medium treatmentled to G0G1 growth arrest in U251 cells

observed inMSCs [25ndash27] But few studies investigatedMSCswithout transducing these targetingmolecules Akimoto et alreported U251 growth inhibition when cocultured with UC-MSCs but growth promotion when cocultured with ASCs[28] Recently Behnan et al reported that MSCs mightcontribute to glioma tumor progression [29] These contra-dictory results might be due to different cell sources beingused (primary tumor cells or tumor cell line) different culturemethods different experimental condition and designs and

so forth Given the fact that MSCs have been widely usedin various studies as tumor-targeting vehicles with obviouseffects it would be problematic if MSCs themselves arepromoting tumor growth Therefore we reasoned MSCsthemselves might have some antitumor properties In ourpreliminary experiments using transwell coculture systemwe did see growth inhibition of U251 human glioma cellswhen coculturedwithMSCsThe same effect can be observedsimply by using conditioned media coculture from MSCs


Table 2 Representative RayBio apoptosis assay overview (in part)

Protein name U251 alone ASC CM UC-MSC-CM Fold change 1 Fold change 2Bad 1005 18161 20300 1807 2020Bax 19200 26505 36300 138 189Bcl-2 43000 20615 17548 048 041Bcl-w 35000 31080 28603 089 082BID 18500 20800 28110 112 152BIM 23100 24051 38715 104 168Caspase 3 23500 93260 88100 397 375Caspase 8 20000 39267 67700 196 339Fas 127150 163843 210005 129 165FasL 27450 26946 17742 098 065p21 17900 20861 18337 117 102p27 24000 33380 23820 139 099p53 25500 31905 25266 125 099SMAC 40750 154860 153500 380 377Survivin 20500 11450 12400 056 060sTNF-R1 9500 27291 41353 287 435sTNF-R2 13200 13807 20446 105 155TNF-alpha 28700 29450 28037 103 098TNF-beta 24600 34948 30632 142 125XIAP 20074 10102 12595 050 063These readouts are the readouts after subtracting the background Signal strength fold change larger than 15 or smaller than 07 is considered statisticallysignificant UC-MSCs and ASCs conditioned media led to significant overexpression of proapoptotic proteins such as bad bax BIM and BID and significantdownregulation of antiapoptotic proteins such as bcl-2 survivin and XIAP


Figure 6 MSCs conditioned media coculture leads to significant differentiation of U251 cells towards a normal glial cell phenotype Phasecontrast image of U251 cells before and after MSCs conditioned media coculture U251 glioma cells are usually with short processes Muchmore and longer processes were seen afterMSCs conditionedmedia induction and these processes connected with neighboring cells formingan interconnected network


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Figure 7 Scratch assay U251 cells were induced with ASC-CM and UC-MSC-CM for two days The cells quickly grew over the scratch forU251 cells cultured alone After 48 hours the scratch was invisible For ASC-CM and UC-MSC-CM culture although there are still somecells growing over the scratch line the scratch is still visible Fewer cells are seen grown over the line

Therefore we designed this ex vivo study For MSCs we com-pared two easily accessible MSCs sources namely humanadipose tissue-derived mesenchymal stem cells (ASCs) andumbilical cord-derived mesenchymal stem cells (UC-MSCs)in parallel We wanted to see whether different sources ofMSCs have impact on growth kinetics apoptotic rate pro-and antiapoptotic gene expressions and so forth

Our study showed conditioned media from both typesof MSCs can significantly inhibit the growth of human U251cell line especially for UC-MSCs which inhibited morethan 50 of growth of U251 cells To find out whetherthis is due to growth retardation of U251 cells or increasedapoptosis we did annexin and PI flow cytometric assayFlow cytometric assay indicated both types of MSCs cansignificantly induce the apoptosis in human U251 cell linewhich is more true for UC-MSCs The susceptibility oftumor cells to induction of apoptosis is usually controlled by

the ratio between proapoptotic and antiapoptotic proteinsInduction of apoptosis usually requires involvement of thebcl-2 family proteins including proapoptotic gene products(eg bad bax bak bcl-XS and bim) and apoptosis inhibitinggene products (eg bcl-2 bcl-XL) [30 31] On the otherhand caspases especially caspase 3 play a key role duringthe execution phase in various forms of apoptosis [32]

To get a comprehensive picture of the apoptotic relatedfactors during induction of apoptosis inU251 cell lines we didRayBio Biotin Label-Based Human Apoptosis Array I (Catnumber AAH-APO-G1-4 Norcross GA) which can detectthe expression levels of 43 human apoptotic related proteinsin cell culture supernatants simultaneously These apoptoticrelated proteins include bcl-2 bax bad caspase 3 caspase8 survivin XIAP p53 p21 Fas TRAIL and TNF-betaAs expected both MSCs conditioned media secreted muchmore prominent apoptotic related proteins especially for


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Figure 8Matrigel invasion assayU251 cells orU87 cells (induced or noninduced)were placed in transwells with 8 120583mpore size polycarbonatefilters precoated with Matrigel The lower wells were filled with DMEMwith 10 fetal bovine serumThe cells were incubated at 37∘C for 24hours Nonmigratory cells on the upper surface of the transwells were removed and themigratory cells from the lower surface of the transwellwere fixed and stained with 1 crystal violet Cells were photographed and average cells per field were calculated Experiments were done intriplicates on three times independently

bad bax bak and bim Survivin and XIAP are significantlylower in MSCs conditioned medium culture as compared toU251 single culture alone Notably we also saw significantoverexpression of TNF receptor after MSC conditionedmedium induction It is plausible that during the inductionof apoptosis byMSCs TNF signalling pathway also played animportant role

Next we did quantitative real-time PCR experiments Wefound significant upregulation of apoptotic genes of bothcaspase 3 and caspase 9 and significant downregulation ofantiapoptotic genes such as survivin and XIAP after MSCconditionedmedium induction in human U251 cell lineThisresult is consistent with our protein array data

The regulation of cell proliferation and terminal dif-ferentiation is a critical aspect of normal development

and homeostasis but is frequently disturbed during tumori-genesis Cell proliferation and differentiation are specificallycontrolled in the G1 phase and the G1S phase transition inthe cell cycle [33] When we did cell cycle analysis on U251cells prior to and after the induction we found significantlymore cells in G0G1 phase after inductionTherefore we havereason to believe MSC conditioned medium might represscell growth via cell cycle arrest in the G0G1 phase

During the induction experiments rather unexpectedlywe found MSC conditioned media induced rapid (as earlyas 6ndash8 hours after induction) and complete differentiation inU251 cells This means MSCs conditioned medium triggerscell transformation indicative of the cellsrsquo differentiation intoa more mature astrocytic state This differentiation potentialis further confirmed by an increased expression of GFAP


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Figure 9 Cellomics high content screening (HCS) system analysis indicated much more prominent cell process outgrowth after MSCsconditioned media induction (a) Representative fluorescent picture of U251 glioma cells before and after conditioned media inductionGFAP positive cells are shown in red Much longer cell processes were seen after conditioned media induction (b) Statistical analysis of cellprocess outgrowth before and after MSCs conditioned media induction Both ASC- and UC-MSC-conditioned media led to more significantoutgrowths of cell processes

a 50 kDa type III intermediate filament protein consideredto be a reliable differentiation marker of normal astrocytes[34] In glial tumors GFAP expression is frequently lostwith increasing grade of malignancy suggesting that GFAPis important for maintaining glial cell morphology or regu-lating astrocytoma cell growth Increased GFAP expressionafter MSC conditioned medium induction is evidenced bysignificant GFAP fluorescent staining and significant upreg-ulation of GFAP genes using real-time PCR (more than 35-fold increase in expression) These results indicate humanmesenchymal stem cells can efficiently induce both apoptosisand differentiation in U251 human glioma cell line WhereasUC-MSCs are more efficient for apoptosis induction thanASCs their capabilities of differentiation induction are notdistinguishable from each other

Differentiation therapy using agents that modifycancer cell differentiation has shown to be effective andbecame the standard therapy for acute promyelocyticleukemia (APL) which uses either all-trans-retinoicacid or the inorganic toxicant arsenic trioxide (AS

2O3)

a well-known environmental carcinogen Both drugstake effect by triggering apoptosis and differentiation ofAPL cells in a dose-dependent manner [35ndash38] Suchexcellent effects however were not reproduced in otherhematological and particularly solid tumors Differentiationagents for malignant gliomas remain a real challengeSome studies have shown arsenic trioxide (AS

2O3) and

forskolin can also trigger apoptosis and differentiationin glioma cells [39 40] In our study we unexpectedlyfound a very strong differentiation effect of conditioned


MSCs medium More impressively the tumor infiltrationcapabilities of U251 cells were greatly reduced afterinduction as is shown in Matrigel infiltration assay Thetumor infiltration capabilities of U87 cells were also reducedafter induction but to a lesser extent We are planning to dofurther experiments to look deeper into this phenomenon

In this study we showed conditioned media from humanmesenchymal stem cells can efficiently induce apoptosisin U251 human glioma cell line Furthermore MSC con-ditioned medium is a powerful astrocytic differentiation-inducing agent in cultured human U251 glioma cell line Ourfindings suggest MSCs themselves have favorable antitumorcharacteristics and should be further explored in futureglioma therapy Future studies however should extend thesefindings to in vivo tumor models

Conflict of Interests

The authors who have taken part in this study declared thatthey have no conflict of interests regarding this paper

Authorsrsquo Contribution

Jingqiong Hu and Shiang Huang contribute equally as corre-sponding authors

Acknowledgment

This work was supported by National Natural Science Foun-dation of China (Grant no 31201100)

References

[1] Y Wang and T Jiang ldquoUnderstanding high grade gliomamolecular mechanism therapy and comprehensive manage-mentrdquo Cancer Letters vol 331 no 2 pp 139ndash146 2013

[2] D Bexell A Svensson and J Bengzon ldquoStem cell-based therapyfor malignant gliomardquo Cancer Treatment Reviews vol 39 no 4pp 358ndash365 2013

[3] M Rahman B Hoh N Kohler E M Dunbar and G J MuradldquoThe future of glioma treatment stem cells nanotechnologyand personalized medicinerdquo Future Oncology vol 8 no 9 pp1149ndash1156 2012

[4] K Sai Q Y Yang D Shen and Z P Chen ldquoChemotherapy forgliomas in mainland China an overviewrdquoOncology Letters vol5 no 5 pp 1448ndash1452 2013

[5] D Szczepanek A Marchel M Moskała M Krupa P Kunertand T Trojanowski ldquoEfficacy of concomitant and adjuvanttemozolomide in glioblastoma treatment A multicentre ran-domized studyrdquo Neurologia i Neurochirurgia Polska vol 47 no2 pp 101ndash108 2013

[6] H M Strik C Marosi B Kaina and B Neyns ldquoTemozolomidedosing regimens for glioma patientsrdquo Current Neurology andNeuroscience Reports vol 12 no 3 pp 286ndash293 2012

[7] C Scaringi R M Enrici and G Minniti ldquoCombining molec-ular targeted agents with radiation therapy for malignantgliomasrdquo Journal of OncoTargets and Therapy vol 6 pp 1079ndash1095 2013

[8] P L Mok C F Leong and S K Cheong ldquoCellular mechanismsof emerging applications of mesenchymal stem cellsrdquo TheMalaysian Journal of Pathology vol 35 no 1 pp 17ndash32 2013

[9] V Neirinckx C Coste B Rogister and S Wislet-GendebienldquoConcise review adult mesenchymal stem cells adult neuralcrest stem cells and therapy of neurological pathologies a stateof playrdquo Stem Cells Translational Medicine vol 2 no 4 pp 284ndash296 2013

[10] X Y Bak D H Lam J Yang et al ldquoHuman embryonicstem cell-derived mesenchymal stem cells as cellular deliveryvehicles for prodrug gene therapy of glioblastomardquo HumanGene Therapy vol 22 no 11 pp 1365ndash1377 2011

[11] S M Kim D-S Kim C H Jeong et al ldquoCXC chemokinereceptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomasrdquoBiochemical and Biophysical Research Communications vol 407no 4 pp 741ndash746 2011

[12] H Jiao F Guan B Yang et al ldquoHuman umbilical cordblood-derived mesenchymal stem cells inhibit C6 glioma viadownregulation of cyclin D1rdquoNeurology India vol 59 no 2 pp241ndash247 2011

[13] S A Choi J Y Lee K-C Wang et al ldquoHuman adipose tissue-derivedmesenchymal stemcells characteristics and therapeuticpotential as cellular vehicles for prodrug gene therapy againstbrainstem gliomasrdquo European Journal of Cancer vol 48 no 1pp 129ndash137 2012

[14] T Doucette G Rao Y Yang et al ldquoMesenchymal stem cells dis-play tumor-specific tropism in an RCASNtv-a glioma modelrdquoNeoplasia vol 13 no 8 pp 716ndash725 2011

[15] J Yin J-K Kim J-H Moon et al ldquoHMSC-mediated con-current delivery of endostatin and carboxylesterase to mousexenografts suppresses glioma initiation and recurrencerdquoMolec-ular Therapy vol 19 no 6 pp 1161ndash1169 2011

[16] S A Park C H Ryu S M Kim et al ldquoCXCR4-transfectedhuman umbilical cord blood-derived mesenchymal stem cellsexhibit enhanced migratory capacity toward gliomasrdquo Interna-tional Journal of Oncology vol 38 no 1 pp 97ndash103 2011

[17] S A Choi S-K Hwang K-C Wang et al ldquoTherapeuticefficacy and safety of TRAIL-producing human adipose tissue-derived mesenchymal stem cells against experimental brain-stem gliomardquo Neuro-Oncology vol 13 no 1 pp 61ndash69 2011

[18] I A Ho H C Toh W H Ng et al ldquoHuman bone marrow-derived mesenchymal stem cells suppress human gliomagrowth through inhibition of angiogenesisrdquo Stem Cells vol 31no 1 pp 146ndash155 2013

[19] L Hu J Hu J Zhao et al ldquoSide-by-side comparison of thebiological characteristics of human umbilical cord and adiposetissue-derivedmesenchymal stem cellsrdquo BioMed Research Inter-national vol 2013 Article ID 438243 12 pages 2013

[20] C EGriguer andC ROliva ldquoBioenergetics pathways and ther-apeutic resistance in gliomas emerging role of mitochondriardquoCurrent Pharmaceutical Design vol 17 no 23 pp 2421ndash24272011

[21] M Gutierrez-Fernandez B Rodrıguez-Frutos L Otero-Ortega J Ramos-Cejudo B Fuentes and E Dıez-TejedorldquoAdipose tissue-derived stem cells in stroke treatment frombench to bedsiderdquoDiscoveryMedicine vol 16 no 86 pp 37ndash432013

[22] F Xu J Shi B Yu W Ni X Wu and Z Gu ldquoChemokinesmediate mesenchymal stem cell migration toward gliomas invitrordquo Oncology Reports vol 23 no 6 pp 1561ndash1567 2010


[23] D-S Kim J H Kim J Kwon Lee et al ldquoOverexpression ofCXC chemokine receptors is required for the superior glioma-tracking property of umbilical cord blood-derived mesenchy-mal stem cellsrdquo Stem Cells and Development vol 18 no 3 pp511ndash519 2009

[24] D Bexell S Gunnarsson A Tormin et al ldquoBonemarrowmulti-potent mesenchymal stroma cells act as pericyte-like migratoryvehicles in experimental gliomasrdquoMolecularTherapy vol 17 no1 pp 183ndash190 2009

[25] S M Kim J S Woo C H Jeong C H Ryu J D Jang and S SJeun ldquoPotential application of temozolomide in mesenchymalstem cell-Based TRAIL gene therapy againstmalignant gliomardquoStemCells TranslationalMedicine vol 3 no 2 pp 172ndash182 2014

[26] S Fei X Qi S Kedong J Guangchun L Jian and QWei ldquoTheantitumor effect of mesenchymal stem cells transduced with alentiviral vector expressing cytosine deaminase in a rat gliomamodelrdquo Journal of Cancer Research and Clinical Oncology vol138 no 2 pp 347ndash357 2012

[27] H Kosaka T Ichikawa K Kurozumi et al ldquoTherapeutic effectof suicide gene-transferred mesenchymal stem cells in a ratmodel of gliomardquo Cancer Gene Therapy vol 19 no 8 pp 572ndash578 2012

[28] K Akimoto K Kimura M Nagano et al ldquoUmbilical cordblood-derived mesenchymal stem cells inhibit but adiposetissue-derived mesenchymal stem cells promote glioblastomamultiforme proliferationrdquo Stem Cells and Development vol 22no 9 pp 1370ndash1386 2013

[29] J Behnan P Isakson M Joel et al ldquoRecruited brain tumor-derived mesenchymal stem cells contribute to brain tumorprogressionrdquo Stem Cells vol 32 no 5 pp 1110ndash123 2013

[30] A H Stegh and R A DePinho ldquoBeyond effector caspaseinhibition Bcl2L12 neutralizes p53 signaling in glioblastomardquoCell Cycle vol 10 no 1 pp 33ndash38 2011

[31] B B Ordys S Launay R F Deighton J McCulloch and I RWhittle ldquoThe role of mitochondria in glioma pathophysiologyrdquoMolecular Neurobiology vol 42 no 1 pp 64ndash75 2010

[32] D Kogel S Fulda and M Mittelbronn ldquoTherapeutic exploita-tion of apoptosis and autophagy for glioblastomardquo Anti-CancerAgents in Medicinal Chemistry vol 10 no 6 pp 438ndash449 2010

[33] B M Alexander N Pinnell P Y Wen and A DrsquoAndrealdquoTargeting DNA repair and the cell cycle in glioblastomardquoJournal of Neuro-Oncology vol 107 no 3 pp 463ndash477 2012

[34] M C Ku S A Wolf D Respondek et al ldquoGDNF mediatesglioblastoma-inducedmicroglia attraction but not astrogliosisrdquoActa Neuropathologica vol 125 no 4 pp 609ndash620 2013

[35] V Balik PMirossay P Bohus I Sulla LMirossay andM Saris-sky ldquoFlow cytometry analysis of neural differentiation markersexpression in human glioblastomas may predict their responseto chemotherapyrdquo Cellular and Molecular Neurobiology vol 29no 6-7 pp 845ndash858 2009

[36] E-K Noh H Kim M J Park et al ldquoGefitinib enhances arsenictrioxide (AS

2

O3

)-induced differentiation of acute promyelo-cytic leukemia cell linerdquo Leukemia Research vol 34 no 11 pp1501ndash1505 2010

[37] J Fang S-J Chen J-H Tong Z-G Wang G-Q Chen and ZChen ldquoTreatment of acute promyelocytic leukemia with ATRAand AS

2

O3

a model of molecular target-based cancer therapyrdquoCancer Biology andTherapy vol 1 no 6 pp 614ndash620 2002

[38] G C de Santis M D B Tamarozzi R B Sousa et al ldquoAdhesionmolecules and differentiation syndrome phenotypic and func-tional analysis of the effect of ATRA AS

2

O3

phenylbutyrate

and G-CSF in acute promyelocytic leukemiardquo Haematologicavol 92 no 12 pp 1615ndash1622 2007

[39] X Song Z Chen C Wu and S Zhao ldquoAbrogating HSPresponse augments cell death induced by AS

2

O3

in glioma celllinesrdquo Canadian Journal of Neurological Sciences vol 37 no 4pp 504ndash511 2010

[40] S He W Zhu Y Zhou et al ldquoTranscriptional and post-transcriptional down-regulation of cyclin D1 contributes toC6 glioma cell differentiation induced by forskolinrdquo Journal ofCellular Biochemistry vol 112 no 9 pp 2241ndash2249 2011


[5 6] with side effects Novel therapy approaches includecombinations of molecular targeted agents such as epidermalgrowth factor receptor vascular endothelial growth factorreceptor and mammalian target of rapamycin But theseagain have only very limited effects [7] Overall gliomas havea poor prognosis and developing novel modalities to increaseantiglioma effects and decrease side effects is necessary

Mesenchymal stem cells (MSCs) are a population of stemcells with self-renewal andmultipotentiality which hold greatpromise for regenerative medicine [8 9] In recent yearsMSCs have gained increased attention because it has beenshown that these cells have an intrinsic property for homingtowards tumor sites and can be used as tumor-tropic vectorsfor tumor therapy [10ndash17] Tumor cell-derived substancesand factors associated with tumor-induced inflammationand tumor neovascularization can specifically attract stemcells to invasive gliomas Injected mesenchymal stem cellsengineered to produce antitumor substances have shownstrong therapeutic effects in experimental gliomamodels Butvery limited studies investigated the antitumor properties ofMSCs themselves There are some preliminary experimentsshowing that MSCs can suppress human glioma growth [18]but the exact mechanisms remain poorly studied

To further explore this phenomenon we examined theinteraction of two different types of mesenchymal stem cellsnamely human adipose tissue-derived mesenchymal stemcells (ASCs) and umbilical cord-derived mesenchymal stemcells (UC-MSCs) with the U251 human glioma cell lineBoth types of MSCs are easily accessible and have com-parative characteristics We found that conditioned mediafrom both types of MSCs can significantly induce apoptosisin U251 cells Interestingly we found a strong effect ofMSC conditioned medium on induction of differentiationof U251 glioma cells Induced U251 cells are evidenced bya morphology similar to normal glial cells and dramaticallydecreased tumor infiltration ability These results indicatehuman mesenchymal stem cells can efficiently induce bothapoptosis and differentiation in the U251 human gliomacell line Our findings suggest that MSCs themselves havefavorable antitumor characteristics and should be furtherexplored in future glioma therapy

2 Material and Methods

21 Isolation Culture and Phenotyping of ASCs and UC-MSCs Human subcutaneous adipose tissues and umbilicalcords were obtained frommothers (18ndash30 years old) planningon cesarean sections after obtaining written informed con-sent and approval by the Ethics Committee of Wuhan UnionHospital ASCs and UC-MSCs were isolated and amplified asdescribed elsewhere [19] ASCs and UC-MSCs were analyzedby multichannel flow cytometry using a standard Becton-Dickinson FACS Aria instrument and the CellQuest Prosoftware (BD Biosciences)

22 Multidifferentiation Capabilities of ASCs and UC-MSCsAdipogenic osteogenic and neurogenic differentiationexperiments were performed as described elsewhere [19]

Briefly for adipogenic differentiation ASCs and UC-MSCswere cultured in DMEMF12 supplemented with 10 FBS1mM dexamethasone 05mM methylisobutylxanthine10mgmL insulin and 100mM indomethacin (all fromSigma) for 3 weeks and analyzed by Oil Red O (Sigma)staining

For osteogenic differentiation ASCs and UC-MSCs werecultured in DMEMF12 supplemented with 10 FBS 01mMdexamethasone 10mM b-glycerophosphate and 50mMascorbic acid (all from Sigma) for about 3 weeks and assayedby alizarin red (Sigma) staining

For neurogenic differentiation ASCs andUC-MSCswerecultured in DMEMF12 supplemented with 5120583molL retinoicacids (RA) for 6ndash10 days and analyzed by immunofluores-cence microscopy [19]

23 U251 Cell Line Cultivation The U251 cell line waspurchased from the cell bank of the Chinese Academyof Sciences Shanghai China The cells were maintainedin DMEMF12 supplemented with 10 fetal bovine serum(FBS) (Hyclone Logan UT) and 1 penicillin-streptomycin(Invitrogen Carlsbad CA)

24 Harvest of ASC-Conditioned Medium and UC-MSC-ConditionedMedium ASCs andUC-MSCs at passage 3 werecultured in mesenchymal stem cell growth medium (whichis comprised of DMEMF12 supplemented with 10 fetalbovine serum) until cells were approximately 80 confluentat that time the medium was replaced with serum-freeDMEMF12 Following incubation in serum-free mediumfor 48 h the conditioned medium was collected as ASC-conditioned medium (ASC-CM) and UC-MSC-conditionedmedium (UC-MSC-CM) The conditioned medium was cen-trifuged at 1000 rpm for 5min filtered through a 022120583msyringe filter and conserved at 4∘C until use

25 CCK-8 Cell Proliferation Assay U251 cells weretrypsinized and adjusted to a concentration of 2 times 104mLand then 100 120583L of cell suspension (5000 cells per well) wasseeded in 96-well plates (Costar) 6 hours later the mediumwas replaced with either ASC-conditioned medium or UC-MSC-conditionedmedium 24 hours later CCK-8 (10 120583Lwell)was added to experimental wells and control wells (U251cells cultured alone) and after 4 hours of incubation theirabsorbance values at 450 nm were measured by enzymeimmunoassay analyzer and the mean values were calculatedThe experiment was performed three times (119899 = 3) with 3wells per condition each time

26 Flow Cytometric Assay for Apoptosis Using AnnexinV and Propidium Iodide U251 cells were plated in 6-wellplates and grown until confluencyThemediumwas replacedwith serum-free ASC-conditioned medium or UC-MSC-conditioned medium and the induction was carried out fortwo consecutive days The experiment was performed threetimes (119899 = 3) with 3 wells per condition each time 48hours after induction U251 cells were digested with 02trypsin for 10 minutes at 37∘C Dissociated cells were washed















3 Results







0010203040506070809

10000 7500 5000

Prol

ifera

tion

( ce

lls)

ASC-CMUC-MSC-CM


0

02

04

06

08

1

12

HT29 A549 MCF-7

Prol

ifera

tion

( co

ntro

l)








Annexin V

PI


100

101

102

103

104

100

101

102

103

104

Annexin VPI

100

101

102

103

104

100

101

102

103

104

Annexin V

PI

100

101

102

103

104

100

101

102

103

104

010 029

9745 215

459 2499

4891 2150

324 1054

2600 6023

(a)

020406080

100


Apop

totic

cells

()

(b)











Caspase 3

0123456

Caspase 9

02468

1012

Survivin

002040608

112

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

XIAP

002040608

112

GFAP

Caspase 3 Caspase 9

Survivin XIAP

GFAP

05

101520253035



4 Discussion





ASC

UC-MSCU251


G2M 1214 plusmn 147

S 3262 plusmn 524


G2M 1012 plusmn 259

S 2862 plusmn 321


G2M 850 plusmn 098

S 2123 plusmn 228

0 40 80 12020 60 100 0 40 80 120 160

0 40 80 120 160


Channels (FL2-A)

0

80

160

240

320

400

Num

ber

Num

ber

Num

ber

0

70

140

210

280

0

300

600

900

1200


Dip G2

Dip S


Dip G2


Dip G2

Dip S

An1 G1










U251 0h

ASC-CM 0h

UC-MSC-CM0h

U251 24h

ASC-CM 24h

UC-MSC-CM24h

U251 48h

ASC-CM 48h

UC-MSC-CM48h







U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3

















3 Results







0010203040506070809

10000 7500 5000

Prol

ifera

tion

( ce

lls)

ASC-CMUC-MSC-CM


0

02

04

06

08

1

12

HT29 A549 MCF-7

Prol

ifera

tion

( co

ntro

l)








Annexin V

PI


100

101

102

103

104

100

101

102

103

104

Annexin VPI

100

101

102

103

104

100

101

102

103

104

Annexin V

PI

100

101

102

103

104

100

101

102

103

104

010 029

9745 215

459 2499

4891 2150

324 1054

2600 6023

(a)

020406080

100


Apop

totic

cells

()

(b)











Caspase 3

0123456

Caspase 9

02468

1012

Survivin

002040608

112

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

XIAP

002040608

112

GFAP

Caspase 3 Caspase 9

Survivin XIAP

GFAP

05

101520253035



4 Discussion





ASC

UC-MSCU251


G2M 1214 plusmn 147

S 3262 plusmn 524


G2M 1012 plusmn 259

S 2862 plusmn 321


G2M 850 plusmn 098

S 2123 plusmn 228

0 40 80 12020 60 100 0 40 80 120 160

0 40 80 120 160


Channels (FL2-A)

0

80

160

240

320

400

Num

ber

Num

ber

Num

ber

0

70

140

210

280

0

300

600

900

1200


Dip G2

Dip S


Dip G2


Dip G2

Dip S

An1 G1










U251 0h

ASC-CM 0h

UC-MSC-CM0h

U251 24h

ASC-CM 24h

UC-MSC-CM24h

U251 48h

ASC-CM 48h

UC-MSC-CM48h







U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3





3 Results







0010203040506070809

10000 7500 5000

Prol

ifera

tion

( ce

lls)

ASC-CMUC-MSC-CM


0

02

04

06

08

1

12

HT29 A549 MCF-7

Prol

ifera

tion

( co

ntro

l)








Annexin V

PI


100

101

102

103

104

100

101

102

103

104

Annexin VPI

100

101

102

103

104

100

101

102

103

104

Annexin V

PI

100

101

102

103

104

100

101

102

103

104

010 029

9745 215

459 2499

4891 2150

324 1054

2600 6023

(a)

020406080

100


Apop

totic

cells

()

(b)











Caspase 3

0123456

Caspase 9

02468

1012

Survivin

002040608

112

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

XIAP

002040608

112

GFAP

Caspase 3 Caspase 9

Survivin XIAP

GFAP

05

101520253035



4 Discussion





ASC

UC-MSCU251


G2M 1214 plusmn 147

S 3262 plusmn 524


G2M 1012 plusmn 259

S 2862 plusmn 321


G2M 850 plusmn 098

S 2123 plusmn 228

0 40 80 12020 60 100 0 40 80 120 160

0 40 80 120 160


Channels (FL2-A)

0

80

160

240

320

400

Num

ber

Num

ber

Num

ber

0

70

140

210

280

0

300

600

900

1200


Dip G2

Dip S


Dip G2


Dip G2

Dip S

An1 G1










U251 0h

ASC-CM 0h

UC-MSC-CM0h

U251 24h

ASC-CM 24h

UC-MSC-CM24h

U251 48h

ASC-CM 48h

UC-MSC-CM48h







U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3




Annexin V

PI


100

101

102

103

104

100

101

102

103

104

Annexin VPI

100

101

102

103

104

100

101

102

103

104

Annexin V

PI

100

101

102

103

104

100

101

102

103

104

010 029

9745 215

459 2499

4891 2150

324 1054

2600 6023

(a)

020406080

100


Apop

totic

cells

()

(b)











Caspase 3

0123456

Caspase 9

02468

1012

Survivin

002040608

112

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

XIAP

002040608

112

GFAP

Caspase 3 Caspase 9

Survivin XIAP

GFAP

05

101520253035



4 Discussion





ASC

UC-MSCU251


G2M 1214 plusmn 147

S 3262 plusmn 524


G2M 1012 plusmn 259

S 2862 plusmn 321


G2M 850 plusmn 098

S 2123 plusmn 228

0 40 80 12020 60 100 0 40 80 120 160

0 40 80 120 160


Channels (FL2-A)

0

80

160

240

320

400

Num

ber

Num

ber

Num

ber

0

70

140

210

280

0

300

600

900

1200


Dip G2

Dip S


Dip G2


Dip G2

Dip S

An1 G1










U251 0h

ASC-CM 0h

UC-MSC-CM0h

U251 24h

ASC-CM 24h

UC-MSC-CM24h

U251 48h

ASC-CM 48h

UC-MSC-CM48h







U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3




Caspase 3

0123456

Caspase 9

02468

1012

Survivin

002040608

112

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

Before CMinduction


After ASCinduction

XIAP

002040608

112

GFAP

Caspase 3 Caspase 9

Survivin XIAP

GFAP

05

101520253035



4 Discussion





ASC

UC-MSCU251


G2M 1214 plusmn 147

S 3262 plusmn 524


G2M 1012 plusmn 259

S 2862 plusmn 321


G2M 850 plusmn 098

S 2123 plusmn 228

0 40 80 12020 60 100 0 40 80 120 160

0 40 80 120 160


Channels (FL2-A)

0

80

160

240

320

400

Num

ber

Num

ber

Num

ber

0

70

140

210

280

0

300

600

900

1200


Dip G2

Dip S


Dip G2


Dip G2

Dip S

An1 G1










U251 0h

ASC-CM 0h

UC-MSC-CM0h

U251 24h

ASC-CM 24h

UC-MSC-CM24h

U251 48h

ASC-CM 48h

UC-MSC-CM48h







U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3




ASC

UC-MSCU251


G2M 1214 plusmn 147

S 3262 plusmn 524


G2M 1012 plusmn 259

S 2862 plusmn 321


G2M 850 plusmn 098

S 2123 plusmn 228

0 40 80 12020 60 100 0 40 80 120 160

0 40 80 120 160


Channels (FL2-A)

0

80

160

240

320

400

Num

ber

Num

ber

Num

ber

0

70

140

210

280

0

300

600

900

1200


Dip G2

Dip S


Dip G2


Dip G2

Dip S

An1 G1










U251 0h

ASC-CM 0h

UC-MSC-CM0h

U251 24h

ASC-CM 24h

UC-MSC-CM24h

U251 48h

ASC-CM 48h

UC-MSC-CM48h







U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3









U251 0h

ASC-CM 0h

UC-MSC-CM0h

U251 24h

ASC-CM 24h

UC-MSC-CM24h

U251 48h

ASC-CM 48h

UC-MSC-CM48h







U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3




U251 0h

ASC-CM 0h

UC-MSC-CM0h

U251 24h

ASC-CM 24h

UC-MSC-CM24h

U251 48h

ASC-CM 48h

UC-MSC-CM48h







U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3




U87U251

U87U251

DMEM

ASC-CM

UC-MSC-CM


20

40

60

80

100

120

140

160

Cell

num

ber p

er fi

eld








U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3




U251

ASC-CM

UC-MSC-CM

(a)

010203040506070


0

1

2

3

4

5

6

7

8

9


0

5

10

15

20

25





(b)




2O3)


2O3) and









Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3










Acknowledgment


References






































2

O3



2

O3



2

O3

phenylbutyrate



2

O3


















2

O3



2

O3



2

O3

phenylbutyrate



2

O3



Documents

Conditioned Media from Human Adipose Tissue-Derived … C Biomed Res Int. 2014.pdf · 2014. 8. 8. · ResearchArticle Conditioned Media from Human Adipose Tissue-Derived Mesenchymal