Research ArticleStromal Derived Factor-1CXCR4 Axis Involved in Bone MarrowMesenchymal Stem Cells Recruitment to Injured Liver

Kuai Xiao Ling1 Li Peng2 Zhang Jian Feng1 Cao Wei1 Yuan Wei Yan1 Shao Nan1

Guan Cheng Qi1 and Wang Zhi Wei2

1Department of Gastroenterology Nantong University Affiliated Hospital Nantong Jiangsu 226001 China2Department of General Surgery Nantong University Affiliated Hospital Nantong Jiangsu 226001 China

Correspondence should be addressed to Wang Zhi Wei alexxs139com

Received 1 June 2015 Revised 10 October 2015 Accepted 15 October 2015

Academic Editor Shinn-Zong Lin

Copyright copy 2016 Kuai Xiao Ling et alThis 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

The molecular mechanism of bone marrow mesenchymal stromal stem cells (BMSCs) mobilization and migration to the liver waspoorly understood Stromal cell-derived factor-1 (SDF-1) participates in BMSCs homing and migration into injury organs Wetry to investigate the role of SDF-1 signaling in BMSCs migration towards injured liver The expression of CXCR4 in BMSCs atmRNA level and protein level was confirmed by RT-PCR flow cytometry and immunocytochemistry The SDF-1 or liver lysatesinduced BMSCsmigrationwas detected by transwell inserts CXCR4 antagonist AMD3100 and anti-CXCR4 antibodywere used toinhibit the migrationThe Sprague-Dawley rat liver injurymodel was established by intraperitoneal injection of thioacetamideTheconcentration of SDF-1 increased as modeling time extended which was determined by ELISA method The Dir-labeled BMSCswere injected into the liver of the rats through portal vein The cell migration in the liver was tracked by in vivo imaging systemand the fluorescent intensity was measured In vivo BMSCs migrated into injured liver which was partially blocked by AMD3100or anti-CXCR4 antibody Taken together the results demonstrated that the migration of BMSCs was regulated by SDF-1CXCR4signaling which involved in BMSCs recruitment to injured liver

1 Introduction

Bone marrow mesenchymal stem cells (BMSCs) are non-hematopoietic progenitor cells capable of differentiating intobone adipose and cartilage [1] They also support the sur-vival and proliferation of hematopoietic stem cells [2] Fur-thermore studies indicated that BMSCs were able to recruitto the site of injury and contribute to the repair process suchas bone heart lung and liver [3ndash6] BMSCs are currentlybeing investigated in numerous clinical trials of tissue repairand various immunological disorders based on their abilityto secrete trophic factors and to modulate inflammatoryresponses BMSCs migration and recruitment are crucial tothe success of BMSCs-based therapies Migratory mecha-nisms need to be elucidated before BMSCs can be exploitedtherapeutically

Chemokines are the most important factors control-ling cellular migration Stromal derived factor-1 (SDF-1 also

called CXCL-12) acting via its receptor CXCR4 play animportant role in BMSCs homing to bone marrow Mobiliza-tion of BMSCs from bone marrow to peripheral blood andthence to injured tissues may be down an SDF-1 concentra-tion gradient [7 8] A number of studies have shown thatSDF-1 is critical for BMSCs homing in injured tissue throughinteraction with its receptor CXCR4 [9ndash12]

It was reported that the injured liver releases chemokinessuch as stroma-derived factor-1 (SDF-1) hepatocellulargrowth factor (HGF) and others to participate in the concertof extrahepatic cells homing to the liver [13 14] It was shownthat levels of SDF-1 in injured liver increasedwhich attractingCD133+ BMSC that are positive for the SDF-1 receptorCXCR4 [15]

In this study we hypothesized that SDF-1 of injured liverpromotes BMSCs migration towards the liver via its receptorCXCR4 We explored the expression of CXCR4 in BMSCsIn vitro SDF-1 induced BMSCs migration was investigated

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 8906945 10 pageshttpdxdoiorg10115520168906945

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and in vivo BMSCs migration towards injured liver was alsotested Our results demonstrated the role of SDF-1CXCR4axis in BMSCs migration towards injured liver

2 Material and Methods

21 Cell Lines and Cell Culture BMSCs were from CyagenBiosciences Inc (Santa Clara CA USA httpwwwcyagencom) and maintained in alpha minimal essential medium(aMEM Gibco Invitrogen Rockville MD httpwwwlife-technologiescom) supplemented with 10 fetal bovineserum (FBS) 100UmL penicillin 100mgmL streptomycinand 2mM L-glutamine (Gibco Invitrogen) as describedpreviously [16] 293 T cells were from ATCC (Beijing Chinahttpwwwatccorg) and cultured in Dulbeccorsquos ModifiedEaglersquos Medium (DMEM Gibco) with 10 FBS 2mM L-glutamine and 1 penicillinstreptomycin

22 Reverse Transcription PCR Total cellular RNA was iso-lated using a RNeasyMini Kit (Qiagen Valencia CA httpswwwqiagencom) and treatedwith aDNA-free kit (AmbionAustin TX httpwwwlifetechnologiescom) to removepotential contamination of genomic DNA A total of 500 ngof RNA was used as a template for reverse transcriptionwith Reverse Transcription System (Promega Madison WIhttpwwwpromegacom) 100 ng of cDNA was used for astandard PCR reaction A housekeeping gene glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) was used as acontrol for the PCR efficiency of each sample The PCRstep was performed using PCR Master Mix kit (PromegaMadison WI) and the PCR products were detected andanalyzed by 2 agarose gel electrophoresis 293 T cells wereas negative control

CXCR4 primers were as follows forward 51015840-ATG GAGGGG ATC AGT ATA TAC AC-31015840 and reverse 51015840-TGG AGTGTG CTA TGT TGGCGT CT-31015840 (product 668 bp) GAPDHprimers were forward 51015840-ACC-ACA-GTC-CAT-GCC-ATC-AC-31015840 and reverse 51015840-TCC-ACC-ACC-CTG-TTG-CTG-TA-31015840 (product 450 bp)

23 Flow Cytometry BMSCs were fixed with 4 paraformal-dehyde (Sigma-Aldrich Saint Louis MO USA httpwwwsigmaaldrichcom) permeabilized with 05 Triton X-100(Sigma-Aldrich) and stained with mouse monoclonal anti-human CXCR4 antibody (RampD Systems) at this step PBSand isotype antibody (RampD Systems) were used as negativecontrol and then followed by anti-mouse IgG (FITC Sigma-Aldrich) according to the manufacturerrsquos instructions Cellswere analyzed on a FACSCalibur with CellQuest software(BD Biosciences)

24 Immunocytochemistry Cells were cultured in chamberslides and then were fixed in 4 paraformaldehyde in PBSfor 15 minutes permeabilized with 01 Triton X-100 for 10minutes and then blocked for 1 hour at room temperature inPBS containing 5 goat serum (Invitrogen Rockville MD)Samples were then incubated in blocking buffer containingmouse monoclonal anti-human CXCR4 antibody or isotypeantibody (RampD Systems) for 2 hours at room temperature

and washed three times with PBS for 15 minutes Cells werethen incubated with secondary anti-mouse antibody conju-gated with FITC (1 1000 Molecular Probes Eugene ORhttpwwwlifetechnologiescom) for 1 hour at room tem-perature The samples were washed as above and mountedwith 6-diamidino-2-phenylindole (DAPI DAKO Carpinte-ria CA httpwwwdakocom) containing mounting solu-tion The cells were examined under a Nikon Eclipse E600fluorescence microscope (Nikon Tokyo Japan httpwwwnikoncom)

25 Chemotaxis Assays BD FluoroBlok inserts (BD Fal-con Labware Franklin Lakes NJ httpwwwbdbiosciencescom) contain fluorescence blocking PET track-etchedmembranes with 80120583m pores This invasion system allowedfor real-time viewing of cell invasion without the needto end the experiment and process the membranes TheFluoroBlok membrane prevents the transmission of light tocells on top of the membrane thus only invaded fluorescent-expressing cells can be viewed Cells were serum-starvedovernight before being harvested for invasion assays BMSCswere labeled with PKH26GL dye (Sigma-Aldrich) and thenwere plated on top of the chamber layer at a concentrationof 4 times 105 cellsmL In the bottom chamber SDF-1 (RampDSystems Minneapolis MN httpwwwrndsystemscom) orliver lysates (taken at the 12th week after TAA injection)were used as chemoattractant For neutralization studiescells were incubated with anti-CXCR4 monoclonal anti-body (RampD Systems) at 20 120583gmL 40 120583gmL 80120583gmL and96 120583gmL or AMD3100 (Plerixafor) at 24120583gmL 48 120583gmLand 96 120583gmL (Sigma-Aldrich) AMD3100 is an antagonistof CXCR4 which disrupted binding of SDF-1 to CXCR4by competing binding site thus blocking the physiologicalfunction of SDF-1CXCR4 axis Six duplicated wells were ineach experiment Invaded cells with red fluorescence wereviewed at 48 hours after chemoattractant addition under aNikon Eclipse E600 fluorescence microscope (Nikon TokyoJapan httpwwwnikoncom) Cells were counted fromentire membranes at 40x The migrated cell number wasexpressed as mean plusmn SD

26 Animal Studies

261 Animals Six- to eight-week-old between 160 and200 g wide-type female Sprague-Dawley (SD) rats wereobtained from the Animal Research Center of Fudan Univer-sity in China They were randomized and divided into differ-ent groups six rats in each group Rats weremaintained in theanimal care facility of our institution All experiments wereapproved by the Animal Experimentation Ethics Committee(AEEC) of Nantong University Affiliated Hospital

262 Liver Injury Model Liver injury model was induced inrats by intraperitoneal (IP) injections with sterile solutions ofthioacetamide (TAA) (Sigma-Aldrich) which was dissolvedin 09 saline administered twice weekly During the initialweek rats were IP injected with TAA 025 gkg body weightbiweekly During the following 11 weeks rats were IP injectedwith TAA 020 gkg body weight biweekly [17] The serum

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Alanine aminotransferase (ALT) and aspartate aminotrans-ferase (AST) levels of rats after TAA injection were detectedby automatic biochemistry analyzer (Beckman Coulter) toconfirm liver injury at 0 weeks 4 weeks 8 weeks and 12weeks And the histological sections of livers were stainedwith hematoxylin-eosin staining (HE staining) to observepathological changes of the livers

263 BMSCs Injection Rats were placed under anesthesiausing isoflurane through inhalation Before cell injectionBMSCs were labeled with 11-dioctadecyl-3333-tetramethy-lindotricarbocyanine iodide (DiR) dye (Invitrogen) Dir-labeled BMSCs before injection were as positive control andrats without cell injection were as negative control First theways of cells injection were compared between portal veininjection (PVI) and tail vein injection (TVI) More migratedcells were found in liver through PVI so PVI was selected inthe following cellmigration study Six rats were in each groupEach rat was injected with 1 times 106 BMSCs through portalvein using a 16G syringe The cells migration in normal andinjured liver was also compared For neutralization studiescells were incubated with anti-CXCR4 monoclonal antibody(RampD Systems) before injection or with AMD3100 duringinjection At the end a laparotomy was performed and thelivers were dissected

264 In Vivo Distribution of the Transplanted Cells Thelocation and the fluorescent strength of the transplanted cellslabeled with dye DiR (excitationemission 748780 nm) weredetected by the Kodak In-VivoMultispectral Imaging SystemFX (excitation filteremission filter ex730em750WA) at 24 hafter cell injection First the fluorescent intensity of wholerats was checked Then the rats were executed and the liverswere perfused with saline (PBS) to remove contaminatingblood which might contain unmigrated BMSCs And thenthe fluorescent intensity of dissected liver wasmeasured Dir-labeled BMSCs before injection were as positive control andrats without cell injection were as negative control At theend of each acquisition a photographic image was obtainedThe data were analyzed with Photovision software whichsuperimposes the signal on the photographic image Themost intense fluorescent signal detected is shown in redwhereas the weakest signal is shown in blue

27 Enzyme-Linked Immunosorbent Assay for SDF-1 Level ofLiver Lysates Liver extracts at 0 weeks 4 weeks 8 weeksand 12 weeks after TAA injection were prepared by cell lysiswith 25mM Tris (pH 75) 1 Triton X-100 05mM EDTA150mM NaCl 10mM NaF and protease inhibitor cocktail(1 PMSF Sigma-Aldrich) Total proteins in these extractswere quantified by Bradford assay and equal protein amountswere assayed for SDF-1 level with SDF-1 enzyme-linkedimmunosorbent assay (ELISA) kit (RampD Systems) accordingto the manufacturerrsquos instructions

28 Statistics Statistical analysis and graphing were per-formed using SPSS 12 All results are expressed as mean plusmnSEM Statistical significance was determined by Studentrsquos 119905-test Significance was defined as 119875 lt 005

3 Results

31 Expression of CXCR4 in BMSCs The expression ofCXCR4 in BMSCs was examined at mRNA and protein levelfrom passages 1 to 8 with RT-PCR immunocytochemistryand flow cytometry CXCR4 was stably expressed in BMSCsat mRNA level from passages 1 to 8 293 T cells were asnegative control (Figure 1(a)) Before staining with CXCR4antibody BMSCswere permeabilizedwith 01TritonX-100so the immunocytochemistry results indicated all the CXCR4expression on the membrane and intracellularly CXCR4were found in 100 of cells (Figure 1(b)) Only membraneCXCR4 positive BMSCs could be detected by flow cytometryThe result of flow cytometry indicated 42 of cells weremembrane CXCR4 positive (Figure 1(c))

32 SDF-1 Induce BMSCs Migration In Vitro In cell tran-swell system SDF-1 added in bottom chamber was at0 ngmL 50 ngmL 100 ngmL 200 ngmL 400 ngmL and800 ngmL the number of migrated cells were countedunder fluorescence microscopes which were 52 plusmn 327 27 plusmn579 644 plusmn 1761 502 plusmn 1514 416 plusmn 75 and 364 plusmn568 respectively The concentration of SDF-1 lower than100 ngmL the number of migrated cells increased as theconcentration increased and the differences were statisticallysignificant (all 119875 lt 005) However after the concentrationwas above 100 ngmL the number of migrated cells decreasedbecause too much SDF-1 would complete the binding site(Figure 2(a))

To demonstrate that the migration was an SDF-1dependent effect CXCR4 antagonist AMD3100 or anti-CXCR4 antibody was added The concentration of SDF-1 at100 ngmL after adding AMD3100 at 24120583gmL 48 120583gmLand 96120583gmL the number of migrated cells decreased from586 plusmn 963 to 725 plusmn 206 325 plusmn 05 and 325 plusmn 171and the differences were statistically significant (all 119875 lt005) (Figure 2(b)) After adding anti-CXCR4 antibody at20120583gmL 40 120583gmL 80120583gmL and 96 120583gmL the numberof migrated cells decreased from 608 plusmn 963 to 1275 plusmn 1257plusmn258 675plusmn096 and 8plusmn141 respectivelyThe differenceswere statistically significant (all 119875 lt 005) (Figure 2(c))These results indicated that SDF-1CXCR4 was involved inthe hBMSCs migration

33 Liver Injury after TAA Injection and SDF-1 Levels of RatsLiver Lysates Liver injury model was induced in rats byIP injections of TAA After TAA injection the serum levelof ALT at 0 weeks 4 weeks 8 weeks and 12 weeks was4117 plusmn 823UL 7835 plusmn 467UL 18656 plusmn 1568UL and31698 plusmn 2085UL respectively and that of the controls was4133 plusmn 484UL 4437 plusmn 662UL 4286 plusmn 398UL and4652 plusmn 566UL respectively The differences in ALT levelbetween TAA injection group and control group at 4 weeks8 weeks and 12 weeks were statistically significant (119875 lt 005)(Figure 3(a))The serum level of AST was 15083plusmn1865UL19678plusmn2462UL 25698plusmn2676UL and 45012plusmn3568ULat 0weeks 4weeks 8weeks and 12weeks afterTAA injectionrespectively and that of the controls was 14582 plusmn 2078UL13267plusmn1896UL 16478plusmn2267UL and 16832plusmn201UL

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BMSCs P 1 2 3 4 5 6 7 8 293 T cells

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Figure 1 Expression of CXCR4 in BMSCs (a) RT-PCR results of CXCR4 expression in BMSCs from passages 1 to 8 CXCR4 was stablyexpressed 293T cells as negative control (b) immunocytochemistry results of CXCR4 expression which indicated both membrane andintracellular CXCR4 expression (c) the result of flow cytometry indicated only 42 of cells were positive for membrane CXCR4

respectively The differences between two groups at 4 weeks8 weeks and 12 weeks were statistically significant (119875 lt 005)(Figure 3(a)) After TAA injection the serum level of AST andALT significantly increased (Figure 3(a)) which indicatedliver injury The livers of rats were collected at 0 4 8 and12 weeks after TAA injection The pathological results alsoindicated liver injury there were vacuole degeneration (at8 weeks) microvascular disintegration (at 8 weeks) tissuenecrosis and disruption of general architecture (at 12 weeks)(Figure 3(b)) The SDF-1 concentrations of livers lysates weredetermined by ELISA The SDF-1 concentrations of liverslysates at 0 4 8 and 12 weeks were 0714 plusmn 0267 ngmL0845 plusmn 0420 ngmL 0937 plusmn 0060 ngmL and 1536 plusmn0339 ngmL (Figure 3(c)) These results indicated that SDF-1level increased in TAA induced liver injury

34 BMSCs Migration Induced by Liver Extracts In Vitro Toexplore whether SDF-1 of liver lysates is involved in recruit-ment of BMSCs a migration experiment was performedin vitro with BMSCs and liver lysates from rats treated byTAA for 12 weeks The number of migrated cells causedby liver lysates from untreated normal rats was 76 plusmn 282and that by TAA induced injured liver lysates was 188 plusmn

396 the difference was statistically significant (119875 lt 005)(Figure 3(d)) As we demonstrated SDF-1 level of liver lysatesfrom rats treated by TAA increased so the question waswhether the increased cell number was caused by SDF-1CXCR4 antagonist AMD3100 or anti-CXCR4 antibody wasadded in the medium in the following study SDF-1 level ofliver lysates was 1536 plusmn 0339 ngmL at 12 weeks Accordingto the results of SDF-1 induced MSCs migration in vitrofor neutralization studies the concentration of AMD3100 waschosen at 24120583gmL and anti-CXCR4 antibody at 20 120583gmLAfter adding AMD3100 (24 120583gmL) or anti-CXCR4 antibody(20120583gmL) the number of migrated cells decreased to 138plusmn277 and 114 plusmn 270 (Figure 3(d)) however still higherthan the number of migrated cells caused by liver lysatesfrom untreated normal rats These results demonstrated thatSDF-1CXCR4 axis involved in injured liver lysates inducedBMSCs migration but not the only pathway

35 SDF-1CXCR4 Mediate BMSCs Migration in Rats Wefirst compare theways of cells injectionThe cells were labeledwith Dir dye which gave out fluorescence in the liver tissuesafter cells injection and the fluorescence could be capturedby in vivo imaging systemThefluorescent intensity of injured

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0120583gmL 20120583gmL

80120583gmL 160120583gmL

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(c)

Figure 2 SDF-1 induces BMSCsmigration in vitro (a) SDF-1 at different concentration the migrated cells were observed under fluorescencemicroscope and the graph indicated the number of migrated cells (b) SDF-1 at 100 ngmL AMD3100 was added at different concentrationthemigrated cells were observed and counted under fluorescencemicroscope and the graph indicated the number of migrated cells (c) SDF-1at 100 ngmL anti-CXCR4 antibody was added at different concentration the migrated cells were observed and counted

livers with cells injection through tail vein (TVI) was 51610plusmn11560 (Figures 4(a) 4(b) and 4(g)) and that through portalvein (PVI) was 85998 plusmn 12780 (Figures 4(c) 4(d) and 4(g))which was significantly higher (fold change 167plusmn025) (119875 lt005) Dir-labeled BMSCs before injection (Figure 4(e)) wereas positive control and rats without cell injection were asnegative control (Figure 4(f))This result indicated that morecells migrated into liver through portal vein injection So PVIinjection was selected in the following transplantation study

We also compared the cell migration between normalliver and injured liver After cells injection the fluorescentintensity of normal livers (17451 plusmn 7682) (Figures 5(a)5(b) and 5(k)) was statistically significantly lower than thatof injured livers (85998 plusmn 12780) (compared with normal

control fold change 494plusmn073) (Figures 5(c) 5(d) and 5(k))(119875 lt 005) More cells migrated into injured liver The SDF-1level of injured liver increased So the question is whetherthe migration was regulated by SDF-1CXCR4 axis FirstCXCR4 antagonist AMD3100 was injected into liver throughPV and then followed by BMSCs injection The fluorescentintensity of the livers declined to 63679 plusmn 19790 (comparedwith normal control fold change 366 plusmn 113) (Figures 5(e)5(f) and 5(k)) which indicated that AMD3100 significantlyinhibited cells migration into the liver And then the cellswere incubated with anti-CXCR4 antibody to neutralizeCXCR4 receptor before injection The cell migration wasalso significantly inhibited by CXCR4 neutralization thefluorescent intensity of the livers decreased to 27267 plusmn 6137

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050

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114 plusmn 270138 plusmn 277

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(d)

Figure 3 Injured liver lysates induce BMSCsmigration in vitro (a) Changes of serumALT and AST level after TAA injection (b) HE stainedliver tissue sections after TAA injection (c) the concentrations of SDF-1 of liver lysates detected by ELISA at different time point of TAAinduced liver injury (d) the migrated cells induced by normal liver lysates and TAA injured liver lysates More cell migration was induced byTAA injured liver lysates which could be partially blocked by AMD3100 (24 120583gmL) or anti-CXCR4 antibody (20120583gmL)The migrated cellswere observed under fluorescence microscope and the graph indicated the number of migrated cells

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50000 129074 208148 287223 36629750000 129074 208148 287223 366297

85998 plusmn 12780

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Figure 4 Difference between portal vein injection and tail vein injection (a)The fluorescent intensity of whole rats the BMSCs were injectedthrough tail vein (b) the fluorescent intensity of dissected livers the BMSCs were injected through tail vein (c) the fluorescent intensity ofwhole rats the BMSCs were injected through portal vein (d) the fluorescent intensity of dissected livers the BMSCs were injected throughportal vein (e) Dir-labeled BMSCs before injection were as positive control (f) rats without cell injection were as negative control (g) thefluorescent intensity value and fold changes of the dissected livers of two groups the difference was statistically significant

(compared with normal control fold change 156 plusmn 08)(119875 lt 005) (Figures 5(g) 5(h) and 5(k)) Dir-labeled BMSCsbefore injection (Figure 5(i)) were as positive control and ratswithout cell injection were as negative control (Figure 5(j))These findings suggested that SDF-1CXCR4 plays a role inthe migration of BMSCs into injured liver

4 Discussion

Recently studies have demonstrated the potential therapeuticfunction of BMSCs transplantation to treat acute and chronicliver injury Because BMSCs produce new hepatocytes [18]secrete trophic factors to promote liver tissue repair [19]

8 Stem Cells International

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Liver fluorescence of liver injured group Liver fluorescence of AMD3100 group Liver fluorescence of anti-CXCR4 group

Fluorescence X-rayNormal group Liver injured group

Merge Fluorescence X-ray MergeAMD3100 group

Fluorescence X-ray MergeAnti-CXCR4 group

Fluorescence X-ray Merge

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Cell number 4 4000 40000Cell fluorescence before injection as positive controls

400

Fluorescence X-ray MergeWithout cell injection as a negative control

(i)

(k)

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40 400000

50000 129074 208148 287223 366297 50000 129074 208148 287223 366297

17451 plusmn 7682 27267 plusmn 6137

85998 plusmn12780 63679 plusmn

19790

P lt 005 P lt 005P lt 005

P lt 005P lt 005

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1 plusmn 044

494 plusmn 073366 plusmn 113

156 plusmn 08

Figure 5 BMSCsmigration in injured liver inhibited by anti-CXCR4 antibody andAMD3100 (a)Thefluorescent intensity of normal rats afterDir-labeled BMSCs injection (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection (c) the fluorescentintensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection (d) the fluorescent intensity of dissected TAA injured liversafter Dir-labeled BMSCs injection (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100injection at the same time (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at thesame time (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCsinjection (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection(i) Dir-labeled BMSCs before injection were as positive control (j) rats without cell injection were as negative control (k) the fluorescentintensity value and fold changes of the dissected livers of each group the differences were statistically significant

and modulate inflammatory responses [20] BMSCs trans-plantation is a promising candidate for cell therapy in liverdiseases Cell homing and engraftment into the host liver areintegral to cell-based therapies The mechanism of BMSCsmigration to injured liver is complicated and still not fullyunderstood In the current study we try to investigate the roleof SDF-1CXCR4 axis in the recruitment of BMSCsmigrationinto TAA induced injured liver We revealed SDF-1 induced

BMSC migration in vitro study Our study found SDF-1 levelgradually increased in rat model of TAA induced liver injuryAndwe demonstrated SDF-1CXCR4 axis involved in BMSCsmigration into the injured liver in vitro and in vivo

CXCR4 is a G protein-linked seven-transmembranespanning receptor which is expressed on the surface of asmall proportion of MSCs Some studies indicated that mostCXCR4 are intracellular storage only small part of functional

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 Stem Cells International

and in vivo BMSCs migration towards injured liver was alsotested Our results demonstrated the role of SDF-1CXCR4axis in BMSCs migration towards injured liver

2 Material and Methods

21 Cell Lines and Cell Culture BMSCs were from CyagenBiosciences Inc (Santa Clara CA USA httpwwwcyagencom) and maintained in alpha minimal essential medium(aMEM Gibco Invitrogen Rockville MD httpwwwlife-technologiescom) supplemented with 10 fetal bovineserum (FBS) 100UmL penicillin 100mgmL streptomycinand 2mM L-glutamine (Gibco Invitrogen) as describedpreviously [16] 293 T cells were from ATCC (Beijing Chinahttpwwwatccorg) and cultured in Dulbeccorsquos ModifiedEaglersquos Medium (DMEM Gibco) with 10 FBS 2mM L-glutamine and 1 penicillinstreptomycin

22 Reverse Transcription PCR Total cellular RNA was iso-lated using a RNeasyMini Kit (Qiagen Valencia CA httpswwwqiagencom) and treatedwith aDNA-free kit (AmbionAustin TX httpwwwlifetechnologiescom) to removepotential contamination of genomic DNA A total of 500 ngof RNA was used as a template for reverse transcriptionwith Reverse Transcription System (Promega Madison WIhttpwwwpromegacom) 100 ng of cDNA was used for astandard PCR reaction A housekeeping gene glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) was used as acontrol for the PCR efficiency of each sample The PCRstep was performed using PCR Master Mix kit (PromegaMadison WI) and the PCR products were detected andanalyzed by 2 agarose gel electrophoresis 293 T cells wereas negative control

CXCR4 primers were as follows forward 51015840-ATG GAGGGG ATC AGT ATA TAC AC-31015840 and reverse 51015840-TGG AGTGTG CTA TGT TGGCGT CT-31015840 (product 668 bp) GAPDHprimers were forward 51015840-ACC-ACA-GTC-CAT-GCC-ATC-AC-31015840 and reverse 51015840-TCC-ACC-ACC-CTG-TTG-CTG-TA-31015840 (product 450 bp)

23 Flow Cytometry BMSCs were fixed with 4 paraformal-dehyde (Sigma-Aldrich Saint Louis MO USA httpwwwsigmaaldrichcom) permeabilized with 05 Triton X-100(Sigma-Aldrich) and stained with mouse monoclonal anti-human CXCR4 antibody (RampD Systems) at this step PBSand isotype antibody (RampD Systems) were used as negativecontrol and then followed by anti-mouse IgG (FITC Sigma-Aldrich) according to the manufacturerrsquos instructions Cellswere analyzed on a FACSCalibur with CellQuest software(BD Biosciences)

24 Immunocytochemistry Cells were cultured in chamberslides and then were fixed in 4 paraformaldehyde in PBSfor 15 minutes permeabilized with 01 Triton X-100 for 10minutes and then blocked for 1 hour at room temperature inPBS containing 5 goat serum (Invitrogen Rockville MD)Samples were then incubated in blocking buffer containingmouse monoclonal anti-human CXCR4 antibody or isotypeantibody (RampD Systems) for 2 hours at room temperature

and washed three times with PBS for 15 minutes Cells werethen incubated with secondary anti-mouse antibody conju-gated with FITC (1 1000 Molecular Probes Eugene ORhttpwwwlifetechnologiescom) for 1 hour at room tem-perature The samples were washed as above and mountedwith 6-diamidino-2-phenylindole (DAPI DAKO Carpinte-ria CA httpwwwdakocom) containing mounting solu-tion The cells were examined under a Nikon Eclipse E600fluorescence microscope (Nikon Tokyo Japan httpwwwnikoncom)

25 Chemotaxis Assays BD FluoroBlok inserts (BD Fal-con Labware Franklin Lakes NJ httpwwwbdbiosciencescom) contain fluorescence blocking PET track-etchedmembranes with 80120583m pores This invasion system allowedfor real-time viewing of cell invasion without the needto end the experiment and process the membranes TheFluoroBlok membrane prevents the transmission of light tocells on top of the membrane thus only invaded fluorescent-expressing cells can be viewed Cells were serum-starvedovernight before being harvested for invasion assays BMSCswere labeled with PKH26GL dye (Sigma-Aldrich) and thenwere plated on top of the chamber layer at a concentrationof 4 times 105 cellsmL In the bottom chamber SDF-1 (RampDSystems Minneapolis MN httpwwwrndsystemscom) orliver lysates (taken at the 12th week after TAA injection)were used as chemoattractant For neutralization studiescells were incubated with anti-CXCR4 monoclonal anti-body (RampD Systems) at 20 120583gmL 40 120583gmL 80120583gmL and96 120583gmL or AMD3100 (Plerixafor) at 24120583gmL 48 120583gmLand 96 120583gmL (Sigma-Aldrich) AMD3100 is an antagonistof CXCR4 which disrupted binding of SDF-1 to CXCR4by competing binding site thus blocking the physiologicalfunction of SDF-1CXCR4 axis Six duplicated wells were ineach experiment Invaded cells with red fluorescence wereviewed at 48 hours after chemoattractant addition under aNikon Eclipse E600 fluorescence microscope (Nikon TokyoJapan httpwwwnikoncom) Cells were counted fromentire membranes at 40x The migrated cell number wasexpressed as mean plusmn SD

26 Animal Studies

261 Animals Six- to eight-week-old between 160 and200 g wide-type female Sprague-Dawley (SD) rats wereobtained from the Animal Research Center of Fudan Univer-sity in China They were randomized and divided into differ-ent groups six rats in each group Rats weremaintained in theanimal care facility of our institution All experiments wereapproved by the Animal Experimentation Ethics Committee(AEEC) of Nantong University Affiliated Hospital

262 Liver Injury Model Liver injury model was induced inrats by intraperitoneal (IP) injections with sterile solutions ofthioacetamide (TAA) (Sigma-Aldrich) which was dissolvedin 09 saline administered twice weekly During the initialweek rats were IP injected with TAA 025 gkg body weightbiweekly During the following 11 weeks rats were IP injectedwith TAA 020 gkg body weight biweekly [17] The serum

Stem Cells International 3

Alanine aminotransferase (ALT) and aspartate aminotrans-ferase (AST) levels of rats after TAA injection were detectedby automatic biochemistry analyzer (Beckman Coulter) toconfirm liver injury at 0 weeks 4 weeks 8 weeks and 12weeks And the histological sections of livers were stainedwith hematoxylin-eosin staining (HE staining) to observepathological changes of the livers

263 BMSCs Injection Rats were placed under anesthesiausing isoflurane through inhalation Before cell injectionBMSCs were labeled with 11-dioctadecyl-3333-tetramethy-lindotricarbocyanine iodide (DiR) dye (Invitrogen) Dir-labeled BMSCs before injection were as positive control andrats without cell injection were as negative control First theways of cells injection were compared between portal veininjection (PVI) and tail vein injection (TVI) More migratedcells were found in liver through PVI so PVI was selected inthe following cellmigration study Six rats were in each groupEach rat was injected with 1 times 106 BMSCs through portalvein using a 16G syringe The cells migration in normal andinjured liver was also compared For neutralization studiescells were incubated with anti-CXCR4 monoclonal antibody(RampD Systems) before injection or with AMD3100 duringinjection At the end a laparotomy was performed and thelivers were dissected

264 In Vivo Distribution of the Transplanted Cells Thelocation and the fluorescent strength of the transplanted cellslabeled with dye DiR (excitationemission 748780 nm) weredetected by the Kodak In-VivoMultispectral Imaging SystemFX (excitation filteremission filter ex730em750WA) at 24 hafter cell injection First the fluorescent intensity of wholerats was checked Then the rats were executed and the liverswere perfused with saline (PBS) to remove contaminatingblood which might contain unmigrated BMSCs And thenthe fluorescent intensity of dissected liver wasmeasured Dir-labeled BMSCs before injection were as positive control andrats without cell injection were as negative control At theend of each acquisition a photographic image was obtainedThe data were analyzed with Photovision software whichsuperimposes the signal on the photographic image Themost intense fluorescent signal detected is shown in redwhereas the weakest signal is shown in blue

27 Enzyme-Linked Immunosorbent Assay for SDF-1 Level ofLiver Lysates Liver extracts at 0 weeks 4 weeks 8 weeksand 12 weeks after TAA injection were prepared by cell lysiswith 25mM Tris (pH 75) 1 Triton X-100 05mM EDTA150mM NaCl 10mM NaF and protease inhibitor cocktail(1 PMSF Sigma-Aldrich) Total proteins in these extractswere quantified by Bradford assay and equal protein amountswere assayed for SDF-1 level with SDF-1 enzyme-linkedimmunosorbent assay (ELISA) kit (RampD Systems) accordingto the manufacturerrsquos instructions

28 Statistics Statistical analysis and graphing were per-formed using SPSS 12 All results are expressed as mean plusmnSEM Statistical significance was determined by Studentrsquos 119905-test Significance was defined as 119875 lt 005

3 Results

31 Expression of CXCR4 in BMSCs The expression ofCXCR4 in BMSCs was examined at mRNA and protein levelfrom passages 1 to 8 with RT-PCR immunocytochemistryand flow cytometry CXCR4 was stably expressed in BMSCsat mRNA level from passages 1 to 8 293 T cells were asnegative control (Figure 1(a)) Before staining with CXCR4antibody BMSCswere permeabilizedwith 01TritonX-100so the immunocytochemistry results indicated all the CXCR4expression on the membrane and intracellularly CXCR4were found in 100 of cells (Figure 1(b)) Only membraneCXCR4 positive BMSCs could be detected by flow cytometryThe result of flow cytometry indicated 42 of cells weremembrane CXCR4 positive (Figure 1(c))

32 SDF-1 Induce BMSCs Migration In Vitro In cell tran-swell system SDF-1 added in bottom chamber was at0 ngmL 50 ngmL 100 ngmL 200 ngmL 400 ngmL and800 ngmL the number of migrated cells were countedunder fluorescence microscopes which were 52 plusmn 327 27 plusmn579 644 plusmn 1761 502 plusmn 1514 416 plusmn 75 and 364 plusmn568 respectively The concentration of SDF-1 lower than100 ngmL the number of migrated cells increased as theconcentration increased and the differences were statisticallysignificant (all 119875 lt 005) However after the concentrationwas above 100 ngmL the number of migrated cells decreasedbecause too much SDF-1 would complete the binding site(Figure 2(a))

To demonstrate that the migration was an SDF-1dependent effect CXCR4 antagonist AMD3100 or anti-CXCR4 antibody was added The concentration of SDF-1 at100 ngmL after adding AMD3100 at 24120583gmL 48 120583gmLand 96120583gmL the number of migrated cells decreased from586 plusmn 963 to 725 plusmn 206 325 plusmn 05 and 325 plusmn 171and the differences were statistically significant (all 119875 lt005) (Figure 2(b)) After adding anti-CXCR4 antibody at20120583gmL 40 120583gmL 80120583gmL and 96 120583gmL the numberof migrated cells decreased from 608 plusmn 963 to 1275 plusmn 1257plusmn258 675plusmn096 and 8plusmn141 respectivelyThe differenceswere statistically significant (all 119875 lt 005) (Figure 2(c))These results indicated that SDF-1CXCR4 was involved inthe hBMSCs migration

33 Liver Injury after TAA Injection and SDF-1 Levels of RatsLiver Lysates Liver injury model was induced in rats byIP injections of TAA After TAA injection the serum levelof ALT at 0 weeks 4 weeks 8 weeks and 12 weeks was4117 plusmn 823UL 7835 plusmn 467UL 18656 plusmn 1568UL and31698 plusmn 2085UL respectively and that of the controls was4133 plusmn 484UL 4437 plusmn 662UL 4286 plusmn 398UL and4652 plusmn 566UL respectively The differences in ALT levelbetween TAA injection group and control group at 4 weeks8 weeks and 12 weeks were statistically significant (119875 lt 005)(Figure 3(a))The serum level of AST was 15083plusmn1865UL19678plusmn2462UL 25698plusmn2676UL and 45012plusmn3568ULat 0weeks 4weeks 8weeks and 12weeks afterTAA injectionrespectively and that of the controls was 14582 plusmn 2078UL13267plusmn1896UL 16478plusmn2267UL and 16832plusmn201UL

4 Stem Cells International

BMSCs P 1 2 3 4 5 6 7 8 293 T cells

CXCR4

GAPDH

(a)

DAPI CXCR4 CXCR4DAPI CXCR4 isotypeDAPI

(b)100

80

60

40

20

0

101 102 103 104 105

Max

()

Iso

Ctr CXCR4

42

AL488-A CXCR4(c)

Figure 1 Expression of CXCR4 in BMSCs (a) RT-PCR results of CXCR4 expression in BMSCs from passages 1 to 8 CXCR4 was stablyexpressed 293T cells as negative control (b) immunocytochemistry results of CXCR4 expression which indicated both membrane andintracellular CXCR4 expression (c) the result of flow cytometry indicated only 42 of cells were positive for membrane CXCR4

respectively The differences between two groups at 4 weeks8 weeks and 12 weeks were statistically significant (119875 lt 005)(Figure 3(a)) After TAA injection the serum level of AST andALT significantly increased (Figure 3(a)) which indicatedliver injury The livers of rats were collected at 0 4 8 and12 weeks after TAA injection The pathological results alsoindicated liver injury there were vacuole degeneration (at8 weeks) microvascular disintegration (at 8 weeks) tissuenecrosis and disruption of general architecture (at 12 weeks)(Figure 3(b)) The SDF-1 concentrations of livers lysates weredetermined by ELISA The SDF-1 concentrations of liverslysates at 0 4 8 and 12 weeks were 0714 plusmn 0267 ngmL0845 plusmn 0420 ngmL 0937 plusmn 0060 ngmL and 1536 plusmn0339 ngmL (Figure 3(c)) These results indicated that SDF-1level increased in TAA induced liver injury

34 BMSCs Migration Induced by Liver Extracts In Vitro Toexplore whether SDF-1 of liver lysates is involved in recruit-ment of BMSCs a migration experiment was performedin vitro with BMSCs and liver lysates from rats treated byTAA for 12 weeks The number of migrated cells causedby liver lysates from untreated normal rats was 76 plusmn 282and that by TAA induced injured liver lysates was 188 plusmn

396 the difference was statistically significant (119875 lt 005)(Figure 3(d)) As we demonstrated SDF-1 level of liver lysatesfrom rats treated by TAA increased so the question waswhether the increased cell number was caused by SDF-1CXCR4 antagonist AMD3100 or anti-CXCR4 antibody wasadded in the medium in the following study SDF-1 level ofliver lysates was 1536 plusmn 0339 ngmL at 12 weeks Accordingto the results of SDF-1 induced MSCs migration in vitrofor neutralization studies the concentration of AMD3100 waschosen at 24120583gmL and anti-CXCR4 antibody at 20 120583gmLAfter adding AMD3100 (24 120583gmL) or anti-CXCR4 antibody(20120583gmL) the number of migrated cells decreased to 138plusmn277 and 114 plusmn 270 (Figure 3(d)) however still higherthan the number of migrated cells caused by liver lysatesfrom untreated normal rats These results demonstrated thatSDF-1CXCR4 axis involved in injured liver lysates inducedBMSCs migration but not the only pathway

35 SDF-1CXCR4 Mediate BMSCs Migration in Rats Wefirst compare theways of cells injectionThe cells were labeledwith Dir dye which gave out fluorescence in the liver tissuesafter cells injection and the fluorescence could be capturedby in vivo imaging systemThefluorescent intensity of injured

Stem Cells International 5

020406080

100

0 50 100 200 400 800

Cell

num

ber

field

SDF-1 concentration (ngmL)

SDF-1 induced hBMSCs migration

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

52 plusmn 327

27 plusmn 579

644 plusmn 1761 502 plusmn 1514416 plusmn 75 364 plusmn 568

0ngmL 50ngmL 100ngmL

200ngmL 400ngmL 800ngmL

(a)

0

20

40

60

80

0 24 48 96

Cel

l num

ber

SDF-1 induced MSCs migration inhibited by AMD3100P lt 005

P lt 005

P lt 005

586 plusmn 963

725 plusmn 206 325 plusmn 05 325 plusmn 171

AMD3100 concentration (120583gmL)

0120583gmL 24120583gmL

48120583gmL 96120583gmL

(b)

0

20

40

60

80

0 20 40 80 160

Cel

l num

ber

SDF-1 induced MSCs migration inhibited by CXCR4 antibodyP lt 005

P lt 005

P lt 005

P lt 005

608 plusmn 963

1275 plusmn 125 7 plusmn 258 675 plusmn 096 8 plusmn 141

CXCR4 antibody concentration (120583gmL)

0120583gmL 20120583gmL

80120583gmL 160120583gmL

40120583gmL

(c)

Figure 2 SDF-1 induces BMSCsmigration in vitro (a) SDF-1 at different concentration the migrated cells were observed under fluorescencemicroscope and the graph indicated the number of migrated cells (b) SDF-1 at 100 ngmL AMD3100 was added at different concentrationthemigrated cells were observed and counted under fluorescencemicroscope and the graph indicated the number of migrated cells (c) SDF-1at 100 ngmL anti-CXCR4 antibody was added at different concentration the migrated cells were observed and counted

livers with cells injection through tail vein (TVI) was 51610plusmn11560 (Figures 4(a) 4(b) and 4(g)) and that through portalvein (PVI) was 85998 plusmn 12780 (Figures 4(c) 4(d) and 4(g))which was significantly higher (fold change 167plusmn025) (119875 lt005) Dir-labeled BMSCs before injection (Figure 4(e)) wereas positive control and rats without cell injection were asnegative control (Figure 4(f))This result indicated that morecells migrated into liver through portal vein injection So PVIinjection was selected in the following transplantation study

We also compared the cell migration between normalliver and injured liver After cells injection the fluorescentintensity of normal livers (17451 plusmn 7682) (Figures 5(a)5(b) and 5(k)) was statistically significantly lower than thatof injured livers (85998 plusmn 12780) (compared with normal

control fold change 494plusmn073) (Figures 5(c) 5(d) and 5(k))(119875 lt 005) More cells migrated into injured liver The SDF-1level of injured liver increased So the question is whetherthe migration was regulated by SDF-1CXCR4 axis FirstCXCR4 antagonist AMD3100 was injected into liver throughPV and then followed by BMSCs injection The fluorescentintensity of the livers declined to 63679 plusmn 19790 (comparedwith normal control fold change 366 plusmn 113) (Figures 5(e)5(f) and 5(k)) which indicated that AMD3100 significantlyinhibited cells migration into the liver And then the cellswere incubated with anti-CXCR4 antibody to neutralizeCXCR4 receptor before injection The cell migration wasalso significantly inhibited by CXCR4 neutralization thefluorescent intensity of the livers decreased to 27267 plusmn 6137

6 Stem Cells International

050

100150200250300350400

ALT level

Normal controlTAA injection group

Normal controlTAA injection group

0100200300400500600

AST level(U

L)

(UL

)

P lt 005

P lt 005

P lt 005

P lt 005P lt 005

P lt 005

4133 plusmn

484 4117 plusmn823

4437 plusmn626

4286 plusmn398

4652 plusmn566

31698 plusmn2085

18656 plusmn1568

14582 plusmn2078 13267 plusmn

1896

15083 plusmn1865

19678 plusmn2462 16478 plusmn

2267

25698 plusmn2676 15832 plusmn

201

45012 plusmn3568

7835 plusmn467

0 4 8 12

(week) (week)0 4 8 12

(a)

Normal liver TAA injection4 weeks

TAA injection8 weeks

TAA injection12 weeks

(b)

002040608

112141618

2

0 4 8 12

SDF-

1 co

ncen

trat

ion

(ng

mL)

The concentration of SDF-1 in injured liver induced by thioacetamide

0714 plusmn

02670845 plusmn

04200937 plusmn

0060

1536 plusmn

0339

P lt 005

P lt 005

P lt 005

Modeling time (week)

(c)

Liver lysis induced BMSCs migration

0

5

10

15

20

25

Normal liverlysis

TAA injuredliver lysis

Anti-CXCR4 AMD3100

Cel

l num

ber

P lt 005 P lt 005

P lt 005

76 plusmn 282

188 plusmn 396

114 plusmn 270138 plusmn 277

Normal liver lysis TAA injured liver lysis

Anti-CXCR4 antibody treated AMD3100 treated

(d)

Figure 3 Injured liver lysates induce BMSCsmigration in vitro (a) Changes of serumALT and AST level after TAA injection (b) HE stainedliver tissue sections after TAA injection (c) the concentrations of SDF-1 of liver lysates detected by ELISA at different time point of TAAinduced liver injury (d) the migrated cells induced by normal liver lysates and TAA injured liver lysates More cell migration was induced byTAA injured liver lysates which could be partially blocked by AMD3100 (24 120583gmL) or anti-CXCR4 antibody (20120583gmL)The migrated cellswere observed under fluorescence microscope and the graph indicated the number of migrated cells

Stem Cells International 7

0200400600800

10001200

PVI TVI

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0

05

1

15

2

25

PVI TVI

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

Without cell injection as a negative control

(a) (c)

(b) (d)

(e) (f)

(g)

Fluorescence X-rayTVI group PVI group

Rat 1

Rat 1

Rat 2

Rat 3

Rat 2

Rat 3

Liver fluorescence of TVI group Liver fluorescence of PVI group

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Cell fluorescence before injection as positive controls

Merge Fluorescence X-ray Merge

Fluorescence X-ray Merge

Cell number 4 4000 4000040040 400000

50000 129074 208148 287223 36629750000 129074 208148 287223 366297

85998 plusmn 12780

51610 plusmn 11560

P lt 005 P lt 005

1 plusmn 022

167 plusmn 025

Figure 4 Difference between portal vein injection and tail vein injection (a)The fluorescent intensity of whole rats the BMSCs were injectedthrough tail vein (b) the fluorescent intensity of dissected livers the BMSCs were injected through tail vein (c) the fluorescent intensity ofwhole rats the BMSCs were injected through portal vein (d) the fluorescent intensity of dissected livers the BMSCs were injected throughportal vein (e) Dir-labeled BMSCs before injection were as positive control (f) rats without cell injection were as negative control (g) thefluorescent intensity value and fold changes of the dissected livers of two groups the difference was statistically significant

(compared with normal control fold change 156 plusmn 08)(119875 lt 005) (Figures 5(g) 5(h) and 5(k)) Dir-labeled BMSCsbefore injection (Figure 5(i)) were as positive control and ratswithout cell injection were as negative control (Figure 5(j))These findings suggested that SDF-1CXCR4 plays a role inthe migration of BMSCs into injured liver

4 Discussion

Recently studies have demonstrated the potential therapeuticfunction of BMSCs transplantation to treat acute and chronicliver injury Because BMSCs produce new hepatocytes [18]secrete trophic factors to promote liver tissue repair [19]

8 Stem Cells International

0200400600800

10001200

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0123456

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

(a) (c) (e) (g)

(b) (d) (f) (h)

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Liver fluorescence of liver injured group Liver fluorescence of AMD3100 group Liver fluorescence of anti-CXCR4 group

Fluorescence X-rayNormal group Liver injured group

Merge Fluorescence X-ray MergeAMD3100 group

Fluorescence X-ray MergeAnti-CXCR4 group

Fluorescence X-ray Merge

Liver fluorescence of normal group

Cell number 4 4000 40000Cell fluorescence before injection as positive controls

400

Fluorescence X-ray MergeWithout cell injection as a negative control

(i)

(k)

(j)

40 400000

50000 129074 208148 287223 366297 50000 129074 208148 287223 366297

17451 plusmn 7682 27267 plusmn 6137

85998 plusmn12780 63679 plusmn

19790

P lt 005 P lt 005P lt 005

P lt 005P lt 005

P lt 005

P lt 005P lt 005

1 plusmn 044

494 plusmn 073366 plusmn 113

156 plusmn 08

Figure 5 BMSCsmigration in injured liver inhibited by anti-CXCR4 antibody andAMD3100 (a)Thefluorescent intensity of normal rats afterDir-labeled BMSCs injection (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection (c) the fluorescentintensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection (d) the fluorescent intensity of dissected TAA injured liversafter Dir-labeled BMSCs injection (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100injection at the same time (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at thesame time (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCsinjection (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection(i) Dir-labeled BMSCs before injection were as positive control (j) rats without cell injection were as negative control (k) the fluorescentintensity value and fold changes of the dissected livers of each group the differences were statistically significant

and modulate inflammatory responses [20] BMSCs trans-plantation is a promising candidate for cell therapy in liverdiseases Cell homing and engraftment into the host liver areintegral to cell-based therapies The mechanism of BMSCsmigration to injured liver is complicated and still not fullyunderstood In the current study we try to investigate the roleof SDF-1CXCR4 axis in the recruitment of BMSCsmigrationinto TAA induced injured liver We revealed SDF-1 induced

BMSC migration in vitro study Our study found SDF-1 levelgradually increased in rat model of TAA induced liver injuryAndwe demonstrated SDF-1CXCR4 axis involved in BMSCsmigration into the injured liver in vitro and in vivo

CXCR4 is a G protein-linked seven-transmembranespanning receptor which is expressed on the surface of asmall proportion of MSCs Some studies indicated that mostCXCR4 are intracellular storage only small part of functional

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 3

Alanine aminotransferase (ALT) and aspartate aminotrans-ferase (AST) levels of rats after TAA injection were detectedby automatic biochemistry analyzer (Beckman Coulter) toconfirm liver injury at 0 weeks 4 weeks 8 weeks and 12weeks And the histological sections of livers were stainedwith hematoxylin-eosin staining (HE staining) to observepathological changes of the livers

263 BMSCs Injection Rats were placed under anesthesiausing isoflurane through inhalation Before cell injectionBMSCs were labeled with 11-dioctadecyl-3333-tetramethy-lindotricarbocyanine iodide (DiR) dye (Invitrogen) Dir-labeled BMSCs before injection were as positive control andrats without cell injection were as negative control First theways of cells injection were compared between portal veininjection (PVI) and tail vein injection (TVI) More migratedcells were found in liver through PVI so PVI was selected inthe following cellmigration study Six rats were in each groupEach rat was injected with 1 times 106 BMSCs through portalvein using a 16G syringe The cells migration in normal andinjured liver was also compared For neutralization studiescells were incubated with anti-CXCR4 monoclonal antibody(RampD Systems) before injection or with AMD3100 duringinjection At the end a laparotomy was performed and thelivers were dissected

264 In Vivo Distribution of the Transplanted Cells Thelocation and the fluorescent strength of the transplanted cellslabeled with dye DiR (excitationemission 748780 nm) weredetected by the Kodak In-VivoMultispectral Imaging SystemFX (excitation filteremission filter ex730em750WA) at 24 hafter cell injection First the fluorescent intensity of wholerats was checked Then the rats were executed and the liverswere perfused with saline (PBS) to remove contaminatingblood which might contain unmigrated BMSCs And thenthe fluorescent intensity of dissected liver wasmeasured Dir-labeled BMSCs before injection were as positive control andrats without cell injection were as negative control At theend of each acquisition a photographic image was obtainedThe data were analyzed with Photovision software whichsuperimposes the signal on the photographic image Themost intense fluorescent signal detected is shown in redwhereas the weakest signal is shown in blue

27 Enzyme-Linked Immunosorbent Assay for SDF-1 Level ofLiver Lysates Liver extracts at 0 weeks 4 weeks 8 weeksand 12 weeks after TAA injection were prepared by cell lysiswith 25mM Tris (pH 75) 1 Triton X-100 05mM EDTA150mM NaCl 10mM NaF and protease inhibitor cocktail(1 PMSF Sigma-Aldrich) Total proteins in these extractswere quantified by Bradford assay and equal protein amountswere assayed for SDF-1 level with SDF-1 enzyme-linkedimmunosorbent assay (ELISA) kit (RampD Systems) accordingto the manufacturerrsquos instructions

28 Statistics Statistical analysis and graphing were per-formed using SPSS 12 All results are expressed as mean plusmnSEM Statistical significance was determined by Studentrsquos 119905-test Significance was defined as 119875 lt 005

3 Results

31 Expression of CXCR4 in BMSCs The expression ofCXCR4 in BMSCs was examined at mRNA and protein levelfrom passages 1 to 8 with RT-PCR immunocytochemistryand flow cytometry CXCR4 was stably expressed in BMSCsat mRNA level from passages 1 to 8 293 T cells were asnegative control (Figure 1(a)) Before staining with CXCR4antibody BMSCswere permeabilizedwith 01TritonX-100so the immunocytochemistry results indicated all the CXCR4expression on the membrane and intracellularly CXCR4were found in 100 of cells (Figure 1(b)) Only membraneCXCR4 positive BMSCs could be detected by flow cytometryThe result of flow cytometry indicated 42 of cells weremembrane CXCR4 positive (Figure 1(c))

32 SDF-1 Induce BMSCs Migration In Vitro In cell tran-swell system SDF-1 added in bottom chamber was at0 ngmL 50 ngmL 100 ngmL 200 ngmL 400 ngmL and800 ngmL the number of migrated cells were countedunder fluorescence microscopes which were 52 plusmn 327 27 plusmn579 644 plusmn 1761 502 plusmn 1514 416 plusmn 75 and 364 plusmn568 respectively The concentration of SDF-1 lower than100 ngmL the number of migrated cells increased as theconcentration increased and the differences were statisticallysignificant (all 119875 lt 005) However after the concentrationwas above 100 ngmL the number of migrated cells decreasedbecause too much SDF-1 would complete the binding site(Figure 2(a))

To demonstrate that the migration was an SDF-1dependent effect CXCR4 antagonist AMD3100 or anti-CXCR4 antibody was added The concentration of SDF-1 at100 ngmL after adding AMD3100 at 24120583gmL 48 120583gmLand 96120583gmL the number of migrated cells decreased from586 plusmn 963 to 725 plusmn 206 325 plusmn 05 and 325 plusmn 171and the differences were statistically significant (all 119875 lt005) (Figure 2(b)) After adding anti-CXCR4 antibody at20120583gmL 40 120583gmL 80120583gmL and 96 120583gmL the numberof migrated cells decreased from 608 plusmn 963 to 1275 plusmn 1257plusmn258 675plusmn096 and 8plusmn141 respectivelyThe differenceswere statistically significant (all 119875 lt 005) (Figure 2(c))These results indicated that SDF-1CXCR4 was involved inthe hBMSCs migration

33 Liver Injury after TAA Injection and SDF-1 Levels of RatsLiver Lysates Liver injury model was induced in rats byIP injections of TAA After TAA injection the serum levelof ALT at 0 weeks 4 weeks 8 weeks and 12 weeks was4117 plusmn 823UL 7835 plusmn 467UL 18656 plusmn 1568UL and31698 plusmn 2085UL respectively and that of the controls was4133 plusmn 484UL 4437 plusmn 662UL 4286 plusmn 398UL and4652 plusmn 566UL respectively The differences in ALT levelbetween TAA injection group and control group at 4 weeks8 weeks and 12 weeks were statistically significant (119875 lt 005)(Figure 3(a))The serum level of AST was 15083plusmn1865UL19678plusmn2462UL 25698plusmn2676UL and 45012plusmn3568ULat 0weeks 4weeks 8weeks and 12weeks afterTAA injectionrespectively and that of the controls was 14582 plusmn 2078UL13267plusmn1896UL 16478plusmn2267UL and 16832plusmn201UL

4 Stem Cells International

BMSCs P 1 2 3 4 5 6 7 8 293 T cells

CXCR4

GAPDH

(a)

DAPI CXCR4 CXCR4DAPI CXCR4 isotypeDAPI

(b)100

80

60

40

20

0

101 102 103 104 105

Max

()

Iso

Ctr CXCR4

42

AL488-A CXCR4(c)

Figure 1 Expression of CXCR4 in BMSCs (a) RT-PCR results of CXCR4 expression in BMSCs from passages 1 to 8 CXCR4 was stablyexpressed 293T cells as negative control (b) immunocytochemistry results of CXCR4 expression which indicated both membrane andintracellular CXCR4 expression (c) the result of flow cytometry indicated only 42 of cells were positive for membrane CXCR4

respectively The differences between two groups at 4 weeks8 weeks and 12 weeks were statistically significant (119875 lt 005)(Figure 3(a)) After TAA injection the serum level of AST andALT significantly increased (Figure 3(a)) which indicatedliver injury The livers of rats were collected at 0 4 8 and12 weeks after TAA injection The pathological results alsoindicated liver injury there were vacuole degeneration (at8 weeks) microvascular disintegration (at 8 weeks) tissuenecrosis and disruption of general architecture (at 12 weeks)(Figure 3(b)) The SDF-1 concentrations of livers lysates weredetermined by ELISA The SDF-1 concentrations of liverslysates at 0 4 8 and 12 weeks were 0714 plusmn 0267 ngmL0845 plusmn 0420 ngmL 0937 plusmn 0060 ngmL and 1536 plusmn0339 ngmL (Figure 3(c)) These results indicated that SDF-1level increased in TAA induced liver injury

34 BMSCs Migration Induced by Liver Extracts In Vitro Toexplore whether SDF-1 of liver lysates is involved in recruit-ment of BMSCs a migration experiment was performedin vitro with BMSCs and liver lysates from rats treated byTAA for 12 weeks The number of migrated cells causedby liver lysates from untreated normal rats was 76 plusmn 282and that by TAA induced injured liver lysates was 188 plusmn

396 the difference was statistically significant (119875 lt 005)(Figure 3(d)) As we demonstrated SDF-1 level of liver lysatesfrom rats treated by TAA increased so the question waswhether the increased cell number was caused by SDF-1CXCR4 antagonist AMD3100 or anti-CXCR4 antibody wasadded in the medium in the following study SDF-1 level ofliver lysates was 1536 plusmn 0339 ngmL at 12 weeks Accordingto the results of SDF-1 induced MSCs migration in vitrofor neutralization studies the concentration of AMD3100 waschosen at 24120583gmL and anti-CXCR4 antibody at 20 120583gmLAfter adding AMD3100 (24 120583gmL) or anti-CXCR4 antibody(20120583gmL) the number of migrated cells decreased to 138plusmn277 and 114 plusmn 270 (Figure 3(d)) however still higherthan the number of migrated cells caused by liver lysatesfrom untreated normal rats These results demonstrated thatSDF-1CXCR4 axis involved in injured liver lysates inducedBMSCs migration but not the only pathway

35 SDF-1CXCR4 Mediate BMSCs Migration in Rats Wefirst compare theways of cells injectionThe cells were labeledwith Dir dye which gave out fluorescence in the liver tissuesafter cells injection and the fluorescence could be capturedby in vivo imaging systemThefluorescent intensity of injured

Stem Cells International 5

020406080

100

0 50 100 200 400 800

Cell

num

ber

field

SDF-1 concentration (ngmL)

SDF-1 induced hBMSCs migration

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

52 plusmn 327

27 plusmn 579

644 plusmn 1761 502 plusmn 1514416 plusmn 75 364 plusmn 568

0ngmL 50ngmL 100ngmL

200ngmL 400ngmL 800ngmL

(a)

0

20

40

60

80

0 24 48 96

Cel

l num

ber

SDF-1 induced MSCs migration inhibited by AMD3100P lt 005

P lt 005

P lt 005

586 plusmn 963

725 plusmn 206 325 plusmn 05 325 plusmn 171

AMD3100 concentration (120583gmL)

0120583gmL 24120583gmL

48120583gmL 96120583gmL

(b)

0

20

40

60

80

0 20 40 80 160

Cel

l num

ber

SDF-1 induced MSCs migration inhibited by CXCR4 antibodyP lt 005

P lt 005

P lt 005

P lt 005

608 plusmn 963

1275 plusmn 125 7 plusmn 258 675 plusmn 096 8 plusmn 141

CXCR4 antibody concentration (120583gmL)

0120583gmL 20120583gmL

80120583gmL 160120583gmL

40120583gmL

(c)

Figure 2 SDF-1 induces BMSCsmigration in vitro (a) SDF-1 at different concentration the migrated cells were observed under fluorescencemicroscope and the graph indicated the number of migrated cells (b) SDF-1 at 100 ngmL AMD3100 was added at different concentrationthemigrated cells were observed and counted under fluorescencemicroscope and the graph indicated the number of migrated cells (c) SDF-1at 100 ngmL anti-CXCR4 antibody was added at different concentration the migrated cells were observed and counted

livers with cells injection through tail vein (TVI) was 51610plusmn11560 (Figures 4(a) 4(b) and 4(g)) and that through portalvein (PVI) was 85998 plusmn 12780 (Figures 4(c) 4(d) and 4(g))which was significantly higher (fold change 167plusmn025) (119875 lt005) Dir-labeled BMSCs before injection (Figure 4(e)) wereas positive control and rats without cell injection were asnegative control (Figure 4(f))This result indicated that morecells migrated into liver through portal vein injection So PVIinjection was selected in the following transplantation study

We also compared the cell migration between normalliver and injured liver After cells injection the fluorescentintensity of normal livers (17451 plusmn 7682) (Figures 5(a)5(b) and 5(k)) was statistically significantly lower than thatof injured livers (85998 plusmn 12780) (compared with normal

control fold change 494plusmn073) (Figures 5(c) 5(d) and 5(k))(119875 lt 005) More cells migrated into injured liver The SDF-1level of injured liver increased So the question is whetherthe migration was regulated by SDF-1CXCR4 axis FirstCXCR4 antagonist AMD3100 was injected into liver throughPV and then followed by BMSCs injection The fluorescentintensity of the livers declined to 63679 plusmn 19790 (comparedwith normal control fold change 366 plusmn 113) (Figures 5(e)5(f) and 5(k)) which indicated that AMD3100 significantlyinhibited cells migration into the liver And then the cellswere incubated with anti-CXCR4 antibody to neutralizeCXCR4 receptor before injection The cell migration wasalso significantly inhibited by CXCR4 neutralization thefluorescent intensity of the livers decreased to 27267 plusmn 6137

6 Stem Cells International

050

100150200250300350400

ALT level

Normal controlTAA injection group

Normal controlTAA injection group

0100200300400500600

AST level(U

L)

(UL

)

P lt 005

P lt 005

P lt 005

P lt 005P lt 005

P lt 005

4133 plusmn

484 4117 plusmn823

4437 plusmn626

4286 plusmn398

4652 plusmn566

31698 plusmn2085

18656 plusmn1568

14582 plusmn2078 13267 plusmn

1896

15083 plusmn1865

19678 plusmn2462 16478 plusmn

2267

25698 plusmn2676 15832 plusmn

201

45012 plusmn3568

7835 plusmn467

0 4 8 12

(week) (week)0 4 8 12

(a)

Normal liver TAA injection4 weeks

TAA injection8 weeks

TAA injection12 weeks

(b)

002040608

112141618

2

0 4 8 12

SDF-

1 co

ncen

trat

ion

(ng

mL)

The concentration of SDF-1 in injured liver induced by thioacetamide

0714 plusmn

02670845 plusmn

04200937 plusmn

0060

1536 plusmn

0339

P lt 005

P lt 005

P lt 005

Modeling time (week)

(c)

Liver lysis induced BMSCs migration

0

5

10

15

20

25

Normal liverlysis

TAA injuredliver lysis

Anti-CXCR4 AMD3100

Cel

l num

ber

P lt 005 P lt 005

P lt 005

76 plusmn 282

188 plusmn 396

114 plusmn 270138 plusmn 277

Normal liver lysis TAA injured liver lysis

Anti-CXCR4 antibody treated AMD3100 treated

(d)

Figure 3 Injured liver lysates induce BMSCsmigration in vitro (a) Changes of serumALT and AST level after TAA injection (b) HE stainedliver tissue sections after TAA injection (c) the concentrations of SDF-1 of liver lysates detected by ELISA at different time point of TAAinduced liver injury (d) the migrated cells induced by normal liver lysates and TAA injured liver lysates More cell migration was induced byTAA injured liver lysates which could be partially blocked by AMD3100 (24 120583gmL) or anti-CXCR4 antibody (20120583gmL)The migrated cellswere observed under fluorescence microscope and the graph indicated the number of migrated cells

Stem Cells International 7

0200400600800

10001200

PVI TVI

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0

05

1

15

2

25

PVI TVI

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

Without cell injection as a negative control

(a) (c)

(b) (d)

(e) (f)

(g)

Fluorescence X-rayTVI group PVI group

Rat 1

Rat 1

Rat 2

Rat 3

Rat 2

Rat 3

Liver fluorescence of TVI group Liver fluorescence of PVI group

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Cell fluorescence before injection as positive controls

Merge Fluorescence X-ray Merge

Fluorescence X-ray Merge

Cell number 4 4000 4000040040 400000

50000 129074 208148 287223 36629750000 129074 208148 287223 366297

85998 plusmn 12780

51610 plusmn 11560

P lt 005 P lt 005

1 plusmn 022

167 plusmn 025

Figure 4 Difference between portal vein injection and tail vein injection (a)The fluorescent intensity of whole rats the BMSCs were injectedthrough tail vein (b) the fluorescent intensity of dissected livers the BMSCs were injected through tail vein (c) the fluorescent intensity ofwhole rats the BMSCs were injected through portal vein (d) the fluorescent intensity of dissected livers the BMSCs were injected throughportal vein (e) Dir-labeled BMSCs before injection were as positive control (f) rats without cell injection were as negative control (g) thefluorescent intensity value and fold changes of the dissected livers of two groups the difference was statistically significant

(compared with normal control fold change 156 plusmn 08)(119875 lt 005) (Figures 5(g) 5(h) and 5(k)) Dir-labeled BMSCsbefore injection (Figure 5(i)) were as positive control and ratswithout cell injection were as negative control (Figure 5(j))These findings suggested that SDF-1CXCR4 plays a role inthe migration of BMSCs into injured liver

4 Discussion

Recently studies have demonstrated the potential therapeuticfunction of BMSCs transplantation to treat acute and chronicliver injury Because BMSCs produce new hepatocytes [18]secrete trophic factors to promote liver tissue repair [19]

8 Stem Cells International

0200400600800

10001200

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0123456

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

(a) (c) (e) (g)

(b) (d) (f) (h)

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Liver fluorescence of liver injured group Liver fluorescence of AMD3100 group Liver fluorescence of anti-CXCR4 group

Fluorescence X-rayNormal group Liver injured group

Merge Fluorescence X-ray MergeAMD3100 group

Fluorescence X-ray MergeAnti-CXCR4 group

Fluorescence X-ray Merge

Liver fluorescence of normal group

Cell number 4 4000 40000Cell fluorescence before injection as positive controls

400

Fluorescence X-ray MergeWithout cell injection as a negative control

(i)

(k)

(j)

40 400000

50000 129074 208148 287223 366297 50000 129074 208148 287223 366297

17451 plusmn 7682 27267 plusmn 6137

85998 plusmn12780 63679 plusmn

19790

P lt 005 P lt 005P lt 005

P lt 005P lt 005

P lt 005

P lt 005P lt 005

1 plusmn 044

494 plusmn 073366 plusmn 113

156 plusmn 08

Figure 5 BMSCsmigration in injured liver inhibited by anti-CXCR4 antibody andAMD3100 (a)Thefluorescent intensity of normal rats afterDir-labeled BMSCs injection (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection (c) the fluorescentintensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection (d) the fluorescent intensity of dissected TAA injured liversafter Dir-labeled BMSCs injection (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100injection at the same time (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at thesame time (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCsinjection (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection(i) Dir-labeled BMSCs before injection were as positive control (j) rats without cell injection were as negative control (k) the fluorescentintensity value and fold changes of the dissected livers of each group the differences were statistically significant

and modulate inflammatory responses [20] BMSCs trans-plantation is a promising candidate for cell therapy in liverdiseases Cell homing and engraftment into the host liver areintegral to cell-based therapies The mechanism of BMSCsmigration to injured liver is complicated and still not fullyunderstood In the current study we try to investigate the roleof SDF-1CXCR4 axis in the recruitment of BMSCsmigrationinto TAA induced injured liver We revealed SDF-1 induced

BMSC migration in vitro study Our study found SDF-1 levelgradually increased in rat model of TAA induced liver injuryAndwe demonstrated SDF-1CXCR4 axis involved in BMSCsmigration into the injured liver in vitro and in vivo

CXCR4 is a G protein-linked seven-transmembranespanning receptor which is expressed on the surface of asmall proportion of MSCs Some studies indicated that mostCXCR4 are intracellular storage only small part of functional

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Stem Cells International

BMSCs P 1 2 3 4 5 6 7 8 293 T cells

CXCR4

GAPDH

(a)

DAPI CXCR4 CXCR4DAPI CXCR4 isotypeDAPI

(b)100

80

60

40

20

0

101 102 103 104 105

Max

()

Iso

Ctr CXCR4

42

AL488-A CXCR4(c)

Figure 1 Expression of CXCR4 in BMSCs (a) RT-PCR results of CXCR4 expression in BMSCs from passages 1 to 8 CXCR4 was stablyexpressed 293T cells as negative control (b) immunocytochemistry results of CXCR4 expression which indicated both membrane andintracellular CXCR4 expression (c) the result of flow cytometry indicated only 42 of cells were positive for membrane CXCR4

respectively The differences between two groups at 4 weeks8 weeks and 12 weeks were statistically significant (119875 lt 005)(Figure 3(a)) After TAA injection the serum level of AST andALT significantly increased (Figure 3(a)) which indicatedliver injury The livers of rats were collected at 0 4 8 and12 weeks after TAA injection The pathological results alsoindicated liver injury there were vacuole degeneration (at8 weeks) microvascular disintegration (at 8 weeks) tissuenecrosis and disruption of general architecture (at 12 weeks)(Figure 3(b)) The SDF-1 concentrations of livers lysates weredetermined by ELISA The SDF-1 concentrations of liverslysates at 0 4 8 and 12 weeks were 0714 plusmn 0267 ngmL0845 plusmn 0420 ngmL 0937 plusmn 0060 ngmL and 1536 plusmn0339 ngmL (Figure 3(c)) These results indicated that SDF-1level increased in TAA induced liver injury

34 BMSCs Migration Induced by Liver Extracts In Vitro Toexplore whether SDF-1 of liver lysates is involved in recruit-ment of BMSCs a migration experiment was performedin vitro with BMSCs and liver lysates from rats treated byTAA for 12 weeks The number of migrated cells causedby liver lysates from untreated normal rats was 76 plusmn 282and that by TAA induced injured liver lysates was 188 plusmn

396 the difference was statistically significant (119875 lt 005)(Figure 3(d)) As we demonstrated SDF-1 level of liver lysatesfrom rats treated by TAA increased so the question waswhether the increased cell number was caused by SDF-1CXCR4 antagonist AMD3100 or anti-CXCR4 antibody wasadded in the medium in the following study SDF-1 level ofliver lysates was 1536 plusmn 0339 ngmL at 12 weeks Accordingto the results of SDF-1 induced MSCs migration in vitrofor neutralization studies the concentration of AMD3100 waschosen at 24120583gmL and anti-CXCR4 antibody at 20 120583gmLAfter adding AMD3100 (24 120583gmL) or anti-CXCR4 antibody(20120583gmL) the number of migrated cells decreased to 138plusmn277 and 114 plusmn 270 (Figure 3(d)) however still higherthan the number of migrated cells caused by liver lysatesfrom untreated normal rats These results demonstrated thatSDF-1CXCR4 axis involved in injured liver lysates inducedBMSCs migration but not the only pathway

35 SDF-1CXCR4 Mediate BMSCs Migration in Rats Wefirst compare theways of cells injectionThe cells were labeledwith Dir dye which gave out fluorescence in the liver tissuesafter cells injection and the fluorescence could be capturedby in vivo imaging systemThefluorescent intensity of injured

Stem Cells International 5

020406080

100

0 50 100 200 400 800

Cell

num

ber

field

SDF-1 concentration (ngmL)

SDF-1 induced hBMSCs migration

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

52 plusmn 327

27 plusmn 579

644 plusmn 1761 502 plusmn 1514416 plusmn 75 364 plusmn 568

0ngmL 50ngmL 100ngmL

200ngmL 400ngmL 800ngmL

(a)

0

20

40

60

80

0 24 48 96

Cel

l num

ber

SDF-1 induced MSCs migration inhibited by AMD3100P lt 005

P lt 005

P lt 005

586 plusmn 963

725 plusmn 206 325 plusmn 05 325 plusmn 171

AMD3100 concentration (120583gmL)

0120583gmL 24120583gmL

48120583gmL 96120583gmL

(b)

0

20

40

60

80

0 20 40 80 160

Cel

l num

ber

SDF-1 induced MSCs migration inhibited by CXCR4 antibodyP lt 005

P lt 005

P lt 005

P lt 005

608 plusmn 963

1275 plusmn 125 7 plusmn 258 675 plusmn 096 8 plusmn 141

CXCR4 antibody concentration (120583gmL)

0120583gmL 20120583gmL

80120583gmL 160120583gmL

40120583gmL

(c)

Figure 2 SDF-1 induces BMSCsmigration in vitro (a) SDF-1 at different concentration the migrated cells were observed under fluorescencemicroscope and the graph indicated the number of migrated cells (b) SDF-1 at 100 ngmL AMD3100 was added at different concentrationthemigrated cells were observed and counted under fluorescencemicroscope and the graph indicated the number of migrated cells (c) SDF-1at 100 ngmL anti-CXCR4 antibody was added at different concentration the migrated cells were observed and counted

livers with cells injection through tail vein (TVI) was 51610plusmn11560 (Figures 4(a) 4(b) and 4(g)) and that through portalvein (PVI) was 85998 plusmn 12780 (Figures 4(c) 4(d) and 4(g))which was significantly higher (fold change 167plusmn025) (119875 lt005) Dir-labeled BMSCs before injection (Figure 4(e)) wereas positive control and rats without cell injection were asnegative control (Figure 4(f))This result indicated that morecells migrated into liver through portal vein injection So PVIinjection was selected in the following transplantation study

We also compared the cell migration between normalliver and injured liver After cells injection the fluorescentintensity of normal livers (17451 plusmn 7682) (Figures 5(a)5(b) and 5(k)) was statistically significantly lower than thatof injured livers (85998 plusmn 12780) (compared with normal

control fold change 494plusmn073) (Figures 5(c) 5(d) and 5(k))(119875 lt 005) More cells migrated into injured liver The SDF-1level of injured liver increased So the question is whetherthe migration was regulated by SDF-1CXCR4 axis FirstCXCR4 antagonist AMD3100 was injected into liver throughPV and then followed by BMSCs injection The fluorescentintensity of the livers declined to 63679 plusmn 19790 (comparedwith normal control fold change 366 plusmn 113) (Figures 5(e)5(f) and 5(k)) which indicated that AMD3100 significantlyinhibited cells migration into the liver And then the cellswere incubated with anti-CXCR4 antibody to neutralizeCXCR4 receptor before injection The cell migration wasalso significantly inhibited by CXCR4 neutralization thefluorescent intensity of the livers decreased to 27267 plusmn 6137

6 Stem Cells International

050

100150200250300350400

ALT level

Normal controlTAA injection group

Normal controlTAA injection group

0100200300400500600

AST level(U

L)

(UL

)

P lt 005

P lt 005

P lt 005

P lt 005P lt 005

P lt 005

4133 plusmn

484 4117 plusmn823

4437 plusmn626

4286 plusmn398

4652 plusmn566

31698 plusmn2085

18656 plusmn1568

14582 plusmn2078 13267 plusmn

1896

15083 plusmn1865

19678 plusmn2462 16478 plusmn

2267

25698 plusmn2676 15832 plusmn

201

45012 plusmn3568

7835 plusmn467

0 4 8 12

(week) (week)0 4 8 12

(a)

Normal liver TAA injection4 weeks

TAA injection8 weeks

TAA injection12 weeks

(b)

002040608

112141618

2

0 4 8 12

SDF-

1 co

ncen

trat

ion

(ng

mL)

The concentration of SDF-1 in injured liver induced by thioacetamide

0714 plusmn

02670845 plusmn

04200937 plusmn

0060

1536 plusmn

0339

P lt 005

P lt 005

P lt 005

Modeling time (week)

(c)

Liver lysis induced BMSCs migration

0

5

10

15

20

25

Normal liverlysis

TAA injuredliver lysis

Anti-CXCR4 AMD3100

Cel

l num

ber

P lt 005 P lt 005

P lt 005

76 plusmn 282

188 plusmn 396

114 plusmn 270138 plusmn 277

Normal liver lysis TAA injured liver lysis

Anti-CXCR4 antibody treated AMD3100 treated

(d)

Figure 3 Injured liver lysates induce BMSCsmigration in vitro (a) Changes of serumALT and AST level after TAA injection (b) HE stainedliver tissue sections after TAA injection (c) the concentrations of SDF-1 of liver lysates detected by ELISA at different time point of TAAinduced liver injury (d) the migrated cells induced by normal liver lysates and TAA injured liver lysates More cell migration was induced byTAA injured liver lysates which could be partially blocked by AMD3100 (24 120583gmL) or anti-CXCR4 antibody (20120583gmL)The migrated cellswere observed under fluorescence microscope and the graph indicated the number of migrated cells

Stem Cells International 7

0200400600800

10001200

PVI TVI

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0

05

1

15

2

25

PVI TVI

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

Without cell injection as a negative control

(a) (c)

(b) (d)

(e) (f)

(g)

Fluorescence X-rayTVI group PVI group

Rat 1

Rat 1

Rat 2

Rat 3

Rat 2

Rat 3

Liver fluorescence of TVI group Liver fluorescence of PVI group

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Cell fluorescence before injection as positive controls

Merge Fluorescence X-ray Merge

Fluorescence X-ray Merge

Cell number 4 4000 4000040040 400000

50000 129074 208148 287223 36629750000 129074 208148 287223 366297

85998 plusmn 12780

51610 plusmn 11560

P lt 005 P lt 005

1 plusmn 022

167 plusmn 025

Figure 4 Difference between portal vein injection and tail vein injection (a)The fluorescent intensity of whole rats the BMSCs were injectedthrough tail vein (b) the fluorescent intensity of dissected livers the BMSCs were injected through tail vein (c) the fluorescent intensity ofwhole rats the BMSCs were injected through portal vein (d) the fluorescent intensity of dissected livers the BMSCs were injected throughportal vein (e) Dir-labeled BMSCs before injection were as positive control (f) rats without cell injection were as negative control (g) thefluorescent intensity value and fold changes of the dissected livers of two groups the difference was statistically significant

(compared with normal control fold change 156 plusmn 08)(119875 lt 005) (Figures 5(g) 5(h) and 5(k)) Dir-labeled BMSCsbefore injection (Figure 5(i)) were as positive control and ratswithout cell injection were as negative control (Figure 5(j))These findings suggested that SDF-1CXCR4 plays a role inthe migration of BMSCs into injured liver

4 Discussion

Recently studies have demonstrated the potential therapeuticfunction of BMSCs transplantation to treat acute and chronicliver injury Because BMSCs produce new hepatocytes [18]secrete trophic factors to promote liver tissue repair [19]

8 Stem Cells International

0200400600800

10001200

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0123456

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

(a) (c) (e) (g)

(b) (d) (f) (h)

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Liver fluorescence of liver injured group Liver fluorescence of AMD3100 group Liver fluorescence of anti-CXCR4 group

Fluorescence X-rayNormal group Liver injured group

Merge Fluorescence X-ray MergeAMD3100 group

Fluorescence X-ray MergeAnti-CXCR4 group

Fluorescence X-ray Merge

Liver fluorescence of normal group

Cell number 4 4000 40000Cell fluorescence before injection as positive controls

400

Fluorescence X-ray MergeWithout cell injection as a negative control

(i)

(k)

(j)

40 400000

50000 129074 208148 287223 366297 50000 129074 208148 287223 366297

17451 plusmn 7682 27267 plusmn 6137

85998 plusmn12780 63679 plusmn

19790

P lt 005 P lt 005P lt 005

P lt 005P lt 005

P lt 005

P lt 005P lt 005

1 plusmn 044

494 plusmn 073366 plusmn 113

156 plusmn 08

Figure 5 BMSCsmigration in injured liver inhibited by anti-CXCR4 antibody andAMD3100 (a)Thefluorescent intensity of normal rats afterDir-labeled BMSCs injection (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection (c) the fluorescentintensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection (d) the fluorescent intensity of dissected TAA injured liversafter Dir-labeled BMSCs injection (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100injection at the same time (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at thesame time (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCsinjection (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection(i) Dir-labeled BMSCs before injection were as positive control (j) rats without cell injection were as negative control (k) the fluorescentintensity value and fold changes of the dissected livers of each group the differences were statistically significant

and modulate inflammatory responses [20] BMSCs trans-plantation is a promising candidate for cell therapy in liverdiseases Cell homing and engraftment into the host liver areintegral to cell-based therapies The mechanism of BMSCsmigration to injured liver is complicated and still not fullyunderstood In the current study we try to investigate the roleof SDF-1CXCR4 axis in the recruitment of BMSCsmigrationinto TAA induced injured liver We revealed SDF-1 induced

BMSC migration in vitro study Our study found SDF-1 levelgradually increased in rat model of TAA induced liver injuryAndwe demonstrated SDF-1CXCR4 axis involved in BMSCsmigration into the injured liver in vitro and in vivo

CXCR4 is a G protein-linked seven-transmembranespanning receptor which is expressed on the surface of asmall proportion of MSCs Some studies indicated that mostCXCR4 are intracellular storage only small part of functional

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 5

020406080

100

0 50 100 200 400 800

Cell

num

ber

field

SDF-1 concentration (ngmL)

SDF-1 induced hBMSCs migration

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

52 plusmn 327

27 plusmn 579

644 plusmn 1761 502 plusmn 1514416 plusmn 75 364 plusmn 568

0ngmL 50ngmL 100ngmL

200ngmL 400ngmL 800ngmL

(a)

0

20

40

60

80

0 24 48 96

Cel

l num

ber

SDF-1 induced MSCs migration inhibited by AMD3100P lt 005

P lt 005

P lt 005

586 plusmn 963

725 plusmn 206 325 plusmn 05 325 plusmn 171

AMD3100 concentration (120583gmL)

0120583gmL 24120583gmL

48120583gmL 96120583gmL

(b)

0

20

40

60

80

0 20 40 80 160

Cel

l num

ber

SDF-1 induced MSCs migration inhibited by CXCR4 antibodyP lt 005

P lt 005

P lt 005

P lt 005

608 plusmn 963

1275 plusmn 125 7 plusmn 258 675 plusmn 096 8 plusmn 141

CXCR4 antibody concentration (120583gmL)

0120583gmL 20120583gmL

80120583gmL 160120583gmL

40120583gmL

(c)

Figure 2 SDF-1 induces BMSCsmigration in vitro (a) SDF-1 at different concentration the migrated cells were observed under fluorescencemicroscope and the graph indicated the number of migrated cells (b) SDF-1 at 100 ngmL AMD3100 was added at different concentrationthemigrated cells were observed and counted under fluorescencemicroscope and the graph indicated the number of migrated cells (c) SDF-1at 100 ngmL anti-CXCR4 antibody was added at different concentration the migrated cells were observed and counted

livers with cells injection through tail vein (TVI) was 51610plusmn11560 (Figures 4(a) 4(b) and 4(g)) and that through portalvein (PVI) was 85998 plusmn 12780 (Figures 4(c) 4(d) and 4(g))which was significantly higher (fold change 167plusmn025) (119875 lt005) Dir-labeled BMSCs before injection (Figure 4(e)) wereas positive control and rats without cell injection were asnegative control (Figure 4(f))This result indicated that morecells migrated into liver through portal vein injection So PVIinjection was selected in the following transplantation study

We also compared the cell migration between normalliver and injured liver After cells injection the fluorescentintensity of normal livers (17451 plusmn 7682) (Figures 5(a)5(b) and 5(k)) was statistically significantly lower than thatof injured livers (85998 plusmn 12780) (compared with normal

control fold change 494plusmn073) (Figures 5(c) 5(d) and 5(k))(119875 lt 005) More cells migrated into injured liver The SDF-1level of injured liver increased So the question is whetherthe migration was regulated by SDF-1CXCR4 axis FirstCXCR4 antagonist AMD3100 was injected into liver throughPV and then followed by BMSCs injection The fluorescentintensity of the livers declined to 63679 plusmn 19790 (comparedwith normal control fold change 366 plusmn 113) (Figures 5(e)5(f) and 5(k)) which indicated that AMD3100 significantlyinhibited cells migration into the liver And then the cellswere incubated with anti-CXCR4 antibody to neutralizeCXCR4 receptor before injection The cell migration wasalso significantly inhibited by CXCR4 neutralization thefluorescent intensity of the livers decreased to 27267 plusmn 6137

6 Stem Cells International

050

100150200250300350400

ALT level

Normal controlTAA injection group

Normal controlTAA injection group

0100200300400500600

AST level(U

L)

(UL

)

P lt 005

P lt 005

P lt 005

P lt 005P lt 005

P lt 005

4133 plusmn

484 4117 plusmn823

4437 plusmn626

4286 plusmn398

4652 plusmn566

31698 plusmn2085

18656 plusmn1568

14582 plusmn2078 13267 plusmn

1896

15083 plusmn1865

19678 plusmn2462 16478 plusmn

2267

25698 plusmn2676 15832 plusmn

201

45012 plusmn3568

7835 plusmn467

0 4 8 12

(week) (week)0 4 8 12

(a)

Normal liver TAA injection4 weeks

TAA injection8 weeks

TAA injection12 weeks

(b)

002040608

112141618

2

0 4 8 12

SDF-

1 co

ncen

trat

ion

(ng

mL)

The concentration of SDF-1 in injured liver induced by thioacetamide

0714 plusmn

02670845 plusmn

04200937 plusmn

0060

1536 plusmn

0339

P lt 005

P lt 005

P lt 005

Modeling time (week)

(c)

Liver lysis induced BMSCs migration

0

5

10

15

20

25

Normal liverlysis

TAA injuredliver lysis

Anti-CXCR4 AMD3100

Cel

l num

ber

P lt 005 P lt 005

P lt 005

76 plusmn 282

188 plusmn 396

114 plusmn 270138 plusmn 277

Normal liver lysis TAA injured liver lysis

Anti-CXCR4 antibody treated AMD3100 treated

(d)

Figure 3 Injured liver lysates induce BMSCsmigration in vitro (a) Changes of serumALT and AST level after TAA injection (b) HE stainedliver tissue sections after TAA injection (c) the concentrations of SDF-1 of liver lysates detected by ELISA at different time point of TAAinduced liver injury (d) the migrated cells induced by normal liver lysates and TAA injured liver lysates More cell migration was induced byTAA injured liver lysates which could be partially blocked by AMD3100 (24 120583gmL) or anti-CXCR4 antibody (20120583gmL)The migrated cellswere observed under fluorescence microscope and the graph indicated the number of migrated cells

Stem Cells International 7

0200400600800

10001200

PVI TVI

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0

05

1

15

2

25

PVI TVI

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

Without cell injection as a negative control

(a) (c)

(b) (d)

(e) (f)

(g)

Fluorescence X-rayTVI group PVI group

Rat 1

Rat 1

Rat 2

Rat 3

Rat 2

Rat 3

Liver fluorescence of TVI group Liver fluorescence of PVI group

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Cell fluorescence before injection as positive controls

Merge Fluorescence X-ray Merge

Fluorescence X-ray Merge

Cell number 4 4000 4000040040 400000

50000 129074 208148 287223 36629750000 129074 208148 287223 366297

85998 plusmn 12780

51610 plusmn 11560

P lt 005 P lt 005

1 plusmn 022

167 plusmn 025

Figure 4 Difference between portal vein injection and tail vein injection (a)The fluorescent intensity of whole rats the BMSCs were injectedthrough tail vein (b) the fluorescent intensity of dissected livers the BMSCs were injected through tail vein (c) the fluorescent intensity ofwhole rats the BMSCs were injected through portal vein (d) the fluorescent intensity of dissected livers the BMSCs were injected throughportal vein (e) Dir-labeled BMSCs before injection were as positive control (f) rats without cell injection were as negative control (g) thefluorescent intensity value and fold changes of the dissected livers of two groups the difference was statistically significant

(compared with normal control fold change 156 plusmn 08)(119875 lt 005) (Figures 5(g) 5(h) and 5(k)) Dir-labeled BMSCsbefore injection (Figure 5(i)) were as positive control and ratswithout cell injection were as negative control (Figure 5(j))These findings suggested that SDF-1CXCR4 plays a role inthe migration of BMSCs into injured liver

4 Discussion

Recently studies have demonstrated the potential therapeuticfunction of BMSCs transplantation to treat acute and chronicliver injury Because BMSCs produce new hepatocytes [18]secrete trophic factors to promote liver tissue repair [19]

8 Stem Cells International

0200400600800

10001200

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0123456

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

(a) (c) (e) (g)

(b) (d) (f) (h)

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Liver fluorescence of liver injured group Liver fluorescence of AMD3100 group Liver fluorescence of anti-CXCR4 group

Fluorescence X-rayNormal group Liver injured group

Merge Fluorescence X-ray MergeAMD3100 group

Fluorescence X-ray MergeAnti-CXCR4 group

Fluorescence X-ray Merge

Liver fluorescence of normal group

Cell number 4 4000 40000Cell fluorescence before injection as positive controls

400

Fluorescence X-ray MergeWithout cell injection as a negative control

(i)

(k)

(j)

40 400000

50000 129074 208148 287223 366297 50000 129074 208148 287223 366297

17451 plusmn 7682 27267 plusmn 6137

85998 plusmn12780 63679 plusmn

19790

P lt 005 P lt 005P lt 005

P lt 005P lt 005

P lt 005

P lt 005P lt 005

1 plusmn 044

494 plusmn 073366 plusmn 113

156 plusmn 08

Figure 5 BMSCsmigration in injured liver inhibited by anti-CXCR4 antibody andAMD3100 (a)Thefluorescent intensity of normal rats afterDir-labeled BMSCs injection (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection (c) the fluorescentintensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection (d) the fluorescent intensity of dissected TAA injured liversafter Dir-labeled BMSCs injection (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100injection at the same time (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at thesame time (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCsinjection (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection(i) Dir-labeled BMSCs before injection were as positive control (j) rats without cell injection were as negative control (k) the fluorescentintensity value and fold changes of the dissected livers of each group the differences were statistically significant

and modulate inflammatory responses [20] BMSCs trans-plantation is a promising candidate for cell therapy in liverdiseases Cell homing and engraftment into the host liver areintegral to cell-based therapies The mechanism of BMSCsmigration to injured liver is complicated and still not fullyunderstood In the current study we try to investigate the roleof SDF-1CXCR4 axis in the recruitment of BMSCsmigrationinto TAA induced injured liver We revealed SDF-1 induced

BMSC migration in vitro study Our study found SDF-1 levelgradually increased in rat model of TAA induced liver injuryAndwe demonstrated SDF-1CXCR4 axis involved in BMSCsmigration into the injured liver in vitro and in vivo

CXCR4 is a G protein-linked seven-transmembranespanning receptor which is expressed on the surface of asmall proportion of MSCs Some studies indicated that mostCXCR4 are intracellular storage only small part of functional

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Stem Cells International

050

100150200250300350400

ALT level

Normal controlTAA injection group

Normal controlTAA injection group

0100200300400500600

AST level(U

L)

(UL

)

P lt 005

P lt 005

P lt 005

P lt 005P lt 005

P lt 005

4133 plusmn

484 4117 plusmn823

4437 plusmn626

4286 plusmn398

4652 plusmn566

31698 plusmn2085

18656 plusmn1568

14582 plusmn2078 13267 plusmn

1896

15083 plusmn1865

19678 plusmn2462 16478 plusmn

2267

25698 plusmn2676 15832 plusmn

201

45012 plusmn3568

7835 plusmn467

0 4 8 12

(week) (week)0 4 8 12

(a)

Normal liver TAA injection4 weeks

TAA injection8 weeks

TAA injection12 weeks

(b)

002040608

112141618

2

0 4 8 12

SDF-

1 co

ncen

trat

ion

(ng

mL)

The concentration of SDF-1 in injured liver induced by thioacetamide

0714 plusmn

02670845 plusmn

04200937 plusmn

0060

1536 plusmn

0339

P lt 005

P lt 005

P lt 005

Modeling time (week)

(c)

Liver lysis induced BMSCs migration

0

5

10

15

20

25

Normal liverlysis

TAA injuredliver lysis

Anti-CXCR4 AMD3100

Cel

l num

ber

P lt 005 P lt 005

P lt 005

76 plusmn 282

188 plusmn 396

114 plusmn 270138 plusmn 277

Normal liver lysis TAA injured liver lysis

Anti-CXCR4 antibody treated AMD3100 treated

(d)

Figure 3 Injured liver lysates induce BMSCsmigration in vitro (a) Changes of serumALT and AST level after TAA injection (b) HE stainedliver tissue sections after TAA injection (c) the concentrations of SDF-1 of liver lysates detected by ELISA at different time point of TAAinduced liver injury (d) the migrated cells induced by normal liver lysates and TAA injured liver lysates More cell migration was induced byTAA injured liver lysates which could be partially blocked by AMD3100 (24 120583gmL) or anti-CXCR4 antibody (20120583gmL)The migrated cellswere observed under fluorescence microscope and the graph indicated the number of migrated cells

Stem Cells International 7

0200400600800

10001200

PVI TVI

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0

05

1

15

2

25

PVI TVI

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

Without cell injection as a negative control

(a) (c)

(b) (d)

(e) (f)

(g)

Fluorescence X-rayTVI group PVI group

Rat 1

Rat 1

Rat 2

Rat 3

Rat 2

Rat 3

Liver fluorescence of TVI group Liver fluorescence of PVI group

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Cell fluorescence before injection as positive controls

Merge Fluorescence X-ray Merge

Fluorescence X-ray Merge

Cell number 4 4000 4000040040 400000

50000 129074 208148 287223 36629750000 129074 208148 287223 366297

85998 plusmn 12780

51610 plusmn 11560

P lt 005 P lt 005

1 plusmn 022

167 plusmn 025

Figure 4 Difference between portal vein injection and tail vein injection (a)The fluorescent intensity of whole rats the BMSCs were injectedthrough tail vein (b) the fluorescent intensity of dissected livers the BMSCs were injected through tail vein (c) the fluorescent intensity ofwhole rats the BMSCs were injected through portal vein (d) the fluorescent intensity of dissected livers the BMSCs were injected throughportal vein (e) Dir-labeled BMSCs before injection were as positive control (f) rats without cell injection were as negative control (g) thefluorescent intensity value and fold changes of the dissected livers of two groups the difference was statistically significant

(compared with normal control fold change 156 plusmn 08)(119875 lt 005) (Figures 5(g) 5(h) and 5(k)) Dir-labeled BMSCsbefore injection (Figure 5(i)) were as positive control and ratswithout cell injection were as negative control (Figure 5(j))These findings suggested that SDF-1CXCR4 plays a role inthe migration of BMSCs into injured liver

4 Discussion

Recently studies have demonstrated the potential therapeuticfunction of BMSCs transplantation to treat acute and chronicliver injury Because BMSCs produce new hepatocytes [18]secrete trophic factors to promote liver tissue repair [19]

8 Stem Cells International

0200400600800

10001200

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0123456

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

(a) (c) (e) (g)

(b) (d) (f) (h)

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Liver fluorescence of liver injured group Liver fluorescence of AMD3100 group Liver fluorescence of anti-CXCR4 group

Fluorescence X-rayNormal group Liver injured group

Merge Fluorescence X-ray MergeAMD3100 group

Fluorescence X-ray MergeAnti-CXCR4 group

Fluorescence X-ray Merge

Liver fluorescence of normal group

Cell number 4 4000 40000Cell fluorescence before injection as positive controls

400

Fluorescence X-ray MergeWithout cell injection as a negative control

(i)

(k)

(j)

40 400000

50000 129074 208148 287223 366297 50000 129074 208148 287223 366297

17451 plusmn 7682 27267 plusmn 6137

85998 plusmn12780 63679 plusmn

19790

P lt 005 P lt 005P lt 005

P lt 005P lt 005

P lt 005

P lt 005P lt 005

1 plusmn 044

494 plusmn 073366 plusmn 113

156 plusmn 08

Figure 5 BMSCsmigration in injured liver inhibited by anti-CXCR4 antibody andAMD3100 (a)Thefluorescent intensity of normal rats afterDir-labeled BMSCs injection (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection (c) the fluorescentintensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection (d) the fluorescent intensity of dissected TAA injured liversafter Dir-labeled BMSCs injection (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100injection at the same time (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at thesame time (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCsinjection (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection(i) Dir-labeled BMSCs before injection were as positive control (j) rats without cell injection were as negative control (k) the fluorescentintensity value and fold changes of the dissected livers of each group the differences were statistically significant

and modulate inflammatory responses [20] BMSCs trans-plantation is a promising candidate for cell therapy in liverdiseases Cell homing and engraftment into the host liver areintegral to cell-based therapies The mechanism of BMSCsmigration to injured liver is complicated and still not fullyunderstood In the current study we try to investigate the roleof SDF-1CXCR4 axis in the recruitment of BMSCsmigrationinto TAA induced injured liver We revealed SDF-1 induced

BMSC migration in vitro study Our study found SDF-1 levelgradually increased in rat model of TAA induced liver injuryAndwe demonstrated SDF-1CXCR4 axis involved in BMSCsmigration into the injured liver in vitro and in vivo

CXCR4 is a G protein-linked seven-transmembranespanning receptor which is expressed on the surface of asmall proportion of MSCs Some studies indicated that mostCXCR4 are intracellular storage only small part of functional

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 7

0200400600800

10001200

PVI TVI

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0

05

1

15

2

25

PVI TVI

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

Without cell injection as a negative control

(a) (c)

(b) (d)

(e) (f)

(g)

Fluorescence X-rayTVI group PVI group

Rat 1

Rat 1

Rat 2

Rat 3

Rat 2

Rat 3

Liver fluorescence of TVI group Liver fluorescence of PVI group

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Cell fluorescence before injection as positive controls

Merge Fluorescence X-ray Merge

Fluorescence X-ray Merge

Cell number 4 4000 4000040040 400000

50000 129074 208148 287223 36629750000 129074 208148 287223 366297

85998 plusmn 12780

51610 plusmn 11560

P lt 005 P lt 005

1 plusmn 022

167 plusmn 025

Figure 4 Difference between portal vein injection and tail vein injection (a)The fluorescent intensity of whole rats the BMSCs were injectedthrough tail vein (b) the fluorescent intensity of dissected livers the BMSCs were injected through tail vein (c) the fluorescent intensity ofwhole rats the BMSCs were injected through portal vein (d) the fluorescent intensity of dissected livers the BMSCs were injected throughportal vein (e) Dir-labeled BMSCs before injection were as positive control (f) rats without cell injection were as negative control (g) thefluorescent intensity value and fold changes of the dissected livers of two groups the difference was statistically significant

(compared with normal control fold change 156 plusmn 08)(119875 lt 005) (Figures 5(g) 5(h) and 5(k)) Dir-labeled BMSCsbefore injection (Figure 5(i)) were as positive control and ratswithout cell injection were as negative control (Figure 5(j))These findings suggested that SDF-1CXCR4 plays a role inthe migration of BMSCs into injured liver

4 Discussion

Recently studies have demonstrated the potential therapeuticfunction of BMSCs transplantation to treat acute and chronicliver injury Because BMSCs produce new hepatocytes [18]secrete trophic factors to promote liver tissue repair [19]

8 Stem Cells International

0200400600800

10001200

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0123456

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

(a) (c) (e) (g)

(b) (d) (f) (h)

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Liver fluorescence of liver injured group Liver fluorescence of AMD3100 group Liver fluorescence of anti-CXCR4 group

Fluorescence X-rayNormal group Liver injured group

Merge Fluorescence X-ray MergeAMD3100 group

Fluorescence X-ray MergeAnti-CXCR4 group

Fluorescence X-ray Merge

Liver fluorescence of normal group

Cell number 4 4000 40000Cell fluorescence before injection as positive controls

400

Fluorescence X-ray MergeWithout cell injection as a negative control

(i)

(k)

(j)

40 400000

50000 129074 208148 287223 366297 50000 129074 208148 287223 366297

17451 plusmn 7682 27267 plusmn 6137

85998 plusmn12780 63679 plusmn

19790

P lt 005 P lt 005P lt 005

P lt 005P lt 005

P lt 005

P lt 005P lt 005

1 plusmn 044

494 plusmn 073366 plusmn 113

156 plusmn 08

Figure 5 BMSCsmigration in injured liver inhibited by anti-CXCR4 antibody andAMD3100 (a)Thefluorescent intensity of normal rats afterDir-labeled BMSCs injection (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection (c) the fluorescentintensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection (d) the fluorescent intensity of dissected TAA injured liversafter Dir-labeled BMSCs injection (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100injection at the same time (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at thesame time (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCsinjection (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection(i) Dir-labeled BMSCs before injection were as positive control (j) rats without cell injection were as negative control (k) the fluorescentintensity value and fold changes of the dissected livers of each group the differences were statistically significant

and modulate inflammatory responses [20] BMSCs trans-plantation is a promising candidate for cell therapy in liverdiseases Cell homing and engraftment into the host liver areintegral to cell-based therapies The mechanism of BMSCsmigration to injured liver is complicated and still not fullyunderstood In the current study we try to investigate the roleof SDF-1CXCR4 axis in the recruitment of BMSCsmigrationinto TAA induced injured liver We revealed SDF-1 induced

BMSC migration in vitro study Our study found SDF-1 levelgradually increased in rat model of TAA induced liver injuryAndwe demonstrated SDF-1CXCR4 axis involved in BMSCsmigration into the injured liver in vitro and in vivo

CXCR4 is a G protein-linked seven-transmembranespanning receptor which is expressed on the surface of asmall proportion of MSCs Some studies indicated that mostCXCR4 are intracellular storage only small part of functional

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 Stem Cells International

0200400600800

10001200

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Normalcontrol

TAA injuredliver

AMD3100 CXCR4antibody

Fluo

resc

ent i

nten

sity

Dir-labeled hBMSCs transplanted into liver

0123456

Fold

chan

ge

Dir-labeled hBMSCs transplanted into liver

(a) (c) (e) (g)

(b) (d) (f) (h)

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1

Rat 2

Rat 3

Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3 Rat 1 Rat 2 Rat 3

Liver fluorescence of liver injured group Liver fluorescence of AMD3100 group Liver fluorescence of anti-CXCR4 group

Fluorescence X-rayNormal group Liver injured group

Merge Fluorescence X-ray MergeAMD3100 group

Fluorescence X-ray MergeAnti-CXCR4 group

Fluorescence X-ray Merge

Liver fluorescence of normal group

Cell number 4 4000 40000Cell fluorescence before injection as positive controls

400

Fluorescence X-ray MergeWithout cell injection as a negative control

(i)

(k)

(j)

40 400000

50000 129074 208148 287223 366297 50000 129074 208148 287223 366297

17451 plusmn 7682 27267 plusmn 6137

85998 plusmn12780 63679 plusmn

19790

P lt 005 P lt 005P lt 005

P lt 005P lt 005

P lt 005

P lt 005P lt 005

1 plusmn 044

494 plusmn 073366 plusmn 113

156 plusmn 08

Figure 5 BMSCsmigration in injured liver inhibited by anti-CXCR4 antibody andAMD3100 (a)Thefluorescent intensity of normal rats afterDir-labeled BMSCs injection (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection (c) the fluorescentintensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection (d) the fluorescent intensity of dissected TAA injured liversafter Dir-labeled BMSCs injection (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100injection at the same time (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at thesame time (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCsinjection (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection(i) Dir-labeled BMSCs before injection were as positive control (j) rats without cell injection were as negative control (k) the fluorescentintensity value and fold changes of the dissected livers of each group the differences were statistically significant

and modulate inflammatory responses [20] BMSCs trans-plantation is a promising candidate for cell therapy in liverdiseases Cell homing and engraftment into the host liver areintegral to cell-based therapies The mechanism of BMSCsmigration to injured liver is complicated and still not fullyunderstood In the current study we try to investigate the roleof SDF-1CXCR4 axis in the recruitment of BMSCsmigrationinto TAA induced injured liver We revealed SDF-1 induced

BMSC migration in vitro study Our study found SDF-1 levelgradually increased in rat model of TAA induced liver injuryAndwe demonstrated SDF-1CXCR4 axis involved in BMSCsmigration into the injured liver in vitro and in vivo

CXCR4 is a G protein-linked seven-transmembranespanning receptor which is expressed on the surface of asmall proportion of MSCs Some studies indicated that mostCXCR4 are intracellular storage only small part of functional

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 9

CXCR4 located on the cell surface [21 22] Our study wasconsistent with other studies we found only small part ofBMSCs expressed CXCR4 on the surface After permeabiliza-tion all the cells were CXCR4 positive which included intra-cellular and membrane CXCR4 It is believed that the recep-tor expression is gradually decreased as cells are expanded invitro [23 24] In our study CXCR4 was stably expressed frompassages 1 to 8 And all the cells used in this study were belowpassage 8

Several signal pathways have been implicated in therecruitment of BMSCs into injured organs one of them isSDF-1CXCR4 axis After injury SDF-1 increased in manytissues which recruit BMSCs to the site of injury [25ndash27]And SDF-1CXCR4 pathway is crucial in the migration ofBMSCs into the injured organs In in vitro study we demon-strated that SDF-1 could induce BMSCs migration whichwas blocked by neutralizing anti-CXCR4 antibody or SDF-1 antagonist AMD3100 These results confirmed that SDF-1CXCR4 involved BMSCsmigration in vitroWe found SDF-1 levels of liver tissues increased after TAA induced injuryOther studies also indicated SDF-1 was increased in manytissues after injury [25ndash27] BMSCs migration was observedby injury liver lysates in vitro So the question raised whetherthe BMSCsmigrationwas caused by upregulated SDF-1 in theliver injury In order to answer this question CXCR4 antag-onist AMD3100 or anti-CXCR4 antibody was added in themediumof liver lysates to neutralize the binding site of SDF-1After AMD3100 or anti-CXCR4 antibody was added the cellsmigration induced by the liver lysates was partially inhibitedIn in vivo study after BMSCs were incubated with anti-CXCR4 antibody before injection or AMD3100 was injectedat the same time during BMSCs injection the migration ofBMSCs towards injured liverwas also partially blockedTheseresults demonstrated that SDF-1 of liver injury and SDF-1CXCR4 axis involved in BMSCs migration to injured liverhowever it is not the only pathway Other studies indicatedproteolytic enzymesMMP-2 andMMP-9 and cytokines HGFare also involved in BMSCs migration [28ndash30]

The conventional methods of tracking transplanted cellsrequire histological immune-staining or real-time PCR forgene detection in vitro which are time consuming and of lowefficiencyThe in vivo imaging system is a relatively new opti-cal imaging system that is being widely used for cell trackingallowing noninvasive cell detection and cell migration moni-toring [31 32]There is also no necessity for detecting cells byimmunohistochemistry in thousands of slidesThis is whole-body imaging technique for monitoring transplanted cells Inour preliminary experiment we found that the fluorescentintensity of the liver gradually increased and reached peak at24 h So in our study the fluorescent intensity of the rats waschecked at 24 h after cell injection Not only the fluorescenceintensity of whole rats was checked but the dissected liversas well which were perfused with saline to remove con-taminating blood which might contain unmigrated BMSCsThe results of whole body experiments and dissected liverswere consistent And both of them demonstrated that SDF-1CXCR4 axis was involved in BMSCs migration into theinjured liver

The studies we report here demonstrate that the levelof SDF-1 increased in liver injury and SDF-1CXCR4 axis isinvolved in BMSCs migration toward the injured liver

Abbreviation

BMSCs Bone marrow mesenchymal stromalstem cells

SDF-1 Stromal cell-derived factor-1HGF Hepatocellular growth factorSD rats Sprague-Dawley ratsAEEC Animal Experimentation Ethics

CommitteeTAA ThioacetamideDiR 11-Dioctadecyl-3333-

tetramethylindotricarbocyanine iodideELISA Enzyme-linked immunosorbent assayPVI Portal vein injectionTVI Tail vein injection

Disclaimer

The authors are solely responsible for the content of thispaper

Conflict of Interests

None of the authors has a financial relationship with a com-mercial entity that has an interest in the subject of this paper

Acknowledgments

The research was supported by grants from the ChineseNational Natural Science Grant (no 3060282) Departmentof Health in Jiangsu Province of China (Z201306) andGrantsfor talents from Jiangsu Government (2012-WS-055)

References

[1] D J Prockop ldquoMarrow stromal cells as stem cells for non-hematopoietic tissuesrdquo Science vol 276 no 5309 pp 71ndash741997

[2] W A Noort A B Kruisselbrink P S Inrsquot Anker et al ldquoMes-enchymal stem cells promote engraftment of human umbilicalcord blood-derived CD34+ cells in NODSCID micerdquo Experi-mental Hematology vol 30 no 8 pp 870ndash878 2002

[3] B Short N Brouard T Occhiodoro-Scott A Ramakrishnanand P J Simmons ldquoMesenchymal stem cellsrdquo Archives ofMedical Research vol 34 no 6 pp 565ndash571 2003

[4] Q Li X Zhou Y Shi et al ldquoIn vivo tracking and comparison ofthe therapeutic effects of MSCs and HSCs for liver injuryrdquo PLoSONE vol 8 no 4 Article ID e62363 2013

[5] F Dong J Harvey A Finan K Weber U Agarwal and MS Penn ldquoMyocardial CXCR4 expression is required for mes-enchymal stem cell mediated repair following acute myocardialinfarctionrdquo Circulation vol 126 no 3 pp 314ndash324 2012

[6] K M Akram S Samad M A Spiteri and N R Forsyth ldquoMes-enchymal stem cells promote alveolar epithelial cell woundrepair in vitro through distinct migratory and paracrine mech-anismsrdquo Respiratory Research vol 14 article 9 2013

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 Stem Cells International

[7] L M Wright W Maloney X Yu L Kindle P Collin-Osdobyand P Osdoby ldquoStromal cell-derived factor-1 binding to itschemokine receptor CXCR4 on precursor cells promotes thechemotactic recruitment development and survival of humanosteoclastsrdquo Bone vol 36 no 5 pp 840ndash853 2005

[8] Y Guo G Hangoc H Bian L M Pelus and H E BroxmeyerldquoSDF-1CXCL12 enhances survival and chemotaxis of murineembryonic stem cells and production of primitive and definitivehematopoietic progenitor cellsrdquo Stem Cells vol 23 no 9 pp1324ndash1332 2005

[9] C Cencioni M C Capogrossi and M Napolitano ldquoThe SDF-1CXCR4 axis in stem cell preconditioningrdquo CardiovascularResearch vol 94 no 3 pp 400ndash407 2012

[10] YWu and R C H Zhao ldquoThe role of chemokines inmesenchy-mal stem cell homing to myocardiumrdquo Stem Cell Reviews andReports vol 8 no 1 pp 243ndash250 2012

[11] X Liu B Duan Z Cheng et al ldquoSDF-1CXCR4 axis modulatesbone marrow mesenchymal stem cell apoptosis migration andcytokine secretionrdquo Protein and Cell vol 2 no 10 pp 845ndash8542011

[12] I Petit D Jin and S Rafii ldquoThe SDF-1-CXCR4 signaling path-way a molecular hub modulating neo-angiogenesisrdquo Trends inImmunology vol 28 no 7 pp 299ndash307 2007

[13] H M Hatch D Zheng M L Jorgensen and B E PetersenldquoSDF-1alphaCXCR4 a mechanism for hepatic oval cell activa-tion and bone marrow stem cell recruitment to the injured liverof ratsrdquo Cloning and Stem Cells vol 4 no 4 pp 339ndash351 2002

[14] E Dalakas P N Newsome D J Harrison and J N PlevrisldquoHematopoietic stem cell trafficking in liver injuryrdquoThe FASEBJournal vol 19 no 10 pp 1225ndash1231 2005

[15] M Z Ratajczak M Kucia R Reca M Majka A Janowska-Wieczorek and J Ratajczak ldquoStem cell plasticity revisitedCXCR4-positive cells expressing mRNA for early muscle liverand neural cells lsquohide outrsquo in the bone marrowrdquo Leukemia vol18 no 1 pp 29ndash40 2004

[16] I Sekiya B L Larson J R Smith R Pochampally J-G Cui andD J Prockop ldquoExpansion of human adult stem cells from bonemarrow stroma conditions that maximize the yields of earlyprogenitors and evaluate their qualityrdquo Stem Cells vol 20 no6 pp 530ndash541 2002

[17] H Kawai N Kudo Y Kawashima and A Mitsumoto ldquoEfficacyof urine bile acid as a non-invasive indicator of liver damage inratsrdquo Journal of Toxicological Sciences vol 34 no 1 pp 27ndash382009

[18] D D Houlihan and P N Newsome ldquoCritical review of clinicaltrials of bone marrow stem cells in liver diseaserdquo Gastroenterol-ogy vol 135 no 2 pp 438ndash450 2008

[19] DVanPoll B Parekkadan CHCho et al ldquoMesenchymal stemcell-derived molecules directly modulate hepatocellular deathand regeneration in vitro and in vivordquoHepatology vol 47 no 5pp 1634ndash1643 2008

[20] Y Shi G Hu J Su et al ldquoMesenchymal stem cells a new strat-egy for immunosuppression and tissue repairrdquo Cell Researchvol 20 no 5 pp 510ndash518 2010

[21] Z Ding T B Issekutz G P Downey and T K Waddell ldquoL-selectin stimulation enhances functional expression of surfaceCXCR4 in lymphocytes implications for cellular activationduring adhesion andmigrationrdquoBlood vol 101 no 11 pp 4245ndash4252 2003

[22] P Goichberg A Kalinkovich N Borodovsky et al ldquocAMP-induced PKCzeta activation increases functional CXCR4expression on human CD34+ hematopoietic progenitorsrdquoBlood vol 107 no 3 pp 870ndash879 2006

[23] M Honczarenko Y Le M Swierkowski I Ghiran A MGlodek and L E Silberstein ldquoHuman bone marrow stromalcells express a distinct set of biologically functional chemokinereceptorsrdquo Stem Cells vol 24 no 4 pp 1030ndash1041 2006

[24] R FWynn C AHart C Corradi-Perini et al ldquoA small propor-tion of mesenchymal stem cells strongly expresses functionallyactive CXCR4 receptor capable of promotingmigration to bonemarrowrdquo Blood vol 104 no 9 pp 2643ndash2645 2004

[25] A T Askari S Unzek Z B Popovic et al ldquoEffect of stromal-cell-derived factor 1 on stem-cell homing and tissue regener-ation in ischaemic cardiomyopathyrdquo The Lancet vol 362 no9385 pp 697ndash703 2003

[26] J D Abbott Y Huang D Liu R Hickey D S Krause and FJ Giordano ldquoStromal cell-derived factor-1120572 plays a critical rolein stem cell recruitment to the heart after myocardial infarctionbut is not sufficient to induce homing in the absence of injuryrdquoCirculation vol 110 no 21 pp 3300ndash3305 2004

[27] J Kijowski M Baj-Krzyworzeka M Majka et al ldquoThe SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrinsbut does not affect proliferation and survival in lymphohe-matopoietic cellsrdquo Stem Cells vol 19 no 5 pp 453ndash466 2001

[28] O Kollet S Shivtiel Y-Q Chen et al ldquoHGF SDF-1 andMMP-9 are involved in stress-induced human CD34+ stem cellrecruitment to the liverrdquo Journal of Clinical Investigation vol112 no 2 pp 160ndash169 2003

[29] K Kawai F Xue T Takahara et al ldquoMatrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in theinjured liverrdquo Cell Transplantation vol 21 no 2-3 pp 453ndash4642012

[30] F Belema-Bedada S Uchida A Martire S Kostin and TBraun ldquoEfficient homing of multipotent adult mesenchymalstem cells depends on FROUNT-mediated clustering of CCR2rdquoCell Stem Cell vol 2 no 6 pp 566ndash575 2008

[31] T Ezzat D K Dhar M Malago and S W M Olde DaminkldquoDynamic tracking of stem cells in an acute liver failure modelrdquoWorld Journal of Gastroenterology vol 18 no 6 pp 507ndash5162012

[32] D A Sipkins X Wei J W Wu et al ldquoIn vivo imaging of spe-cialized bone marrow endothelial microdomains for tumourengraftmentrdquo Nature vol 435 no 7044 pp 969ndash973 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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