Intravenous Administration of Adipose-Derived Stem Cell Protein …downloads.hindawi.com/journals/sci/2017/2153629.pdf · 2019. 7. 30. · StemCellsInternational 5 may impact the

Research ArticleIntravenous Administration of Adipose-DerivedStem Cell Protein Extracts Improves NeurologicalDeficits in a Rat Model of Stroke

Kai Zhao1 Rui Li2 Changcong Gu3 Long Liu1 Yulong Jia1 Xize Guo3

Wanping Zhang3 Chunying Pei3 Linlu Tian3 Bo Li3 Jianrong Jia4 Huakun Cheng4

Hongwei Xu3 and Lixian Li1

1Department of Neurosurgery The First Affiliate Hospital of Harbin Medical University Harbin Heilongjiang 150001 China2Neuroinflammation Unit Montreal Neurological Institute McGill University Montreal QC Canada H3A 2B43Department of Immunology Harbin Medical University Harbin Heilongjiang 150086 China4Department of Neurosurgery Heilongjiang Provincial Hospital Harbin Heilongjiang 150001 China

Correspondence should be addressed to Hongwei Xu hongwei56hotmailcom and Lixian Li llxzhdysinacom

Received 5 September 2016 Revised 21 November 2016 Accepted 27 November 2016 Published 19 January 2017

Academic Editor Marc L Turner

Copyright copy 2017 Kai Zhao et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Treatment of adipose-derived stem cell (ADSC) substantially improves the neurological deficits during stroke by reducing neuronalinjury limiting proinflammatory immune responses and promoting neuronal repair which makes ADSC-based therapy anattractive approach for treating stroke However the potential risk of tumorigenicity and low survival rate of the implanted cellslimit the clinical use of ADSC Cell-free extracts from ADSC (ADSC-E) may be a feasible approach that could overcome theselimitations Here we aim to explore the potential usage of ADSC-E in treating rat transient middle cerebral artery occlusion(tMCAO) We demonstrated that intravenous (IV) injection of ADSC-E remarkably reduces the ischemic lesion and number ofapoptotic neurons as compared to other control groups Although ADSC and ADSC-E treatment results in a similar degree ofa long-term clinical beneficial outcome the dynamics between two ADSC-based therapies are different While the injection ofADSC leads to a relatively mild but prolonged therapeutic effect the administration of ADSC-E results in a fast and pronouncedclinical improvement which was associated with a unique change in the molecular signature suggesting that potential mechanismsunderlying different therapeutic approach may be different Together these data provide translational evidence for using proteinextracts from ADSC for treating stroke

1 Introduction

Worldwide stroke is one of the main causes of death and dis-ability in adult and has become a serious public health prob-lem [1] Current therapeutic approaches focus on removingthe blockage of the blood vessel by thrombolysis (eg tPA)and surgery [2 3] However these treatments usually havesmall time window and have no or limiting effect on neuralrepair after the injury and therefore can only benefit 10 ofthe patients [2]There is a call for new therapies that can bothlimit ischemic brain lesion and promote neural repair

Recently several studies using the rodent model of strokehave demonstrated that the administration of mesenchymalstem cells (MSC) can substantially improve the neurological

deficits [4] The potential mechanisms for the therapeuticeffect of MSC on stroke may involve secreting neurotrophicfactors that promote neural cell survival and growth [5ndash7]enhancing neurogenesis [6] angiogenesis [6 8] and mod-ulating neuroinflammatory responses [6 9] In addition tothese beneficial effects MSC do not have issues such astissue-rejection as seen in other cell-transplantation-basedtherapies which furthermakesMSC-based therapy an attrac-tive approach for treating stroke MSC can be isolated frommany tissues such as bone-marrow umbilical cord andadipose tissue [4 10] Compared with other sources adiposetissue derived MSC (ADSC) have an advantage of beingabundant and easy to obtain ADSC can achieve comparablefunctional improvement after stroke as compared to other

HindawiStem Cells InternationalVolume 2017 Article ID 2153629 11 pageshttpsdoiorg10115520172153629

2 Stem Cells International

tissue derived MSC [4 11] However the potential risk oftumorigenicity and low survival rate of the implanted cellspotentially limit the clinical use of ADSC ADSC-basedcell-free approaches such as using conditioned medium orprotein extracts (ADSC-E) may be a feasible approach thatcould overcome these limitations Indeed the therapeuticeffect of ADSC-E has been shown in several disease contexts[7 12ndash15] Im et al showed that ADSC-E can slow downthe progression of Huntingtonrsquos disease by upregulating theexpression of p-CREB and PGC-1120572 two important disease-modifying molecules [13] Furthermore administration ofhuman ADSC-E before epilepticus has been shown to leadto the reduction of BBB leakage and earlier attenuation ofseizure spike activities after treatment with diazepam [12]Emerging evidence has suggested that the intraperitonealadministration of ADSC-E decreased ischemic lesion [7]however the optimal therapeutic regimen such as the deliveryroutes and molecular mechanisms still require further study

The delivery route is important for every cell-basedtherapy [16 17] In some cases the different delivery routesmay result in a completely different clinical outcome In othercases although different delivery routes may lead to similarclinical improvement the underlying mechanisms may becompletely different It has been shown by several studies thateither intravenous (IV) or intracerebral (IC) injection ofMSCcan enhance functional recovery after stroke [1 7 10 18]However there is no direct evidence comparing the impactof different routes of administration on the therapeutic effectof MSC

In this study we first explored the potential impact ofdifferent routes to deliver ADSC-E on stroke We demon-strated that IV injection of ADSC-E significantly improvesthe clinical symptom as compared to other control groupsFurthermore we showed that although ADSC and ADSC-E treatment results in a similar degree of a long-termclinical beneficial outcome the kinetics of these two ADSC-based therapies are quite different Such differences betweenADSC and ADSC-E are associated with unique molecularsignatures Together these data provide translational evidencefor ADSC-E therapy in stroke

2 Materials and Methods

21 Isolation of Adipose-Derived Mesenchymal Stem Cells(ADSC) Adipose tissue surrounding the enterocoelia wascarefully dissected The extracted tissue was washed withsterile PBS three times Next the tissue was enzymaticallydigested with an equal volume of 02 type I collagenase(Sigma-Aldrich) at 37∘C for 60 minutes under shaking con-ditions Collagenase activity was neutralized with an equalvolume of low glucose Dulbeccorsquos modified Eaglersquos medium(L-DMEM) containing 10 fetal bovine serum (FBS) Thefiltered cells were centrifuged at 2000 rpmmin for 10min toisolate the stromal vascular fraction (SVF) The cell pelletwas filtered through a 70mm pore-size filter Cells werethen cultured in L-DMEM 1 penicillinstreptomycin and10 fetal bovine serum Culture medium was changed every24 hrs for 3 days

22 Flow Cytometry (FACS) Surface Staining MSC werevalidated by flow cytometry Briefly ADSC were detachedand then washed once with FACS buffer (PBS + 05 BSA+ 01 sodium azide) The cells were incubated with surfaceantibodies on ice for 20min After two times wash cellswere acquired by flow cytometry (Accuri C6 BD) Thefluorescence conjugated antibodies for FACS include CD31-PE (Cat555027 BD) CD44-FITC (203906 Biolegend)CD45-FITC (202205 Biolegend) and CD90-APC (202507Biolegend) Matched isotype controls are as follows FITCMouse IgG2a 120581 (400207 Biolegend) Alexa Fluor 647MouseIgG1 120581 (400130 Biolegend) and PE Mouse IgG1 120581 (550617BD)

23 Adipogenic Differentiation of ADSC In Vitro

Adipogenic Differentiation and Identification of ADSC Cellswere expanded in 35mm culture plates until passage 3 or 4and then were induced for 14 days in an adipogenic inductionmedium (L-DMEM supplemented with 10 FBS 05mMisobutylmethylxanthine (IBMX) (Sigma-Aldrich) 1mMdex-amethasone 10mM insulin 200mM indomethacin and 1antibioticantimycotic solution) The culture medium waschanged every 3 days At Day 14 cells were fixed in 4formalin and stained with 2 fresh Oil red-O solution(Sigma-Aldrich) to detect lipid droplets in the induced cells

24 Osteogenic Differentiation of ADSC In Vitro ADSCsat passage 3 (P3) were incubated at 1 times 104 cellscm2in a 35mm culture plate The induction medium consist-ing of L-DMEM 10 FBS 01 120583M dexamethasone 10mM120573-glycerophosphate 005mM ascorbate-2- phosphate and1 antibioticantimycotic solution was used to induceosteogenic differentiation in vitro The culture medium waschanged every 3 days At Day 21 cells were fixed with 4paraformaldehyde and then gently washed with deionizedwater and incubated for 1 h in a 5 (wtvol) silver nitratesolution under UV light the cells were washed 3 times withneutralization of 5 sodium thiosulfate Finally the cellswere observed and photographswere taken under an invertedmicroscope

25 ADSC Extract (ADSC-E) Preparation TheADSC-Ewerefreshly prepared before injection Briefly 1 times 106 ADSCwere harvested and washed twice with PBS Cells were thensuspended in lysis buffer (1mM DTT 1mM EDTA proteaseinhibitor cocktail P8340 01DEPC in PBS) and lysedwithina syringe tube with a plugged end by pushing and pullingthe syringe piston several times Then put the tubes intoliquid nitrogen and water at 37∘C about several times Celllysates were then centrifuged at 14000119892 for 15min The finalproducts (around 600 ug) were later filtered through 045 uMfilter

26 Animals and Grouping Adult Sprague-Dawley rats wereused with an average bodyweight range of 250ndash320 gram andaverage age range of 4ndash6 weeks The animals were housedwith free access to food and waterat a room temperature

Stem Cells International 3

All experimental procedures involved were performed basedon protocols that are reviewed and approved by the Institu-tional Animal Care and Use Committee at Harbin MedicalUniversity The animals were randomly assigned to one offive experimental groups (1) the sham-operated group whichunderwent surgery without infarct (2) the control groupwhich underwent surgery with transient middle cerebralartery occlusion (tMCAO) and received PBS infusion in thefemoral vein intraperitoneal or intracerebral (different deliv-ery routes alone without ADSC-E had no effect on cerebralischemia (data not shown)) (3) the ADSC group whichunderwent tMCAO surgery and received ADSC infusion inthe femoral vein (4) the ADSC-E group which underwenttMCAO surgery and received an ADSC-E infusion in thefemoral vein intraperitoneal or intracerebral

27 Transient Middle Cerebral Artery Occlusion (tMCAO)Model Anesthesia was induced by intraperitoneal injectionof a solution of 10 chloral hydrate at a dose of 350mgkgmaintaining their rectal temperature at 37∘C The left com-mon carotid artery (CCA) external carotid artery (ECA) andinternal carotid artery (ICA) of each rat were exposed via amidline neck incision The tip of a 4-0 nylon monofilamentwas rounded by heating over a flame and coatedwith siliconeTo occlude the origin of the middle cerebral artery (MCA)the monofilament was moved carefully into the ICA lumenvia the ECA lumen until a slight resistance was felt After 2 hthe suture was slowly withdrawn to allow reperfusion Therats in the sham group received all the surgical procedures inthe absence of a suture Neurological function was evaluatedby mNSS scaling system and Rogerrsquos functional scale asdescribed before [19 20] by two researcherswhowere blindedto the experimental groups

28 Cell Administration ADSCs cultured at passage 3 to5 were collected by trypsin (025) digestion Thus intra-venous injections of ADSC and ADSC-E heated ADSC-E DNA and RNA derived from equal amounts of ADSC(around 1 times 106) in 650120583l PBS were administered for5min through the femoral vein after common carotid arteryreperfusion and intracerebral injections of ADSC-E in 15 120583lPBS were administered through a Hamilton syringe in astereotactic apparatus (AP-10mm ML-15mm DV-35 to45mm) immediately after CCA reperfusion the process forabout 10 minutes

29 Neurological Function Evaluation Neurological func-tion was evaluated by modified neurological severity scores(mNSS) scaling system and Rogerrsquos functional scale asdescribed before by two researchers who were blinded tothe experimental groups The mNSS was defined as followsraising rat by the tail (3) placing rat on the floor (normal= 0 maximum = 3) sensory test (2) beam balance tests(normal = 0 maximum = 6) reflexes absent and abnormalmovements (4) and maximum points (18) In all 1 to 6indicates mild injury 7 to 12 moderate injury and 13 to 18severe injury A variant of Rogerrsquos functional scale was usedto assign scores as follows 0 no functional deficit 1 failure toextend forepaw fully 2 decreased grip of forelimb while tail

is gently pulled 3 spontaneous movement in all directionscontralateral circling only if pulled by the tail 4 circling 5walking onlywhen stimulated 6 unresponsive to stimulationwith a depressed level of consciousness and 7 dead

210 235-Triphenyltetrazolium Chloride (TTC) StainingThe brain tissues were sliced into seven 2mm thick coronalsections and stained with 1 TTC solution at 37∘C for 15minEach section was then washed with PBS and fixed in 4paraformaldehyde The infarction volume was presented asa percentage of the volume of the brain Image J software wasused to analyze the percentage of TTC-stained tissue

211 TUNEL Staining Cell death was detected by terminaldeoxynucleotidyl transferase (TUNEL) staining followingmanufacturerrsquos protocol Briefly the section was first fixedusing 4 paraformaldehyde and then incubated in thepermeabilization buffer (01 Triton X-100 in 01 sodiumcitrate) on ice for 2min The section was then incubatedin TUNEL reaction mixture (enzyme solution and labelsolution) for 60min at 37∘C After that the reaction wasterminated by adding 1X PBS solution for 15min Nuclei werethen counterstained with DAPI for 5min

212 Real-Time Quantitative PCR Analysis The expressionlevels of IL-6 (forward primer 51015840-CAGGGAGATCTTGGAAATGAG-31015840 reverse primer 51015840-GTTGTTCTTCACAAACTCC-31015840) TNF120572 (forward primer 51015840-GATCGGTCCCAACAAGGAGG-31015840 reverse primer 51015840-GCTGGTACCACCAGTTGGTTG-31015840) IFN120574 (forward primer 51015840-AGGAAAGAGCCTCCTCTTGG-31015840 reverse primer 51015840-TGGGTTGTTCACCTCGAACT-31015840) IL-10 (forward primer 51015840-TAAGGGTTACCTTGGGTTGCCAAGCC-31015840 reverse primer 51015840-AGGGGAGAAATCGATGACAGCGCC-31015840) BDNF (forwardprimer 51015840-CGACGTCCCTGGCTGACACTTTT-31015840 reverseprimer 51015840-AGTAAGGGCCCGAACATACGATTGG-31015840)NGF (forward primer 51015840-ACCTCTTCGGACACTCTGG-31015840reverse primer 51015840-CGTGGCTGTGGTCTTATCTC-31015840)CNTF (forward primer 51015840-TCCAAGAGAACCTCCAGGCTTA-31015840 reverse primer 51015840-GCTGGTAGGCAAAGGCAGAA-31015840) IGF-1 (forward primer 51015840-GACATGCCCAAGACCCAGAAGGA-31015840 reverse primer 51015840-CGGTGGCATGTCACTCTTCACTC-31015840) and 120573-actin (forward primer 51015840-ACGTTGACAT CCGTAAAGAC-31015840 reverse primer 51015840-GAAGGTGGACAGTGAGGC-31015840) were measured by real-time PCR (RT-PCR) Total RNAof each sample was extractedfrom the tissue of the infarcted border zones using Trizol(Invitrogen) according to the manufacturersquos protocol and weconducted RT-PCR using the First-Strand cDNA SynthesisKit (Roche) Quantitative real-time RT-PCR was performedusing SYBR-Green (Roche)

213 Statistic GraphPad Prism 6 was used to perform all thestatistical analysis One-way ANOVA was used for statisticalcomparisons among more than two groups All statisticaltests have been indicated in the figure legends 119901 values of005 or less were considered significant


(a) (b) (c)

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Figure 1 ADSCs characterization and ADSC treatment regime ADSC were isolated from adipose tissue surrounding the enterocoelia AsADSC approached confluence they showed a more spindle-shaped fibroblastic morphology Scale bars 100 um (a) (b) ADSC were culturedin the adipogenic induction medium for 14 days Oil red was used to validate the induction The intracellular lipid droplets were showed aspositive red loci Scale bars 100 um (c) ADSC were cultured in the osteogenic induction medium for 21 days Von Kossa staining showedthat a calcified extracellular matrix of induced ADSC was detected Scale bars 500 um (d) Flow cytometry analysis was used to measure thesurface marker of ADSCs ADSCs were negative for leukocyte markers CD45 (171) and CD31 (021) and positive for mesenchymal stemcell markers CD44 (984) and CD90 (999) (d) ADSCADSC-E were administrated through IV or IC or IP 1 hour after tMCAO (e)

3 Results

31 ADSC Characterization ADSC were isolated from adi-pose tissue surrounding the enterocoelia The primary cul-ture of ADSC appeared as a monolayer of large flat cellsAs the cells approached confluence they showed a morespindle-shaped fibroblasticmorphology (Figure 1(a)) To testadipogenic differentiation of ADSC ADSC were culturedin the adipogenic induction medium for 14 days Oil redwas used to validate the induction As shown in Figure 1(b)the intracellular lipid droplets were showed as positive redloci (Figure 1(b)) To test osteogenic differentiation in vitroADSC were cultured in the osteogenic induction medium

for 21 days Von Kossa staining showed that a calcifiedextracellular matrix of induced ADSC was detected whichconfirmed ADSC could differentiate into osteogenic cells(Figure 1(c)) Flow cytometry analysis of ADSC at passages 3-4 demonstrated that the cells were negative for leukocyte andendotheliocytemarkers CD45 (171) andCD31 (021) andpositive for mesenchymal stem cell markers CD44 (984)and CD90 (999) (Figure 1(d))

32 The Impact of Delivery Routes on the Therapeutic Effectof Protein Extracts from ADSC during Stroke Little is knownhow different delivery routes may influence the therapeuticeffect of ADSC-E To test how different delivery routes


may impact the therapeutic effect of ADSC-E on strokewe induced tMCAO on SD Rat Protein extracts fromprevalidated ADSC were injected either intravenous (IV)or intracerebral (IC) or intraperitoneal (IP) to the tMCAOrats Neurological deficits were graded based on modifiedneurological severity scores (mNSS) scale system from Day0 to Day 28 (Figure 1(e)) TTC staining was used toquantify the volume of infarct lesion at Day 1 and Day7 (Figure 1(e)) Inconsistent with previous work [7] weshowed that injection of ADSC-E through IP did not improveeither neurological deficits or ischemic lesion (Figures 2(a)ndash2(c)) Compared to IC IV injection of ADSC-E showedmuch better improvement of the neurological deficits of bothmNSS scaling system (Figure 2(a)) and Rogerrsquos functionalscale (Supplementary 1A in SupplementaryMaterial availableonline at httpsdoiorg10115520172153629) In keepingwith the clinical evaluation the volume of infarct regionwas also reduced in the IV group and to a lesser extent inthe IC group at both Day 1 and Day 7 (Figures 2(b) and2(c)) Furthermore the number of apoptotic neuron in thepenumbra area of the ischemic lesion is also remarkablydecreased in the IV group (Figures 2(d) and 2(e)) In additionwe noticed that the operation procedure of intracerebralinjection significantly increases the rate of death (data notshown) Taken together compared to other delivery routesadministration of ADSC-E through IV is relatively safe andhighly effective way to deliver ADSC

33 The Impact of Different Components of ADSC duringStroke Previous study has found Neuro2a cells that aretreated with the ADSC-E increased cell viability and reducedcytotoxicity [7] However most of the cells treated with heat-treated ADSC-E DNA RNA or lipids derived from ADSCdid not show similar protection But this has never beentested in vivo To test which kind of the cellular componentsof ADSChave themost robust therapeutic effect on stroke weintravenously inject ADSC-E heat-treated ADSC-E DNARNA or vehicle after tMCAO We found that in the ADSC-E group on the first Day after treatment the neurologicalfunction was restored (Supplementary 2A and 2B) and theinfarct size was significantly reduced atDay 1 (Supplementary2C) However no differences were observed in the othergroups suggesting that the protein component of ADSC-Ewas responsible for the therapeutic effect of ADSC on stroke

34 Different Therapeutic Dynamics between ADSC andADSC-E on Stroke Next wewould like to know the potentialdifferences between adipose-derived stem cell and proteinextracts from ADSC in stroke therapy Either ADSC (1 times106) or ADSC-E (generated from 1 times 106) were injectedthrough IV to the rats that received tMCAO We foundthat although both ADSC and ADSC-E can significantlyimprove the neurological deficits both mNSS scaling system(Figure 3(a)) and Rogerrsquos functional scale (Supplementary1B) and reduce the infarct lesion (Figure 3(c)) the dynamicsof these two treatments are different The administration ofADSC-E results in a much faster neurological improvementwhile the injection of ADSC leads to a relatively modestbut prolonged relief of the clinical symptoms (Figure 3(a))

Consistent with the clinical evaluation TTC staining andTUNEL staining further supported that the treatment ofADSC-E results in less neuronal damage at earlier time points(Days 1sim3) (Figures 3(b) and 3(d)) These together suggestedthat the therapeutic dynamics between ADSC and ADSC-Eon stroke is different

35 Neuroprotective Function of ADSC and Immunomodu-latory Roles of ADSC-E Are Associated with the Differencesof Therapeutic Dynamics between ADSC and ADSC-E Nextwe would like to further explore the potential reason forthe difference of therapeutic dynamics between ADSC andADSC-E Studies have suggested that ldquobystanderrdquo functionsincluding upregulation of neurotrophic factors downregu-lation of proinflammatory cytokines and enhancing angio-genesis are the main contributors to the therapeutic effect ofADSC during the strokeWe asked whether the phenomenonthat we observed above may be due to the differentialmolecularcellular mechanisms underlying different ADSC-based therapeutic approaches To test this hypothesis wemeasured the expression of proinflammatory cytokines andneurotrophic factors by real-time PCR using mRNA isolatedfrom the ipsolateral cortex of the rat with or without ADSC-based therapies either at Day 1 or Day 3 We noticedthat ADSC-E significantly downregulated proinflammatorycytokine (IL-6 TNF120572 and IFN120574) production in the ischemicbrain (Figures 4(a)ndash4(d)) while the injection of ADSC onlyreduced TNF120572 (Figure 4(b)) which suggested that ADSC-Eat the earlier time point showed a stronger immunomodu-latory effect as compared to ADSC In contrast the upreg-ulation of neurotrophic factors (BDNF NGF CNTF andIGF) is much more robust in the ADSC-treated group ascompared to the group which received ADSC-E (Figures4(f)ndash4(h)) at Day 3 which may explain the prolonged effectof ADSC Collectively these data indicated that the differenttherapeutic dynamics between ADSC and ADSC-E mayberelated to their differential modes of action

4 Discussion

Studies have shown that stem cell-based therapies can sub-stantially improve the neurological deficits during stroke[4 11] Our current study explored the potential usage ofa ldquocell-freerdquo ADSC-based approach in stroke therapy Weshowed that IV injection of the protein extract but notinjecting other components of ADSC substantially improvesthe neurological deficits and rescues neuron from death Inaddition compared to ADSC ADSC-E treatment results ina much faster clinical improvement which is associated withits stronger ability to modulate neuroinflammation

The choice of delivery routes is critically important forcell-based therapy which may directly influence outcomes ofthe treatment The ideal delivery route should be relativelysafe cost efficient and easy to apply to the patients withoutcausing any additional issue Furthermore cells administeredby this route should result in benefits to clinical outcomemeasures The delivering routes utilized for MSCADSC-based therapy on stroke include intracerebral (IC) intra-venous (IV) and intraperitoneal (IP) [7 8 10 17 18]


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Figure 2 The impact of delivery routes on the therapeutic effect of ADSC-E during stroke tMCAO was induced using SD rat Proteinextracts fromADSCs (ADSC-E) were injected either through IV IC or IP after the operation (a)The neurological deficits were graded basedon mNSS scaling system (119899 = 8group) (b c) TTC staining was used to quantify the volume of infarct area at either Day 1 (119899 = 6group) (b)or Day 7 (119899 = 6group) (c) (d e) TUNEL staining was used to measure apoptotic neurons Scale bars 100 um (e) Five sections from eachmouse were selected (119899 = 3group) For each section TUNEL positive neural cells were counted from 3 separated yields ANOVA was usedto analyze the data lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001


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Figure 3 Different therapeutic characteristics between ADSC and ADSC-E on stroke tMCAO was induced using SD rat Either ADSC orADSC-E was injected through IV (a)The neurological deficits were graded based onmNSS scaling system (119899 = 8group) (b c) TTC stainingwas used to quantify the volume of infarct area at either Day 1 (119899 = 6group) (b) or Day 7 (119899 = 6group) (c) (d e) TUNEL staining wasused to measure apoptotic neurons Scale bars 100 um (e) Five sections from each rat were selected (119899 = 3group) For each section TUNELpositive neural cells were counted from 3 separated yields ANOVA was used to analyze the data lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001


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Figure 4 Different molecular signatures between ADSC and ADSC-E on stroke therapies tMCAO was induced using SD rat Either ADSCor ADSC-E was injected through IV mRNAwas extracted from the ipsolateral cortex Cytokines (both pro- and anti-inflammatory) ((a)ndash(d)mRNA from Day 1) and neurotrophic factors ((e)ndash(h) mRNA from Day 3) were measured by real-time PCR (119899 = 6group) ANOVA wasused to analyze the data lowast119901 lt 005 lowastlowast119901 lt 001

However no study has directly compared the effectivenessof different delivery routes on ADSC-based therapies duringthe stroke In this study we have demonstrated that thetherapeutic effect of ADSC-based therapies on stroke isstrongly associated with the delivery routes In contrast topreviously published study we did not observe any beneficialeffect of ADSC-E injected through peritoneal administrationThis may be due to the differences of cell number to generateprotein extracts In the current study we injected proteinextract that is isolated from 5 times less ADSC than theprevious study [7] Despite this we still see a substantialclinical beneficial effect in the IV group further suggestingthat IV is a preferable choice for ADSC-E in stroke therapy

There are several advantages of using ldquocell-freerdquo ADSC-based over cell-based ADSC therapy Firstly protein extractsare more readily to use and easier to preserve Secondlyalthough the therapeutic effect of cell-free approach maybetransient due to the short half-life of the effector proteinldquocell-freerdquo approach also eliminated the potential long-termside effect of cell-based approach such as tumorigenesis Inaddition our data suggested that the treatment of ADSC-E results in a faster therapeutic effect in stroke which isextremely valuable and may open therapeutic windows forother combination therapies

Although evidence has shown that ADSC can differenti-ate into neural cells in vitro [21 22] recent studies indicatedthat very fewADSC can actually differentiate into neural cellsin vivo [23 24] whichmay due to the lack of permissive localenvironment for neural differentiation [4] suggesting thatneural cell replacement may not be the primary mechanismunderlying the therapeutic effect of ADSC on stroke Theeffectiveness of using protein extract or conditional mediumfrom ADSC on stroke further supports this [7 25] Immune

modulatory function may be important for the therapeuticfunction of ADSC during stroke [4 9 26 27] The molecularmechanisms may involve immune regulatory molecules suchas TGFbeta [28 29] IDO [30] and PGE2 [31 32] Howeverlittle is known about the place where ADSC regulates theimmune responses peripheral lymphoid tissue or local CNSor both Our data have shown that IV injection of ADSC-E has a stronger impact on limiting ischemic lesion andapoptotic lesion as compared to IC injection of ADSC-Ewhich may indirectly indicate that the effect of ADSC-E onthe peripheral immune responses maybe more importantmechanism underlying the therapeutic effect of ADSC-E

5 Conclusion

Taken together we conclude that IV injection of proteinextracts from ADSC showed a faster clinical outcome ascompared to ADSC Different therapeutic dynamics betweenADSC and ADSC-E may be due to different modes of actionOur study provides the translational basis for using proteinextracts from ADSC in stroke therapies

Competing Interests

The authors have no conflict of interests

Authorsrsquo Contributions

Kai Zhao generated the initial hypothesis performed theexperiment and analyzed the data Rui Li analyzed the dataand wrote the manuscript Changcong Gu and Yulong Jiahelped with establishing the model Xize Guo WanpingZhang helped with rat keeping Chunying Pei and Linlu


Tian helped with the HE staining Lixian Li Bo Li HuakunCheng and Jianrong Jia provided thoughtful research discus-sion Hongwei Xu and Lixian Li provided overall supervisionand financial support for this study Kai Zhao and Rui Licontributed equally to this work

Acknowledgments

The authors thank Professor Hulun Li for project discus-sion and technical support These studies were funded byNational Natural Science Foundation Projects (no 31371080)to Dr Hongwei Xu and National Natural Science FoundationProject (no 81371301) to Dr Lixian Li

References

[1] V L Feigin C M Lawes D A Bennett S L Barker-Collo andV Parag ldquoWorldwide stroke incidence and early case fatalityreported in 56 population-based studies a systematic reviewrdquoThe Lancet Neurology vol 8 no 4 pp 355ndash369 2009

[2] J Emberson K R Lees P Lyden et al ldquoEffect of treatmentdelay age and stroke severity on the effects of intravenousthrombolysis with alteplase for acute ischaemic stroke a meta-analysis of individual patient data from randomised trialsrdquoTheLancet vol 384 no 9958 pp 1929ndash1935 2014

[3] W J Powers C P Derdeyn J Biller et al ldquo2015 AmericanHeart AssociationAmerican stroke association focused updateof the 2013 guidelines for the early management of patientswith acute ischemic stroke regarding endovascular treatment aguideline for healthcare professionals from the American HeartAssociationAmerican stroke associationrdquo Stroke vol 46 no10 pp 3020ndash3035 2015

[4] L Hao Z Zou H Tian Y Zhang H Zhou and L Liu ldquoStemcell-based therapies for ischemic strokerdquo BioMed ResearchInternational vol 2014 Article ID 468748 17 pages 2014

[5] A-Z Chen N Liu H Huang F-F Lin D-S Liu and X-HLin ldquoOutgrowth of neuronal axons on adipose-derived stemcell transplanting for treatment of cerebral infarction in ratsrdquoChinese Journal of Cellular and Molecular Immunology vol 27no 8 pp 868ndash871 2011

[6] S Leu Y-C Lin C-M Yuen et al ldquoAdipose-derivedmesenchy-mal stem cellsmarkedly attenuate brain infarct size and improveneurological function in ratsrdquo Journal of TranslationalMedicinevol 8 article no 63 2010

[7] D Jeon K Chu S-T Lee et al ldquoNeuroprotective effect ofa cell-free extract derived from human adipose stem cells inexperimental stroke modelsrdquo Neurobiology of Disease vol 54pp 414ndash420 2013

[8] H S Nam I Kwon B H Lee et al ldquoEffects of mesenchymalstem cell treatment on the expression of matrix metallopro-teinases and angiogenesis during ischemic stroke recoveryrdquoPLoS ONE vol 10 Article ID e0144218 2015

[9] H-W Du N Liu J-H Wang Y-X Zhang R-H Chen andY-C Xiao ldquoThe effects of adipose-derived stem cell transplan-tation on the expression of IL-10 and TNF-alpha after cerebralischaemia in ratsrdquo Chinese Journal of Cellular and MolecularImmunology vol 25 no 11 pp 998ndash1001 2009

[10] M Gutierrez-Fernandez B Rodrıguez-Frutos J Ramos-Cejudo et al ldquoComparison between xenogeneic and allogeneic

adipose mesenchymal stem cells in the treatment of acute cere-bral infarct proof of concept in ratsrdquo Journal of TranslationalMedicine vol 13 no 1 article no 46 2015

[11] M Gutierrez-Fernandez L Otero-Ortega J Ramos-Cejudo BRodrıguez-Frutos B Fuentes and E Dıez-Tejedor ldquoAdiposetissue-derived mesenchymal stem cells as a strategy to improverecovery after strokerdquoExpert Opinion on BiologicalTherapy vol15 no 6 pp 873ndash881 2015

[12] D Jeon K Chu S-T Lee et al ldquoA cell-free extract from humanadipose stem cells protects mice against epilepsyrdquo Epilepsia vol52 no 9 pp 1617ndash1626 2011

[13] W Im J Ban J Lim et al ldquoExtracts of adipose derived stem cellsslows progression in the R62 model of Huntingtonrsquos diseaserdquoPLoS ONE vol 8 no 4 Article ID e59438 2013

[14] J-J Ban S Yang W Im and M Kim ldquoNeurogenic effects ofcell-free extracts of adipose stem cellsrdquo PLoS ONE vol 11 no 2Article ID e0148691 2016

[15] K Kingwell ldquoEpilepsy extract from human stem cells showspromise inmousemodel of epilepsyrdquoNature ReviewsNeurologyvol 7 no 10 p 536 2011

[16] N G Campbell andK Suzuki ldquoCell delivery routes for stem celltherapy to the heart current and future approachesrdquo Journal ofCardiovascular Translational Research vol 5 no 5 pp 713ndash7262012

[17] T J Kean P Lin A I Caplan and J E Dennis ldquoMSCsdelivery routes and engraftment cell-targeting strategies andimmunemodulationrdquo StemCells International vol 2013 ArticleID 732742 13 pages 2013

[18] S K Kang D H Lee Y C Bae H K Kim S Y Baik and JS Jung ldquoImprovement of neurological deficits by intracerebraltransplantation of human adipose tissue-derived stromal cellsafter cerebral ischemia in ratsrdquoExperimental Neurology vol 183no 2 pp 355ndash366 2003

[19] Y Xiong A Mahmood and M Chopp ldquoAnimal models oftraumatic brain injuryrdquoNature ReviewsNeuroscience vol 14 no2 pp 128ndash142 2013

[20] MGutierrez-Fernandez B Rodrıguez-Frutos J Alvarez-Grechet al ldquoFunctional recovery after hematic administration ofallogenic mesenchymal stem cells in acute ischemic stroke inratsrdquo Neuroscience vol 175 pp 394ndash405 2011

[21] T Huang D He G Kleiner and J Kuluz ldquoNeuron-likedifferentiation of adipose-derived stem cells from infant pigletsin vitrordquo Journal of Spinal Cord Medicine vol 30 no 1 pp S35ndashS40 2007

[22] G Tian J Zhou J Wang et al ldquoNeuronal differentiation ofadipose-derived stem cells and their transplantation for cerebralischemiardquoNeural Regeneration Research vol 7 no 25 pp 1992ndash1999 2012

[23] L E Fox J Shen KMa et al ldquoMembrane properties of neuron-like cells generated from adult human bone-marrow-derivedmesenchymal stem cellsrdquo Stem Cells and Development vol 19no 12 pp 1831ndash1841 2010

[24] G Paul I Ozen N S Christophersen et al ldquoThe adulthuman brain harbors multipotent perivascular mesenchymalstem cellsrdquo PLoS ONE vol 7 no 4 Article ID e35577 2012

[25] Y Egashira S Sugitani Y Suzuki et al ldquoThe conditionedmedium of murine and human adipose-derived stem cellsexerts neuroprotective effects against experimental strokemodelrdquo Brain Research vol 1461 pp 87ndash95 2012

[26] A J Nauta andW E Fibbe ldquoImmunomodulatory properties ofmesenchymal stromal cellsrdquo Blood vol 110 no 10 pp 3499ndash3506 2007


[27] A A Leto Barone S KhalifianW P A Lee and G BrandacherldquoImmunomodulatory effects of adipose-derived stem cells factor fictionrdquo BioMed Research International vol 2013 Article ID383685 8 pages 2013

[28] C Xu P Yu X Han et al ldquoTGF-120573 promotes immune responsesin the presence ofmesenchymal stem cellsrdquo Journal of Immunol-ogy vol 192 no 1 pp 103ndash109 2014

[29] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[30] Q-F Kong S Bo G-Y Wang et al ldquoBM stromal cell same-liorate experimental autoimmunemyasthenia gravis by alteringthe balance ofTh cells through the secretion of IDOrdquo EuropeanJournal of Immunology vol 39 no 3 pp 800ndash809 2009

[31] B Hegyi G Kudlik E Monostori and F Uher ldquoActivatedT-cells and pro-inflammatory cytokines differentially regulateprostaglandin E2 secretion by mesenchymal stem cellsrdquo Bio-chemical and Biophysical Research Communications vol 419 no2 pp 215ndash220 2012

[32] A Rozenberg A Rezk M-N Boivin et al ldquoHuman mes-enchymal stem cells impact Th17 and Th1 responses througha prostaglandin E2 and myeloid-dependent mechanismrdquo StemCells Translational Medicine vol 5 no 11 pp 1506ndash1514 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


tissue derived MSC [4 11] However the potential risk oftumorigenicity and low survival rate of the implanted cellspotentially limit the clinical use of ADSC ADSC-basedcell-free approaches such as using conditioned medium orprotein extracts (ADSC-E) may be a feasible approach thatcould overcome these limitations Indeed the therapeuticeffect of ADSC-E has been shown in several disease contexts[7 12ndash15] Im et al showed that ADSC-E can slow downthe progression of Huntingtonrsquos disease by upregulating theexpression of p-CREB and PGC-1120572 two important disease-modifying molecules [13] Furthermore administration ofhuman ADSC-E before epilepticus has been shown to leadto the reduction of BBB leakage and earlier attenuation ofseizure spike activities after treatment with diazepam [12]Emerging evidence has suggested that the intraperitonealadministration of ADSC-E decreased ischemic lesion [7]however the optimal therapeutic regimen such as the deliveryroutes and molecular mechanisms still require further study

The delivery route is important for every cell-basedtherapy [16 17] In some cases the different delivery routesmay result in a completely different clinical outcome In othercases although different delivery routes may lead to similarclinical improvement the underlying mechanisms may becompletely different It has been shown by several studies thateither intravenous (IV) or intracerebral (IC) injection ofMSCcan enhance functional recovery after stroke [1 7 10 18]However there is no direct evidence comparing the impactof different routes of administration on the therapeutic effectof MSC

In this study we first explored the potential impact ofdifferent routes to deliver ADSC-E on stroke We demon-strated that IV injection of ADSC-E significantly improvesthe clinical symptom as compared to other control groupsFurthermore we showed that although ADSC and ADSC-E treatment results in a similar degree of a long-termclinical beneficial outcome the kinetics of these two ADSC-based therapies are quite different Such differences betweenADSC and ADSC-E are associated with unique molecularsignatures Together these data provide translational evidencefor ADSC-E therapy in stroke

2 Materials and Methods

21 Isolation of Adipose-Derived Mesenchymal Stem Cells(ADSC) Adipose tissue surrounding the enterocoelia wascarefully dissected The extracted tissue was washed withsterile PBS three times Next the tissue was enzymaticallydigested with an equal volume of 02 type I collagenase(Sigma-Aldrich) at 37∘C for 60 minutes under shaking con-ditions Collagenase activity was neutralized with an equalvolume of low glucose Dulbeccorsquos modified Eaglersquos medium(L-DMEM) containing 10 fetal bovine serum (FBS) Thefiltered cells were centrifuged at 2000 rpmmin for 10min toisolate the stromal vascular fraction (SVF) The cell pelletwas filtered through a 70mm pore-size filter Cells werethen cultured in L-DMEM 1 penicillinstreptomycin and10 fetal bovine serum Culture medium was changed every24 hrs for 3 days

22 Flow Cytometry (FACS) Surface Staining MSC werevalidated by flow cytometry Briefly ADSC were detachedand then washed once with FACS buffer (PBS + 05 BSA+ 01 sodium azide) The cells were incubated with surfaceantibodies on ice for 20min After two times wash cellswere acquired by flow cytometry (Accuri C6 BD) Thefluorescence conjugated antibodies for FACS include CD31-PE (Cat555027 BD) CD44-FITC (203906 Biolegend)CD45-FITC (202205 Biolegend) and CD90-APC (202507Biolegend) Matched isotype controls are as follows FITCMouse IgG2a 120581 (400207 Biolegend) Alexa Fluor 647MouseIgG1 120581 (400130 Biolegend) and PE Mouse IgG1 120581 (550617BD)

23 Adipogenic Differentiation of ADSC In Vitro

Adipogenic Differentiation and Identification of ADSC Cellswere expanded in 35mm culture plates until passage 3 or 4and then were induced for 14 days in an adipogenic inductionmedium (L-DMEM supplemented with 10 FBS 05mMisobutylmethylxanthine (IBMX) (Sigma-Aldrich) 1mMdex-amethasone 10mM insulin 200mM indomethacin and 1antibioticantimycotic solution) The culture medium waschanged every 3 days At Day 14 cells were fixed in 4formalin and stained with 2 fresh Oil red-O solution(Sigma-Aldrich) to detect lipid droplets in the induced cells

24 Osteogenic Differentiation of ADSC In Vitro ADSCsat passage 3 (P3) were incubated at 1 times 104 cellscm2in a 35mm culture plate The induction medium consist-ing of L-DMEM 10 FBS 01 120583M dexamethasone 10mM120573-glycerophosphate 005mM ascorbate-2- phosphate and1 antibioticantimycotic solution was used to induceosteogenic differentiation in vitro The culture medium waschanged every 3 days At Day 21 cells were fixed with 4paraformaldehyde and then gently washed with deionizedwater and incubated for 1 h in a 5 (wtvol) silver nitratesolution under UV light the cells were washed 3 times withneutralization of 5 sodium thiosulfate Finally the cellswere observed and photographswere taken under an invertedmicroscope

25 ADSC Extract (ADSC-E) Preparation TheADSC-Ewerefreshly prepared before injection Briefly 1 times 106 ADSCwere harvested and washed twice with PBS Cells were thensuspended in lysis buffer (1mM DTT 1mM EDTA proteaseinhibitor cocktail P8340 01DEPC in PBS) and lysedwithina syringe tube with a plugged end by pushing and pullingthe syringe piston several times Then put the tubes intoliquid nitrogen and water at 37∘C about several times Celllysates were then centrifuged at 14000119892 for 15min The finalproducts (around 600 ug) were later filtered through 045 uMfilter

26 Animals and Grouping Adult Sprague-Dawley rats wereused with an average bodyweight range of 250ndash320 gram andaverage age range of 4ndash6 weeks The animals were housedwith free access to food and waterat a room temperature












(a) (b) (c)

0

Cou

nt

CD44100 102 104 106

CD45100 102 104 106 100 102 104 106 100 102 104 106

Cou

nt

CD31

Cou

nt

CD90

500

984 171 021 999

ADSCIsotype

ADSCIsotype

ADSCIsotype

ADSCIsotype

20K

15K

10K

0

500

20K

15K

10K

25K

Cou

nt

0

500

20K

15K

10K

0

500

15K

10K

25K

(d)

ADSCADSC-EtMCAO

IV or IP or IC

mNSSTUNEL and TTC

mNSS TTC

Day 0 Day 1 Day 7

mNSS

simDay 28

(e)


3 Results










4 Discussion




mN

SS

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

0

5

10

15

20


lowastlowast

Day

0

Day

1

Day

7

Day

14

Day

21

Day

28

(a)

ns

in

farc

t les

ion

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

10

20

30lowastlowast


lowast

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

(b)

in

farc

t les

ion

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

10

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

15


lowast

lowast

lowast

5

(c)

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

(d)

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol


lowast

lowast

100

0

150

200

250

50

Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



mN

SS

0

5

10

15

20

lowastlowastlowast

Iv-ADSC-EIv-ADSC

Control

Day

0

Day

1

Day

3

Day

7

Day

14

Day

21

Day

28

lowastlowastlowast

ns

ns

(a)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol


in

farc

t les

ion

0

10

20

30lowast

(b)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

ns


in

farc

t les

ion

0

10

5

15

20

(c)

Sham

Iv-ADSC-EIv-ADSC

Control

(d)

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

0

50

100

150

200

250

Sham


Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



0

50

100

150 ns


Fold

chan

ge (I

L-6)

lowastlowast

lowast

(a)

0

20

40

60

80


lowastlowast

lowastlowast

Fold

chan

ge (T

NF120572

)

(b)

0

5

10

15

ns


lowastlowast

lowast

Fold

chan

ge (I

FN120574

)

(c)

0

10

20

30

40


Fold

chan

ge (I

L-10

)

(d)

0

10

20

30

40

5050

100

150

ns



Fold

chan

ge (B

DN

F)

(e)

0

10

20

30

40

50


lowast


Fold

chan

ge (N

GF)

(f)

Figure 4 Continued


0

10

20

30

40ns


Fold

chan

ge (C

NTF

)


0

2

4

6

8

10

125

250

ns


Fold

chan

ge (I

GF1

)







5 Conclusion


Competing Interests






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












(a) (b) (c)

0

Cou

nt

CD44100 102 104 106

CD45100 102 104 106 100 102 104 106 100 102 104 106

Cou

nt

CD31

Cou

nt

CD90

500

984 171 021 999

ADSCIsotype

ADSCIsotype

ADSCIsotype

ADSCIsotype

20K

15K

10K

0

500

20K

15K

10K

25K

Cou

nt

0

500

20K

15K

10K

0

500

15K

10K

25K

(d)

ADSCADSC-EtMCAO

IV or IP or IC

mNSSTUNEL and TTC

mNSS TTC

Day 0 Day 1 Day 7

mNSS

simDay 28

(e)


3 Results










4 Discussion




mN

SS

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

0

5

10

15

20


lowastlowast

Day

0

Day

1

Day

7

Day

14

Day

21

Day

28

(a)

ns

in

farc

t les

ion

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

10

20

30lowastlowast


lowast

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

(b)

in

farc

t les

ion

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

10

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

15


lowast

lowast

lowast

5

(c)

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

(d)

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol


lowast

lowast

100

0

150

200

250

50

Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



mN

SS

0

5

10

15

20

lowastlowastlowast

Iv-ADSC-EIv-ADSC

Control

Day

0

Day

1

Day

3

Day

7

Day

14

Day

21

Day

28

lowastlowastlowast

ns

ns

(a)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol


in

farc

t les

ion

0

10

20

30lowast

(b)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

ns


in

farc

t les

ion

0

10

5

15

20

(c)

Sham

Iv-ADSC-EIv-ADSC

Control

(d)

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

0

50

100

150

200

250

Sham


Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



0

50

100

150 ns


Fold

chan

ge (I

L-6)

lowastlowast

lowast

(a)

0

20

40

60

80


lowastlowast

lowastlowast

Fold

chan

ge (T

NF120572

)

(b)

0

5

10

15

ns


lowastlowast

lowast

Fold

chan

ge (I

FN120574

)

(c)

0

10

20

30

40


Fold

chan

ge (I

L-10

)

(d)

0

10

20

30

40

5050

100

150

ns



Fold

chan

ge (B

DN

F)

(e)

0

10

20

30

40

50


lowast


Fold

chan

ge (N

GF)

(f)

Figure 4 Continued


0

10

20

30

40ns


Fold

chan

ge (C

NTF

)


0

2

4

6

8

10

125

250

ns


Fold

chan

ge (I

GF1

)







5 Conclusion


Competing Interests






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

0

Cou

nt

CD44100 102 104 106

CD45100 102 104 106 100 102 104 106 100 102 104 106

Cou

nt

CD31

Cou

nt

CD90

500

984 171 021 999

ADSCIsotype

ADSCIsotype

ADSCIsotype

ADSCIsotype

20K

15K

10K

0

500

20K

15K

10K

25K

Cou

nt

0

500

20K

15K

10K

0

500

15K

10K

25K

(d)

ADSCADSC-EtMCAO

IV or IP or IC

mNSSTUNEL and TTC

mNSS TTC

Day 0 Day 1 Day 7

mNSS

simDay 28

(e)


3 Results










4 Discussion




mN

SS

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

0

5

10

15

20


lowastlowast

Day

0

Day

1

Day

7

Day

14

Day

21

Day

28

(a)

ns

in

farc

t les

ion

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

10

20

30lowastlowast


lowast

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

(b)

in

farc

t les

ion

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

10

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

15


lowast

lowast

lowast

5

(c)

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

(d)

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol


lowast

lowast

100

0

150

200

250

50

Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



mN

SS

0

5

10

15

20

lowastlowastlowast

Iv-ADSC-EIv-ADSC

Control

Day

0

Day

1

Day

3

Day

7

Day

14

Day

21

Day

28

lowastlowastlowast

ns

ns

(a)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol


in

farc

t les

ion

0

10

20

30lowast

(b)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

ns


in

farc

t les

ion

0

10

5

15

20

(c)

Sham

Iv-ADSC-EIv-ADSC

Control

(d)

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

0

50

100

150

200

250

Sham


Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



0

50

100

150 ns


Fold

chan

ge (I

L-6)

lowastlowast

lowast

(a)

0

20

40

60

80


lowastlowast

lowastlowast

Fold

chan

ge (T

NF120572

)

(b)

0

5

10

15

ns


lowastlowast

lowast

Fold

chan

ge (I

FN120574

)

(c)

0

10

20

30

40


Fold

chan

ge (I

L-10

)

(d)

0

10

20

30

40

5050

100

150

ns



Fold

chan

ge (B

DN

F)

(e)

0

10

20

30

40

50


lowast


Fold

chan

ge (N

GF)

(f)

Figure 4 Continued


0

10

20

30

40ns


Fold

chan

ge (C

NTF

)


0

2

4

6

8

10

125

250

ns


Fold

chan

ge (I

GF1

)







5 Conclusion


Competing Interests






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







4 Discussion




mN

SS

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

0

5

10

15

20


lowastlowast

Day

0

Day

1

Day

7

Day

14

Day

21

Day

28

(a)

ns

in

farc

t les

ion

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

10

20

30lowastlowast


lowast

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

(b)

in

farc

t les

ion

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

10

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

15


lowast

lowast

lowast

5

(c)

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

(d)

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol


lowast

lowast

100

0

150

200

250

50

Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



mN

SS

0

5

10

15

20

lowastlowastlowast

Iv-ADSC-EIv-ADSC

Control

Day

0

Day

1

Day

3

Day

7

Day

14

Day

21

Day

28

lowastlowastlowast

ns

ns

(a)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol


in

farc

t les

ion

0

10

20

30lowast

(b)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

ns


in

farc

t les

ion

0

10

5

15

20

(c)

Sham

Iv-ADSC-EIv-ADSC

Control

(d)

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

0

50

100

150

200

250

Sham


Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



0

50

100

150 ns


Fold

chan

ge (I

L-6)

lowastlowast

lowast

(a)

0

20

40

60

80


lowastlowast

lowastlowast

Fold

chan

ge (T

NF120572

)

(b)

0

5

10

15

ns


lowastlowast

lowast

Fold

chan

ge (I

FN120574

)

(c)

0

10

20

30

40


Fold

chan

ge (I

L-10

)

(d)

0

10

20

30

40

5050

100

150

ns



Fold

chan

ge (B

DN

F)

(e)

0

10

20

30

40

50


lowast


Fold

chan

ge (N

GF)

(f)

Figure 4 Continued


0

10

20

30

40ns


Fold

chan

ge (C

NTF

)


0

2

4

6

8

10

125

250

ns


Fold

chan

ge (I

GF1

)







5 Conclusion


Competing Interests






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


mN

SS

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

0

5

10

15

20


lowastlowast

Day

0

Day

1

Day

7

Day

14

Day

21

Day

28

(a)

ns

in

farc

t les

ion

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

10

20

30lowastlowast


lowast

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

(b)

in

farc

t les

ion

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

10

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol

0

15


lowast

lowast

lowast

5

(c)

Ip-ADSC-EIc-ADSC-E

Iv-ADSC-EControl

(d)

ns

Ip-A

DSC

-E

Ic-A

DSC

-E

Iv-A

DSC

-E

Cont

rol


lowast

lowast

100

0

150

200

250

50

Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



mN

SS

0

5

10

15

20

lowastlowastlowast

Iv-ADSC-EIv-ADSC

Control

Day

0

Day

1

Day

3

Day

7

Day

14

Day

21

Day

28

lowastlowastlowast

ns

ns

(a)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol


in

farc

t les

ion

0

10

20

30lowast

(b)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

ns


in

farc

t les

ion

0

10

5

15

20

(c)

Sham

Iv-ADSC-EIv-ADSC

Control

(d)

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

0

50

100

150

200

250

Sham


Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



0

50

100

150 ns


Fold

chan

ge (I

L-6)

lowastlowast

lowast

(a)

0

20

40

60

80


lowastlowast

lowastlowast

Fold

chan

ge (T

NF120572

)

(b)

0

5

10

15

ns


lowastlowast

lowast

Fold

chan

ge (I

FN120574

)

(c)

0

10

20

30

40


Fold

chan

ge (I

L-10

)

(d)

0

10

20

30

40

5050

100

150

ns



Fold

chan

ge (B

DN

F)

(e)

0

10

20

30

40

50


lowast


Fold

chan

ge (N

GF)

(f)

Figure 4 Continued


0

10

20

30

40ns


Fold

chan

ge (C

NTF

)


0

2

4

6

8

10

125

250

ns


Fold

chan

ge (I

GF1

)







5 Conclusion


Competing Interests






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


mN

SS

0

5

10

15

20

lowastlowastlowast

Iv-ADSC-EIv-ADSC

Control

Day

0

Day

1

Day

3

Day

7

Day

14

Day

21

Day

28

lowastlowastlowast

ns

ns

(a)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol


in

farc

t les

ion

0

10

20

30lowast

(b)

lowastlowast

lowastlowast

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

ns


in

farc

t les

ion

0

10

5

15

20

(c)

Sham

Iv-ADSC-EIv-ADSC

Control

(d)

Iv-A

DSC

-E

Iv-A

DSC

Cont

rol

0

50

100

150

200

250

Sham


Num

ber o

f TU

NEL

+ce

lls p

er y

ield

(e)



0

50

100

150 ns


Fold

chan

ge (I

L-6)

lowastlowast

lowast

(a)

0

20

40

60

80


lowastlowast

lowastlowast

Fold

chan

ge (T

NF120572

)

(b)

0

5

10

15

ns


lowastlowast

lowast

Fold

chan

ge (I

FN120574

)

(c)

0

10

20

30

40


Fold

chan

ge (I

L-10

)

(d)

0

10

20

30

40

5050

100

150

ns



Fold

chan

ge (B

DN

F)

(e)

0

10

20

30

40

50


lowast


Fold

chan

ge (N

GF)

(f)

Figure 4 Continued


0

10

20

30

40ns


Fold

chan

ge (C

NTF

)


0

2

4

6

8

10

125

250

ns


Fold

chan

ge (I

GF1

)







5 Conclusion


Competing Interests






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

50

100

150 ns


Fold

chan

ge (I

L-6)

lowastlowast

lowast

(a)

0

20

40

60

80


lowastlowast

lowastlowast

Fold

chan

ge (T

NF120572

)

(b)

0

5

10

15

ns


lowastlowast

lowast

Fold

chan

ge (I

FN120574

)

(c)

0

10

20

30

40


Fold

chan

ge (I

L-10

)

(d)

0

10

20

30

40

5050

100

150

ns



Fold

chan

ge (B

DN

F)

(e)

0

10

20

30

40

50


lowast


Fold

chan

ge (N

GF)

(f)

Figure 4 Continued


0

10

20

30

40ns


Fold

chan

ge (C

NTF

)


0

2

4

6

8

10

125

250

ns


Fold

chan

ge (I

GF1

)







5 Conclusion


Competing Interests






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

10

20

30

40ns


Fold

chan

ge (C

NTF

)


0

2

4

6

8

10

125

250

ns


Fold

chan

ge (I

GF1

)







5 Conclusion


Competing Interests






Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Intravenous Administration of Adipose-Derived Stem Cell Protein …downloads.hindawi.com/journals/sci/2017/2153629.pdf · 2019. 7. 30. · StemCellsInternational 5 may impact the