research article adipose tissue-derived stem cell...

Research ArticleAdipose Tissue-Derived Stem Cell Secreted IGF-1 ProtectsMyoblasts from the Negative Effect of Myostatin

Sebastian Gehmert12 Carina Wenzel1 Markus Loibl13 Gero Brockhoff4

Michaela Huber3 Werner Krutsch3 Michael Nerlich3 Martin Gosau5 Silvan Klein2

Stephan Schreml6 Lukas Prantl2 and Sanga Gehmert14

1 Applied Stem Cell Research Center University Medical Center Regensburg 93053 Regensburg Germany2Department of Trauma Surgery Center of Plastic and Hand Surgery University Medical Center Regensburg93053 Regensburg Germany

3Department of Trauma Surgery University Medical Center Regensburg 93053 Regensburg Germany4Department of Obstetrics and Gynecology University Medical Center Regensburg 93053 Regensburg Germany5Department of Cranio-Maxillofacial Surgery University Medical Center Regensburg 93053 Regensburg Germany6Department of Dermatology University Medical Center Regensburg 93053 Regensburg Germany

Correspondence should be addressed to Sebastian Gehmert gehmertgmailcom

Received 5 October 2013 Accepted 3 December 2013 Published 23 January 2014

Academic Editor Xiaowen Bai

Copyright copy 2014 Sebastian Gehmert et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Myostatin a TGF-120573 family member is associated with inhibition of muscle growth and differentiation and might interact withthe IGF-1 signaling pathway Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs)these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD) But the protective effect of stem cellsecreted IGF-1 on myoblast under high level of myostatin remains unclear In the present study murine myoblasts were exposed tomyostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis The protective effect ofIGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology MyoDexpression was detected to identify impact of IGF-1 onmyoblasts differentiation when exposed tomyostatin IGF-1 was accountablefor 436 of the antiapoptotic impact and 488 for the proliferative effect of ASCs conditioned medium Furthermore IGF-1restored mRNA and protein MyoD expression of myoblasts under risk Beside fusion and transdifferentiation the beneficial effectof ASCs is mediated by paracrine secreted cytokines particularly IGF-1 The present study underlines the potential of ASCs as atherapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases

1 Introduction

Duchenne muscular dystrophy (DMD) is caused by muta-tions in the dystrophin gene and patients suffer fromdecreaseof muscle tissue [1 2] By age of 12 most patients arewheelchair-bound and die early between the age of 20ndash30 due to respiratory failure The progressive fiber dam-age and membrane leakage are caused by the destabilizeddystrophin-associated protein complex (DAPC) due to themissing dystrophin protein In fact a single exposure to high-force eccentric contractions without prior adaptation induce

a loss of DAPC components and apoptosis in normal muscletissue [3 4] Normal muscles tissue repeatedly subjected tomechanical overload respondwith growth and adaptingwitha greater resistance to membrane wounding [5] It has beendemonstrated that endurance training upregulates IGF-1 andIGF2 mRNA as well as IGF regulatory proteins (IGFBP2IGFBP4 IGFBP7 and PRSS11) This is of particular impor-tance since IGF signaling pathway interacts with TFG-bsignaling influencingmuscle cell differentiation and prolifer-ation in a complex manner [6] TGF-120573 protein concentrationhas been shown to be elevated in dystrophic muscle whereas

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 129048 9 pageshttpdxdoiorg1011552014129048

2 BioMed Research International

H2B EGFP 2A SS mCherry GPI pA

HS-GR cassette

WPREcPPTRRE

pLVX-Puro-HS-GR

PCMV PPGKΨ Puror3998400LTR5998400LTR

Figure 1 The pLVX-Puro-HS-GR vector was constructed by cloning HS-GR cassette into CMV driven lentiviral backbone (Clontech)betweenXhoI andXbaI restriction sites H2B histone protein sequence 2A self-cleavage peptide SS endoplasmatic reticulum signal peptideGPI C terminal GPI anchor sequence pA bovine growth hormone polyadenylation signal

this protein did not appear in the exercise training analysisfor normal muscle tissue [7 8] Especially myostatin a newmember of the TGF-120573 superfamily has been reported tobe a negative regulator of skeletal muscle growth [9] Thiswould suggest a role of TGF-120573 in the degeneration of skeletalmuscles in DMD (eg disruption of IGF-related signaling)Indeed application of IGF-1 attenuates the deterioration ofskeletal muscles mass in a murine Duchenne (mdx) model[4 10]

Previous studies demonstrated beneficial effects of IGF-1 produced by mesenchymal stem cells particularly by adi-pose tissue-derived stem cells (ASCs) which can be easilyharvested and expanded in vitro [11] Thus this study wasperformed to investigate whether paracrine factors secretedby ASCs are able to stimulate proliferation of myoblastsand ameliorate myostatin effects on myoblasts Moreover wewanted to elucidate the role of IGF-1 and its protective rolefor myoblasts when exposed to an elevated myostatin level

2 Materials and Methods

21 Cell Culture Adipose tissue was harvested from theinguinal fat pads of five male BALBc mice (Jackson Lab-oratory) at the age of 8ndash12 weeks and pooled for furtherprocessingThe tissuewaswashed twice with prewarmed PBSprior to mincing Two units of LiberaseMNP-S (Roche) weremixed in 1mLprewarmedDulbeccorsquosmodified eaglemedium(DMEM) (Invitrogen) per gram fat and incubated at 37∘C225 rpm shaking for 30 minutes followed by mixing with apipette to release cells Afterwards mixture was centrifugedat 500 g for 10min and cell pellet was washed three times withPBSThe resulting cell pelletwas resuspended andpropagatedin complete ASCs culture medium (DMEM) supplementedwith 20 fetal bovine serum (FBS PAN-Biotech) 100UmLPenicillin (Sigma) and 100 120583gmL streptomycin (Sigma) andincubated at 37∘C in a humidified atmosphere containing 5CO2 C2C12 cells (ATCC) were cultured in growth medium

consisting of DMEM 10 FBS 100UmL Penicillin and100 120583gmL streptomycin Myoblasts were subcultured beforereaching 80 confluency and used for all experiments beforepassage 7

For lentiviral production human embryonic kidney 293Tcells (HEK293T ATCC) were grown in DMEM containing10 FBS and used in passages below 20

22 Real-Time Fusion Assay A plasmid containing H2B-EGFP-2A-mCherry-GPI (HS-GR) was generously providedby Dr Richard Beheringer and Chuan-Wei Jang (MDAnder-son Cancer Center) A lentiviral construct was generated bysubcloning the HS-GR cassette into the pLVX-Puro vector(Clontech) between Xhol and Xbal by restriction digest andligation (Figure 1) The integrity of cloned HS-GR cassettewas confirmed by DNA sequencing and resulting vector wasnamed pLVX-Puro-HS-GR

ASCs were stable transfected by lentiviral infection asdescribed below DAPI dye was added to myoblast cellculture at a final concentration of 50120583gmL in 10 FBSsupplemented DMEM and incubated for 30 minutes DAPIlabeled myoblasts and pLVX-Puro-HS-GR expressing ASCscultured in 20 FBS supplemented DMEM were rinsed sixtimes with PBS and trypsinized for coculture fusion assay8 times 104 DAPI labeled C2C12 and 2 times 104 pLVX-Puro-HS-GR labeled ASCs were mixed and placed in a 6-well plate andincubated until cell culture reached 100 confluency usingmyoblast specific medium (DMEM 10 FBS 100UmLPenicillin and 100 120583gmL streptomycin) Afterwards mediawas switch to fusion media (DMEM supplemented with2 horse serum) and replaced every second day Controlgroup of ASC cell culture was propagated in fusion media tomonitor spontaneous fusion of cells

23 Lentiviral Particle Production and Transduction of ASCsCommercial available short hairpin RNA (shRNA) con-structs were obtained as bacterial glycerol stocks (Sigma)and used to silence murine IGF-I Plasmid DNA of shRNAand pLVX-Puro-HS-GR vector was purified using a plasmidextraction kit (Plasmid Maxi Kit Qiagen) Lentiviral vectorparticles were produced by three plasmid cotransfectiononto HEK 293T with 40 120583g of shRNA or pLVX-Puro-HS-GR vector DNA 30 120583g pCMV-ΔR891 (The Broad InstituteMA USA) and 10 120583g pMD2G (Addgene clone 12259) usinga calcium-phosphate transfection kit (Invitrogen) Lentivi-ral vector concentration was determined by p24 ELISA

BioMed Research International 3

Table 1 Primer sets for SYBR Green assay

Primer Tm Sequence

Actin 55∘C Sense CAGGTCCAGACGCAGGATGGCAntisense CTACAATGAGCTGCGTGTGGC

MyoD 57∘C Sense GGTCTGGGTTCCCTGTTCTGTGTAntisense CCCCGGCGGCAGAATGGCTACG

GAPDH 67∘C Sense AGCCACATCGCTCAGACACCAntisense GTACTCAGCCGCCAGCATCG

IGF-1 60∘C Sense GCTGGTGGAAGCTCTTCAGTTCAntisense AGCTGACTTGGCAGGCTTGAG

(Cell Biolabs) ASCs were transduced in passage 2 with256 times 105 TUmL virus and polybrene (8 120583gmL) (Chemi-con) After 8 hours the medium was replaced by freshDMEM Twenty-four hours later antibiotic selection (13120583Mpuromycin) was initiated for 10 days following additional4-day recovery ASCs transduced with nontarget shRNA(Sigma) served as negative controls Stable silencing of IGF-Iin ASCs was determined by SYBR Green assay (Biorad) andELISA (RnD System)

24 Apoptosis and Proliferation Assay 1 times 103 C2C12 cellswere seeded in quintuplicates into a 96-well plate and treatedwith myostatin after 24 hours resting period followed byfurther 24 hours incubation Apoptosis of myoblasts aftervarious myostatin concentrations (RnD Systems) was inves-tigated by Cell Death Detection Kit (Roche) according toinstruction manual

In order to quantify proliferation of murine C2C12 undermyostatin treatment cells were seeded at concentration of1 times 103 in each well of the 96-well plate and proliferation wasdetected by a BrdU kit (Roche) according to the manufac-turerrsquos instruction In detail cells were seeded in quintuplicateand allowed to attach for 24 hours followed by myostatinexposure under specific treatment conditions for another24 hours Myostatin was added to conditioned medium atindicated final concentration prior to myoblasts incubationIGF-1 receptors ofmyoblasts were blocked by adding receptorantibodies (Abcam) 30 minutes prior to myostatin incuba-tion IGF-1 receptor and IGF-1 neutralizing antibody (RnDSystem) were simultaneously added to myoblasts in additionto myostatin and incubated for 24 hours

25 Conditioned Medium Conditioned medium of ASCswas used to investigate the effect of ASCs paracrine factorsSerum free DMEM medium containing antibiotics (peni-cillin streptomycin) was added to 90 confluent ASCs andcollected after 48 hThe conditionedmediumwas centrifugedat 500 g for 5 minutes and subsequently filtered by a 022micron filter (Millipore) Fresh prepared CM of wild typeASCs IGF-1 shRNA and nontarget shRNA transfected ASCswere used for further experiments

26 Western Blot C2C12 cells were seeded in order to obtain40 confluent cultures prior to adding serum-reduced (25

FBS) medium or conditioned medium (CM) containing075 120583gmL myostatin Total protein content of cell culturewas extracted after 72 hours of myostatin incubation whenculture reached 70 confluency First C2C12 cells wererinsed twice with PBS and trypsinized followed by centrifu-gation at 500 g for 5 minutesThe cell pellet was washed twicewith ice cold PBS subsequently lysed with fresh preparedRIPA-B-buffer and incubated on ice for 15 minutes followedby centrifugation at 13000 g for 20 minutes at 4∘C Proteinconcentration was measured by using Bradford method (DcProtein Assay kit BioRad) and samples were prepared withLaemmli buffer (BioRad) for SDS-PAGE Gels were loadedwith 90120583g total protein and run for 50minutes at 100VAfter-wards the gel was blotted on a nitrocellulose membrane forone hour at 100V The nitrocellulose membrane was blockedin fresh prepared TBS-T5 milk at room temperature forone hour MyoD primary antibody (Santa Cruz sc-71629)was used at a dilution of 1 200 and 120573-Actin primary antibody(cell signaling number 4967) at a dilution of 1 1000 andincubated at 4∘CovernightThenitrocellulosemembranewaswashed four times for five minutes with TBS-T and subjectedto appropriate secondary antibody Anti-mouse IgG (DyLight800 or 680 Cell Signaling) for one hour at room temperatureThe Infrared Imaging System Odysee (Licor) was used toobtain digital reading of Western blot

27 Real-Time PCR Total RNA was extracted from cell cul-ture using RNAqueous-Micro-Kit according tomanufacturesprotocol and stored at minus80∘C until further investigationsUsing iScript cDNA Synthesis Kit (BioRad) RNA was reversetranscribed into cDNA according to manufacturerrsquos instruc-tions To determine MyoD mRNA expression quantitativeanalyses were performed using Eco qPCR system (illumina)with DyNAmo ColorFlash SYBR Green assay (Biozyme)and appropriate primer set (8 nM final concentration) 120573-Actin and GAPDH primer set served as a housekeeping genereference (Table 1)

28 Statistical Analysis Results are shown as mean plusmn SDwith at least 3 replicates In addition all experiments wereperformed 3 times For pair wise comparison a Studentrsquos t-test was carried out and for group wise comparison one-wayANOVA with Scheffe post hoc correction was carried out by


CD44 9544

(a)

CD 90 9717

(b)

CD 105 9854

(c)

HLA-DR 094

(d)

CD11b 025

(e)

CD14 013

(f)

CD34 092

(g)

CD45 026

(h)

Figure 2 Flowcytometry analyses of ASCs for hematopoietic and endothelial cell markers as well as for mesenchymal cells surface proteins

(a) (b) (c)

(d) (e) (f)

Figure 3 Fusion of DAPI labeled murine myoblasts (blue nucleus) and murine ASCs carrying the pLVX-Puro-HS-GR vector (red cellmembrane green nucleus) monitored by immunofluorescence ((a)ndash(c)) and phase contrast microscopy overlay ((d)ndash(f)) The red cellmembrane surrounding blue and green nuclei indicates a fusion between myoblasts and ASCs (white arrow indicating myotubes generatedby fusion)

using SPSS statistical software package (SPSS Inc IL USA)Values at 119875 lt 005 were considered as statistically significant

3 Results

31 Characterization of ASCs Flowcytometry analyses wereperformed on ASCs for hematopoietic and endothelial cellmarkers as well as for mesenchymal cells surface proteinsASCs were positive for CD44 (9544 plusmn 266) CD90 (9717plusmn 405) and CD105 (9854 plusmn 189) and negative for CD11b(025 plusmn 014) CD14 (013 plusmn 009) CD34 (092 plusmn 151)and CD45 (026 plusmn 030) which preclude contamination by

hematopoietic cells HLA-DR a class II antigen of HLA wasalso negative (094 plusmn 068) for ASCs (Figure 2)

32 Fusion of Murine ASCs and Murine Duchenne MyoblastsDAPI labeled murine Duchenne myoblasts and pLVX-Puro-HS-GR labeledmurine ASCs were cocultured andmonitoredfor myotube formation in order to evaluate whether murineASCs contribute to myotubes by fusion After 3ndash5 days DAPIand GFP labeled nuclei were found to be surrounded by a redcell membrane clearly indicating a fusion of both cell types(Figure 3)


15

05Control00

01

05

075 10

lowastlowast

lowastlowastlowastlowast

lowastlowast

Prol

ifera

tion

of m

yobl

ast (

)

01

Myostatin (120583gmL)

(a)

00

04

06

08

10

02

Control 10

lowastlowast

lowastlowast

lowastlowast

Apop

totic

cells

()

01 05001


(b)

Figure 4 (a) Proliferation of murine myoblasts is shown as percentage after exposure to different myostatin concentrations for 24 hIncreasing concentrations of myostatin were associated with decreased proliferation rate of murine myoblasts Myoblasts cultured with10120583gmL myostatin for 24 h showed lowest proliferation (332) in comparison to 075 120583gmL (520) 05 120583gmL (536) and 01 120583gmL(9087) ( lowast119875 lt 0001 in comparison to control group) (b) Apoptotic effect of myostatin on murine myoblasts is shown as percentage ofapoptotic cellsMurinemyoblasts exposed to 10 120583gmLmyostatin for 24 h showed highest apoptotic rate of 924 in comparison to 05 120583gmL(693) 01 120583gmL (486) and 001 120583gmL (139) ( lowastlowast119875 lt 0001 in comparison to control group)

33 Myostatin Induces Apoptosis onMyoblast Cell and InhibitsMyoblast Proliferation To investigate the apoptotic effectof myostatin on myoblasts increasing concentrations (001ndash10 120583gmL) of murine myostatin were added to murinemyoblasts and incubated for 24 h Addition of 001 120583gmLmyostatin did not show a significant increase in apopto-sis of murine myoblast cells Murine myoblasts exposedto 10 120583gmL myostatin for 24 h showed highest apoptoticrate of 924 in comparison to 05 120583gmL (693) and01 120583gmL (486) (119875 lt 0001 in comparison to controlgroup Figure 4(a)) Proliferation of myoblasts was analyzedafter 24 h myostatin exposure by performing a colorimetricBrdU-based ELISA Addition of various myostatin concen-trations resulted in a significant decrease (119875 lt 0001)of myoblasts proliferation whereas myoblasts cultured with10 120583gmL myostatin showed lowest proliferation (332) incomparison to 075 120583gmL (520) 05 120583gmL (536) and01 120583gmL (9087) (lt0001 in comparison to control groupFigure 4(b))

The concentration of 075120583gmL myostatin was selectedfor subsequent experiments due to significant effects onmyoblasts apoptosis and proliferation

34 IGF-1 Is Accountable for Protective Effect of ASCs Con-ditioned Medium Paracrine factors of ASCs were investi-gated for their effect on myoblastsrsquo proliferation exposed tomyostatinThe proliferation decreased bymyostatin to 548(plusmn32) was significantly abrogated by stem cell conditionedmedium and increased to 764 (plusmn27) (Figure 5(a)) Neu-tralization antibodies against IGF-1 and its correspondingreceptor were simultaneously added to conditioned medium

in order to elucidate the protective role of IGF-1 secretedby ASCs Proliferation of myoblasts decreased from 764(plusmn27) to 658 (plusmn29) after blocking IGF-1 ligand-receptor interaction Thus IGF-1 secreted by ASCs accountsfor sim51 of the protective effect on myoblasts proliferationwhen exposed to myostatin

Furthermore CM of ASCs reduced apoptosis rate ofmyostatin treated myoblasts from 755 (plusmn87) to 228(plusmn42) But after blocking the effect of IGF-1 in conditionedmedium of ASCs the apoptosis rate significantly increased to663 (plusmn73) (119875 lt 0001 Figure 5(b)) Thus 825 of theantiapoptotic effect of the CM is due to IGF-1 secreted byASCs

35 IGF-1 Restores MyoD Expression in Myoblasts MyoDexpression at mRNA and protein level was investigatedto evaluate the influence of IGF-1 on murine myoblasttransdifferentiation First the expression of IGF-1 was sup-pressed by a lentiviral shRNA vector construct in ASCsand efficiency was assessed by quantitative real-time PCR(97) and ELISA (85) ASCs showed a high expressionof IGF-1 protein (32833 plusmn 227 pg120583g DNA) which was notaffected by nontarget shRNA transfection MyoD mRNAexpression in myoblasts treated with 075120583gmL myostatinfor 72 h decreased to 228 (plusmn46) in comparison tountreatedmyoblasts Conditionedmediumofwild typeASCscould partially abolish the negative effect of myostatin sinceMyoD mRNA significantly increased to 512 (plusmn59) Thispositive effect was accountable for IGF-1 by 873 since IGF-1depleted CM decreasedMyoDmRNA level to 264 (plusmn34)(Figure 6)


minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

05

10

15

Prol

ifera

tion

of m

yobl

ast (

)

Myostatin

Conditionedmedium

Anti-IGF-1neutralizing

antibodies

(075120583gmL)

(a)

minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

04

06

08

10

02

Apop

totic

cells

()

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(b)

Figure 5 (a) Effect of conditioned medium onmyoblastsrsquo proliferation is shown as percentage after 48 hmyostatin exposure Proliferation ofmurine myoblast cultured in 075 120583gmL myostatin significantly decreased to 548 (plusmn32 lowastlowast119875 lt 0001) whereas conditioned medium ofASCs significantly increased myoblastsrsquo proliferation under 075120583gmLmyostatin incubation to 764 (plusmn27 lowastlowast119875 lt 0001)This protectiveeffect was abolished after neutralization of IGF-1 ligand-receptor interaction and proliferation significantly decreased to 658 (plusmn29 lowastlowast119875 lt0001) (b) Myoblasts exposed to 075120583gmL myostatin showed a significant increase in apoptosis rate to 755 (plusmn87) but were reducedto 228 (plusmn42) due to paracrine factors of ASCs conditioned medium ( lowastlowast119875 lt 0001) The protective effect of ASCs conditioned mediumwas abolished after blocking IGF-1 and its receptor whereas apoptotic rate increased to 663 (plusmn73) ( lowastlowast119875 lt 0001) Thus IGF-1 secretedby ASCs accounts for 825 of the antiapoptotic effect of the CM (119873 = 3 119899 = 3)

The expression of MyoD protein was examined inmyoblast cells under 075 120583gmL myostatin exposure withand without conditioned medium Control samples clearlyshowed MyoD protein expression whereas protein was nolonger detectable after myostatin treatment But addition ofconditioned medium to myoblasts under myostatin treat-ment demonstrated a weak but distinct detection of MyoDprotein expression (Figure 7)

4 Discussion

The aim of the study was to examine the regenerative poten-tial of ASCs mediated by the secretion of paracrine factorsparticularly IGF-1 for dystrophicmuscle diseases consideringmyostatin as a negative regulator of skeletal muscle mass

Stem cell based therapy provides a promising treatmentoption for DMD since fusion between healthy stem cellsand dystrophic myoblasts restored dystrophin expression invitro and in vivo [12 13] We confirmed previous resultsthat dystrophic myoblasts fuse with ASCs but clearly providethe evidence that nuclei from both cell types are apparentin emerged myotubes The nucleus and the cell membrane

of one cell line were labeled with two different fluorescentproteins in the present study andweremonitored in real timeThis represents a method superior to a single fluorescent celllabeling approach for fusion since currently used dyes (egDAPI Dio Dil DiD quantum dots) provide only transientstaining restricted to specific cell organelles (ie nucleuscytoplasm or mitochondria)

Fusion is a feature of myogenic precursor cells occurringafter activation of muscle satellite cells in order to restoreaffectedmuscle tissue But tremendous destruction of musclecells reduces the capacity of satellite cells regarding pro-liferation and self-renewal [14] Thus application of stemcells provides muscle tissue with a cell population presentingintact self-renewal and proliferation potential More recentlyit has been shown thatmouse adipose tissue stem cells restoredystrophin expression in mdx mice [12] but only at the siteof injection [15] Previous studies and our results provideevidence that ASCs possess the capacity to contribute tomuscle regeneration by gene expression modification afterfusion However the paracrine effects of ASCs have notbeen investigated on dystrophic myoblast as previously formyocardial infarction [11] or hind limb ischemia [16] Themajor finding of this study is that IGF-1 secreted by ASCs


minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

00

05

10

15

Myostatin

Conditionedmedium

shRNA IGF-1

(075120583gmL)

Relat

ive e

xpre

ssio

n of

Myo

D

ns

Figure 6 Relative quantification of MyoD mRNA is shown asmean plusmn SD percentage of MyoD mRNA expression in murinemyoblasts after 72 h myostatin treatment Myostatin exposure sig-nificantly reduced MyoD mRNA to 228 (plusmn46 lowastlowast119875 lt 0001) incomparison to untreatedmyoblasts Due to the effect of conditionedmedium fromwild type ASCsMyoD mRNA significantly increasedto 512 (plusmn59 lowastlowast119875 lt 0001) when compared to myostatintreatment But this effect was accountable for IGF-1 by 873 sinceIGF-1 depleted CM decreased MyoD mRNA level to 264 (plusmn34ns compared to myostatin treatment) (119873 = 3 119899 = 3)

increase MyoD expression in myostatin treated myoblastsFurthermore the paracrine factors of ASCs particularlyIGF-1 provide protective effects on murine myoblasts undermyostatin treatment

IGF-1 plays an essential role in muscle developmentmuscle cell proliferation and muscle growth as alreadyreported for insulin [17 18] due to its structural resemblanceHowever the anabolic function of IGF-1 is based on theinteraction between binding proteins and IGF-1 receptor inan autocrinal and hormonal manner [19]

In contrast TGF-120573 acts as an antagonist of IGF-1 by usingthe same signaling cascade (PI3KAktGSK-3120573 pathway)[20] This is of interest since previous studies indicated anelevated TGF-120573 protein level in dystrophic muscles [7 8]Noteworthy myostatin belongs to the TGF-120573 family andis known as an inhibitor of muscle growth and myoblastsrsquoproliferation [21 22] In line in vitro studies providedevidence that application of recombinant myostatin [23] ormyostatin transfected myoblasts [24] was associated withdecreased proliferation ofmuscle cells Recently it was shownthat dystrophic muscle disease symptoms decreased in mdxmice when treated with myostatin antibodies [9]

These findings indicate that myostatin is involved indegeneration of skeletal muscles in DMD and that IGF-1might be able to improve the inhibitory effect of myostatin onmuscle growth We for the first time could demonstrate thatIGF-1 secreted by ASCs improves proliferation and viabilityof myostatin treated myoblasts In line hypertrophic effects

1 2 3 4

Conditionedmedium

Myostatinminus

minus minus

+ +

+

(075120583gmL)

Figure 7 Representative Western blot analysis of MyoD proteinexpression Nontreated cells clearly showed an expression of MyoDprotein (lane 2) whereas no MyoD protein was detected afterincubation with myostatin (lane 3) After incubation with CM ofASCs a weak but significant expression of MyoD was detectable byWestern blot analyses (lane 4) A total protein amount of 90120583g wasloaded on each lane Green arrow indicates the band of MyoD andred arrow indicates 120573-Actin as loading control (119873 = 3)

on skeletal muscles were demonstrated in vivo for IGF-1overexpressing mouse [25] Moreover muscle hypertrophydue to exercise is mediated by upregulation of IGF-1 mRNA[26] But excessive physical muscle activity is critical forDMD patients and results in muscle fiber damage

Gregorevic et al [27] showed an improvement of musclefatigue in EDL (extensor digitorum longus) and soleus mus-cles in IGF-1 treatedmdxmice IGF-1 is able to activatemusclegrowth and hypertrophy and seems to ameliorate the loss ofmusclemass inDMDHowever growth factors have a limitedhalf-life and are small in size which restricts their retentionwithin a tissue for prolonged periods Moreover systemicadministration of growth factors has shown amodest successor unpleasant side effects in a number of in vivo studieseven when using gene transfer [23ndash26] For this reason cellbased therapy is advantageous since mesenchymal stem cellsproduce antiapoptotic factors at a bioactive level [7]

Activation of muscle differentiation requires myogenicregulatory factors (MRFs) consisting of four transcriptionfactors includingMyoDmyogeninMyf5 andMRF4 [28 29]Of importance quiescent satellite cells express no detectablelevels of MRFs and first express MyoD or Myf-5 afteractivation Regeneration inMyoDminusminusmuscle is characterizedby almost complete abolished proliferation of myogenic cells[30] In line Langley et al [31] provided evidence thatMyoD expression is significantly decreased in myoblasts bymyostatin treatmentMoreoverMyoD can be downregulatedby myostatin treatment even after initial induction due tomyogenic differentiation In addition the study revealedthat myostatin treatment increased Smad3 level that coim-munoprecipitated with MyoD suggesting Smad3 binding toMyoD thereby inhibiting muscle differentiation IGF-1 has


been reported to block Smad3 activation via a phosphatidyli-nositol 3-kinase (PI3K)Akt dependent pathway and therebyinhibiting TGF-120573 signaling [32] Moreover Trendelenburget al reported that myostatin inhibits the activation of Aktsignaling and IGF-1 treatment can counteract myostatinrsquosantidifferentiation effects [33]These previous results supportour findings that IGF-1 secreted by ASCs can restore thenegative effect of myostatin on MyoD expression

5 Conclusion

The present study indicates that IGF-1 secreted by ASCsimproves the catabolic effect of myostatin on myoblasts Inaddition our results support that ASCs might represent avaluable cell source for local application in DMD treatmentdue to their high secretion of bioactive IGF-1 These resultsshow the important paracrine action of adipose tissue derivedstem cells in therapy for muscular dystrophies and musclewasting diseases

Conflict of Interests

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

Acknowledgments

This work was supported by the German Research Foun-dation (DFG) within the funding program Open AccessPublishing The project was funded by the University Hos-pital Regensburg within the ReForM-A-program (SebastianGehmert)

References

[1] D J Blake A Weir S E Newey and K E Davies ldquoFunctionand genetics of dystrophin and dystrophin-related proteins inmusclerdquo Physiological Reviews vol 82 no 2 pp 291ndash329 2002

[2] J R Mendell L R Rodino-Klapac and V Malik ldquoMoleculartherapeutic strategies targetingDuchennemuscular dystrophyrdquoJournal of Child Neurology vol 25 no 9 pp 1145ndash1148 2010

[3] R M Lovering and P G De Deyne ldquoContractile functionsarcolemma integrity and the loss of dystrophin after skeletalmuscle eccentric contraction-induced injuryrdquoAmerican Journalof PhysiologymdashCell Physiology vol 286 no 2 pp C230ndashC2382004

[4] L J Beaton M A Tarnopolsky and S M PhillipsldquoContraction-induced muscle damage in humans followingcalcium channel blocker administrationrdquo Journal of Physiologyvol 544 no 3 pp 849ndash859 2002

[5] J L Croisier G Camus I Venneman et al ldquoEffects oftraining on exercise-induced muscle damage and interleukin 6productionrdquoMuscle amp Nerve vol 22 no 2 pp 208ndash212 1999

[6] E Kamanga-Sollo M S Pampusch M E White and W RDayton ldquoRole of insulin-like growth factor binding protein(IGFBP)-3 in TGF-beta- and GDF-8 (myostatin)-induced sup-pression of proliferation in porcine embryonic myogenic cellculturesrdquo Journal of Cellular Physiology vol 197 no 2 pp 225ndash231 2003

[7] J N Haslett D Sanoudou A T Kho et al ldquoGene expressioncomparison of biopsies from Duchenne muscular dystrophy(DMD) and normal skeletal musclerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no23 pp 15000ndash15005 2002

[8] L Passerini P Bernasconi F Baggi et al ldquoFibrogenic cytokinesand extent of fibrosis in muscle of dogs with X-linked goldenretrievermuscular dystrophyrdquoNeuromuscularDisorders vol 12no 9 pp 828ndash835 2002

[9] S Bogdanovich T O B Krag E R Barton et al ldquoFunctionalimprovement of dystrophic muscle by myostatin blockaderdquoNature vol 420 no 6914 pp 418ndash421 2002

[10] P Gregorevic D R Plant K S Leeding L A Bach and G SLynch ldquoImproved contractile function of the mdx dystrophicmouse diaphragm muscle after insulin-like growth factor-Iadministrationrdquo American Journal of Pathology vol 161 no 6pp 2263ndash2272 2002

[11] S Sadat S Gehmert Y-H Song et al ldquoThe cardioprotectiveeffect of mesenchymal stem cells is mediated by IGF-I andVEGFrdquo Biochemical and Biophysical Research Communicationsvol 363 no 3 pp 674ndash679 2007

[12] G Di Rocco M G Iachininoto A Tritarelli et al ldquoMyogenicpotential of adipose-tissue-derived cellsrdquo Journal of Cell Sciencevol 119 no 14 pp 2945ndash2952 2006

[13] N M Vieira V Brandalise E Zucconi et al ldquoHuman multipo-tent adipose-derived stem cells restore dystrophin expression ofDuchenne skeletal-muscle cells in vitrordquo Biology of the Cell vol100 no 4 pp 231ndash241 2008

[14] L Heslop J E Morgan and T A Partridge ldquoEvidence for amyogenic stem cell that is exhausted in dystrophic musclerdquoJournal of Cell Science vol 113 part 12 pp 2299ndash2308 2000

[15] A-M Rodriguez D Pisani C A Dechesne et al ldquoTransplanta-tion of amultipotent cell population fromhuman adipose tissueinduces dystrophin expression in the immunocompetent mdxmouserdquo Journal of Experimental Medicine vol 201 no 9 pp1397ndash1405 2005

[16] J Rehman D Traktuev J Li et al ldquoSecretion of angiogenicand antiapoptotic factors by human adipose stromal cells rdquoCirculation vol 109 no 10 pp 1292ndash1298 2004

[17] C-J Xiong P-F Li Y-L Song et al ldquoInsulin induces C2C12 cellproliferation and apoptosis through regulation of cyclin D1 andBAD expressionrdquo Journal of Cellular Biochemistry vol 114 no12 pp 2708ndash2717 2013

[18] W-Y Li Y-L Song C-J Xiong et al ldquoInsulin induces prolif-eration and cardiac differentiation of P19CL6 cells in a dose-dependent mannerrdquo Development Growth amp Differentiationvol 55 no 7 pp 676ndash686 2013

[19] J R Florini D Z Ewton and S A Coolican ldquoGrowth hormoneand the insulin-like growth factor system in myogenesisrdquoEndocrine Reviews vol 17 no 5 pp 481ndash517 1996

[20] W Yang Y Zhang Y Li Z Wu and D Zhu ldquoMyostatininduces cyclin D1 degradation to cause cell cycle arrest througha phosphatidylinositol 3-kinaseAKTGSK-3120573 pathway and isantagonized by insulin-like growth factorrdquo Journal of BiologicalChemistry vol 282 no 6 pp 3799ndash3808 2007

[21] A C McPherron A M Lawler and S-J Lee ldquoRegulation ofskeletal muscle mass in mice by a new TGF-120573 superfamilymemberrdquo Nature vol 387 no 6628 pp 83ndash90 1997

[22] W E Taylor S Bhasin J Artaza et al ldquoMyostatin inhibitscell proliferation and protein synthesis in C2C12 musclecellsrdquo American Journal of PhysiologymdashEndocrinology andMetabolism vol 280 no 2 pp E221ndashE228 2001


[23] M Thomas B Langley C Berry et al ldquoMyostatin a negativeregulator of muscle growth functions by inhibiting myoblastproliferationrdquo Journal of Biological Chemistry vol 275 no 51pp 40235ndash40243 2000

[24] R Rıos I Carneiro V M Arce and J Devesa ldquoMyostatinregulates cell survival during C2C12 myogenesisrdquo Biochemicaland Biophysical Research Communications vol 280 no 2 pp561ndash566 2001

[25] M E Coleman F DeMayo K C Yin et al ldquoMyogenic vectorexpression of insulin-like growth factor I stimulates muscle celldifferentiation and myofiber hypertrophy in transgenic micerdquoJournal of Biological Chemistry vol 270 no 20 pp 12109ndash121161995

[26] D L DeVol P Rotwein J L Sadow J Novakofski and P JBechtel ldquoActivation of insulin-like growth factor gene expres-sion during work-induced skeletal muscle growthrdquo AmericanJournal of PhysiologymdashEndocrinology and Metabolism vol 259no 1 pp E89ndashE95 1990

[27] P Gregorevic D R Plant and G S Lynch ldquoAdministrationof insulin-like growth factor-I improves fatigue resistance ofskeletal muscles from dystrophic mdx micerdquoMuscle and Nervevol 30 no 3 pp 295ndash304 2004

[28] J D Molkentin and E N Olson ldquoCombinatorial control ofmuscle development by basic helix-loop-helix and MADS-boxtranscription factorsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 93 no 18 pp 9366ndash9373 1996

[29] K Yun and B Wold ldquoSkeletal muscle determination anddifferentiation story of a core regulatory network and itscontextrdquo Current Opinion in Cell Biology vol 8 no 6 pp 877ndash889 1996

[30] L A Megeney B Kablar K Garrett J E Anderson and M ARudnicki ldquoMyoD is required for myogenic stem cell functionin adult skeletal musclerdquoGenes and Development vol 10 no 10pp 1173ndash1183 1996

[31] B Langley M Thomas A Bishop M Sharma S Gilmourand R Kambadur ldquoMyostatin inhibits myoblast differentiationby down-regulating MyoD expressionrdquo Journal of BiologicalChemistry vol 277 no 51 pp 49831ndash49840 2002

[32] K Song H Wang T L Krebs and D Danielpour ldquoNovelroles of Akt and mTOR in suppressing TGF-120573ALK5-mediatedSmad3 activationrdquoEMBO Journal vol 25 no 1 pp 58ndash69 2006

[33] A U Trendelenburg A Meyer D Rohner J Boyle SHatakeyama and D J Glass ldquoMyostatin reduces AktTORC1p70S6K signaling inhibiting myoblast differentiation andmyotube sizerdquoAmerican Journal of PhysiologymdashCell Physiologyvol 296 no 6 pp C1258ndashC1270 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


H2B EGFP 2A SS mCherry GPI pA

HS-GR cassette

WPREcPPTRRE

pLVX-Puro-HS-GR

PCMV PPGKΨ Puror3998400LTR5998400LTR

Figure 1 The pLVX-Puro-HS-GR vector was constructed by cloning HS-GR cassette into CMV driven lentiviral backbone (Clontech)betweenXhoI andXbaI restriction sites H2B histone protein sequence 2A self-cleavage peptide SS endoplasmatic reticulum signal peptideGPI C terminal GPI anchor sequence pA bovine growth hormone polyadenylation signal

this protein did not appear in the exercise training analysisfor normal muscle tissue [7 8] Especially myostatin a newmember of the TGF-120573 superfamily has been reported tobe a negative regulator of skeletal muscle growth [9] Thiswould suggest a role of TGF-120573 in the degeneration of skeletalmuscles in DMD (eg disruption of IGF-related signaling)Indeed application of IGF-1 attenuates the deterioration ofskeletal muscles mass in a murine Duchenne (mdx) model[4 10]

Previous studies demonstrated beneficial effects of IGF-1 produced by mesenchymal stem cells particularly by adi-pose tissue-derived stem cells (ASCs) which can be easilyharvested and expanded in vitro [11] Thus this study wasperformed to investigate whether paracrine factors secretedby ASCs are able to stimulate proliferation of myoblastsand ameliorate myostatin effects on myoblasts Moreover wewanted to elucidate the role of IGF-1 and its protective rolefor myoblasts when exposed to an elevated myostatin level

2 Materials and Methods

21 Cell Culture Adipose tissue was harvested from theinguinal fat pads of five male BALBc mice (Jackson Lab-oratory) at the age of 8ndash12 weeks and pooled for furtherprocessingThe tissuewaswashed twice with prewarmed PBSprior to mincing Two units of LiberaseMNP-S (Roche) weremixed in 1mLprewarmedDulbeccorsquosmodified eaglemedium(DMEM) (Invitrogen) per gram fat and incubated at 37∘C225 rpm shaking for 30 minutes followed by mixing with apipette to release cells Afterwards mixture was centrifugedat 500 g for 10min and cell pellet was washed three times withPBSThe resulting cell pelletwas resuspended andpropagatedin complete ASCs culture medium (DMEM) supplementedwith 20 fetal bovine serum (FBS PAN-Biotech) 100UmLPenicillin (Sigma) and 100 120583gmL streptomycin (Sigma) andincubated at 37∘C in a humidified atmosphere containing 5CO2 C2C12 cells (ATCC) were cultured in growth medium

consisting of DMEM 10 FBS 100UmL Penicillin and100 120583gmL streptomycin Myoblasts were subcultured beforereaching 80 confluency and used for all experiments beforepassage 7

For lentiviral production human embryonic kidney 293Tcells (HEK293T ATCC) were grown in DMEM containing10 FBS and used in passages below 20

22 Real-Time Fusion Assay A plasmid containing H2B-EGFP-2A-mCherry-GPI (HS-GR) was generously providedby Dr Richard Beheringer and Chuan-Wei Jang (MDAnder-son Cancer Center) A lentiviral construct was generated bysubcloning the HS-GR cassette into the pLVX-Puro vector(Clontech) between Xhol and Xbal by restriction digest andligation (Figure 1) The integrity of cloned HS-GR cassettewas confirmed by DNA sequencing and resulting vector wasnamed pLVX-Puro-HS-GR

ASCs were stable transfected by lentiviral infection asdescribed below DAPI dye was added to myoblast cellculture at a final concentration of 50120583gmL in 10 FBSsupplemented DMEM and incubated for 30 minutes DAPIlabeled myoblasts and pLVX-Puro-HS-GR expressing ASCscultured in 20 FBS supplemented DMEM were rinsed sixtimes with PBS and trypsinized for coculture fusion assay8 times 104 DAPI labeled C2C12 and 2 times 104 pLVX-Puro-HS-GR labeled ASCs were mixed and placed in a 6-well plate andincubated until cell culture reached 100 confluency usingmyoblast specific medium (DMEM 10 FBS 100UmLPenicillin and 100 120583gmL streptomycin) Afterwards mediawas switch to fusion media (DMEM supplemented with2 horse serum) and replaced every second day Controlgroup of ASC cell culture was propagated in fusion media tomonitor spontaneous fusion of cells

23 Lentiviral Particle Production and Transduction of ASCsCommercial available short hairpin RNA (shRNA) con-structs were obtained as bacterial glycerol stocks (Sigma)and used to silence murine IGF-I Plasmid DNA of shRNAand pLVX-Puro-HS-GR vector was purified using a plasmidextraction kit (Plasmid Maxi Kit Qiagen) Lentiviral vectorparticles were produced by three plasmid cotransfectiononto HEK 293T with 40 120583g of shRNA or pLVX-Puro-HS-GR vector DNA 30 120583g pCMV-ΔR891 (The Broad InstituteMA USA) and 10 120583g pMD2G (Addgene clone 12259) usinga calcium-phosphate transfection kit (Invitrogen) Lentivi-ral vector concentration was determined by p24 ELISA



Primer Tm Sequence














CD44 9544

(a)

CD 90 9717

(b)

CD 105 9854

(c)

HLA-DR 094

(d)

CD11b 025

(e)

CD14 013

(f)

CD34 092

(g)

CD45 026

(h)


(a) (b) (c)

(d) (e) (f)



3 Results





15

05Control00

01

05

075 10

lowastlowast


lowastlowast

Prol

ifera

tion

of m

yobl

ast (

)

01


(a)

00

04

06

08

10

02

Control 10

lowastlowast

lowastlowast

lowastlowast

Apop

totic

cells

()

01 05001


(b)









minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

05

10

15

Prol

ifera

tion

of m

yobl

ast (

)

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(a)

minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

04

06

08

10

02

Apop

totic

cells

()

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(b)



4 Discussion






minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

00

05

10

15

Myostatin

Conditionedmedium

shRNA IGF-1

(075120583gmL)

Relat

ive e

xpre

ssio

n of

Myo

D

ns






1 2 3 4

Conditionedmedium

Myostatinminus

minus minus

+ +

+

(075120583gmL)







5 Conclusion




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Primer Tm Sequence














CD44 9544

(a)

CD 90 9717

(b)

CD 105 9854

(c)

HLA-DR 094

(d)

CD11b 025

(e)

CD14 013

(f)

CD34 092

(g)

CD45 026

(h)


(a) (b) (c)

(d) (e) (f)



3 Results





15

05Control00

01

05

075 10

lowastlowast


lowastlowast

Prol

ifera

tion

of m

yobl

ast (

)

01


(a)

00

04

06

08

10

02

Control 10

lowastlowast

lowastlowast

lowastlowast

Apop

totic

cells

()

01 05001


(b)









minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

05

10

15

Prol

ifera

tion

of m

yobl

ast (

)

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(a)

minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

04

06

08

10

02

Apop

totic

cells

()

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(b)



4 Discussion






minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

00

05

10

15

Myostatin

Conditionedmedium

shRNA IGF-1

(075120583gmL)

Relat

ive e

xpre

ssio

n of

Myo

D

ns






1 2 3 4

Conditionedmedium

Myostatinminus

minus minus

+ +

+

(075120583gmL)







5 Conclusion




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


CD44 9544

(a)

CD 90 9717

(b)

CD 105 9854

(c)

HLA-DR 094

(d)

CD11b 025

(e)

CD14 013

(f)

CD34 092

(g)

CD45 026

(h)


(a) (b) (c)

(d) (e) (f)



3 Results





15

05Control00

01

05

075 10

lowastlowast


lowastlowast

Prol

ifera

tion

of m

yobl

ast (

)

01


(a)

00

04

06

08

10

02

Control 10

lowastlowast

lowastlowast

lowastlowast

Apop

totic

cells

()

01 05001


(b)









minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

05

10

15

Prol

ifera

tion

of m

yobl

ast (

)

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(a)

minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

04

06

08

10

02

Apop

totic

cells

()

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(b)



4 Discussion






minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

00

05

10

15

Myostatin

Conditionedmedium

shRNA IGF-1

(075120583gmL)

Relat

ive e

xpre

ssio

n of

Myo

D

ns






1 2 3 4

Conditionedmedium

Myostatinminus

minus minus

+ +

+

(075120583gmL)







5 Conclusion




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


15

05Control00

01

05

075 10

lowastlowast


lowastlowast

Prol

ifera

tion

of m

yobl

ast (

)

01


(a)

00

04

06

08

10

02

Control 10

lowastlowast

lowastlowast

lowastlowast

Apop

totic

cells

()

01 05001


(b)









minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

05

10

15

Prol

ifera

tion

of m

yobl

ast (

)

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(a)

minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

04

06

08

10

02

Apop

totic

cells

()

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(b)



4 Discussion






minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

00

05

10

15

Myostatin

Conditionedmedium

shRNA IGF-1

(075120583gmL)

Relat

ive e

xpre

ssio

n of

Myo

D

ns






1 2 3 4

Conditionedmedium

Myostatinminus

minus minus

+ +

+

(075120583gmL)







5 Conclusion




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

05

10

15

Prol

ifera

tion

of m

yobl

ast (

)

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(a)

minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

lowastlowast

00

04

06

08

10

02

Apop

totic

cells

()

Myostatin

Conditionedmedium


antibodies

(075120583gmL)

(b)



4 Discussion






minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

00

05

10

15

Myostatin

Conditionedmedium

shRNA IGF-1

(075120583gmL)

Relat

ive e

xpre

ssio

n of

Myo

D

ns






1 2 3 4

Conditionedmedium

Myostatinminus

minus minus

+ +

+

(075120583gmL)







5 Conclusion




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


minus

minus

minus

minus

minus

+ +

+

+minus

+

+

lowastlowast

lowastlowast

00

05

10

15

Myostatin

Conditionedmedium

shRNA IGF-1

(075120583gmL)

Relat

ive e

xpre

ssio

n of

Myo

D

ns






1 2 3 4

Conditionedmedium

Myostatinminus

minus minus

+ +

+

(075120583gmL)







5 Conclusion




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusion




Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article adipose tissue-derived stem cell...

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