TGF-β1 enhances cardiomyogenic differentiation of skeletal muscle-derived adult primitive cells

Ahmed Abdel-LatifEwa K. Zuba-SurmaJamie CaseSumit TiwariGreg HuntSmita RanjanRobert J. VincentEdward F. SrourRoberto BolliBuddhadeb Dawn

TGF-b1 enhances cardiomyogenicdifferentiation of skeletal muscle-derivedadult primitive cells

Received: 22 December 2007Returned for 1. Revision: 8 January 20081. Revision received: 8 April 2008Accepted: 22 April 2008Published online: 23 May 2008

j Abstract The optimal medium for cardiac differentiation of adultprimitive cells remains to be established. We quantitatively compared theefficacy of IGF-1, dynorphin B, insulin, oxytocin, bFGF, and TGF-b1 ininducing cardiomyogenic differentiation. Adult mouse skeletal muscle-derived Sca1+/CD45-/c-kit-/Thy-1+ (SM+) and Sca1-/CD45-/c-kit-/Thy-1+ (SM-) cells were cultured in basic medium (BM; DMEM, FBS, IGF-1,dynorphin B) alone and BM supplemented with insulin, oxytocin, bFGF,or TGF-b1. Cardiac differentiation was evaluated by the expression ofcardiac-specific markers at the mRNA (qRT-PCR) and protein (immu-nocytochemistry) levels. BM+TGF-b1 upregulated mRNA expression ofNkx2.5 and GATA-4 after 4 days and Myl2 after 9 days. After 30 days,BM+TGF-b1 induced the greatest extent of cardiac differentiation (bymorphology and expression of cardiac markers) in SM- cells. Weconclude that TGF-b1 enhances cardiomyogenic differentiation inskeletal muscle-derived adult primitive cells. This strategy may beutilized to induce cardiac differentiation as well as to examine thecardiomyogenic potential of adult tissue-derived stem/progenitor cells.

j Key words adult stem cell – cardiac differentiation –culture medium – skeletal muscle – growth factor

ORIGINAL CONTRIBUTIONBasic Res Cardiol 103:514–524 (2008)DOI 10.1007/s00395-008-0729-9

BR

C72

9

Ahmed Abdel-Latif and Ewa K. Zuba-Sur-ma contributed equally to this work.

Electronic supplementary material: Theonline version of this article (doi:10.1007/s00395-008-0729-9) contains supplemen-tary material, which is available to autho-rized users.

A. Abdel-Latif, MDE.K. Zuba-Surma, PhD Æ S. Tiwari, MDG. Hunt, BS Æ S. Ranjan, BSR.J. Vincent, BS Æ R. Bolli, MDB. Dawn, MD (&)Division of CardiologyUniversity of Louisville550 S. Jackson St., ACB, 3rd floorLouisville (KY) 40292, USATel.: +1-502/852-7959Fax: +1-502/852-7147E-Mail: [email protected]

J. Case, PhDDept. of MedicineHerman B Wells Center forPediatric ResearchIndiana University School of MedicineIndianapolis (IN), USA

E.F. Srour, PhDDept. of Microbiology/ImmunologyHerman B Wells Center forPediatric ResearchIndiana University School of MedicineIndianapolis (IN), USA

Introduction

Results from numerous studies in animals indicatethat therapy with adult stem/progenitor cells canameliorate left ventricular (LV) remodeling and im-prove LV function after myocardial infarction [9, 18,25]. Based on this phenomenological evidence, severalclinical trials of cell therapy for cardiac repair havebeen completed or are in progress [1]. Althoughcontroversial, differentiation of transplanted cells intocells of cardiac lineages has been proposed as one ofthe mechanisms underlying these beneficial effects.Therefore, it is crucial to determine the cardiomyo-genic potential of transplanted cells in vitro prior totransplantation in vivo. In this regard, it is importantto formulate culture conditions that can predictablyinduce cardiomyocytic differentiation in candidatecells for cardiac repair in vivo. However, the impact ofdifferent growth factors/cytokines on the cardiomy-ogenic differentiation of adult stem/progenitors invitro has not been systematically investigated.

We have previously reported that specific subsetsof adult skeletal muscle-derived primitive cells canundergo cardiomyocytic differentiation in vitro [35].In this study, we quantitatively examined the ability ofsix specific growth factors/cytokines in five combi-nations to induce cardiomyocytic differentiation intwo subsets (Sca-1+/CD45-/c-kit-/Thy-1+ [SM+] cellsand Sca-1-/CD45-/c-kit-/Thy-1+ [SM-] cells) of skel-etal muscle-derived adult primitive cells in vitro.These factors (insulin-like growth factor [IGF]-1 [5],dynorphin B [32], insulin [29], oxytocin [22], basicfibroblast growth factor [bFGF] [28], and transform-ing growth [TGF]-b1 [6, 21] were selected because oftheir known involvement in cardiac development and/or their ability to induce cardiac differentiation invarious stem cell lines. To our knowledge, this is thefirst systematic comparison of the ability of differentgrowth factors and cytokines to induce cardiomyo-cytic differentiation in adult primitive cells in vitro ina controlled setting.

Materials and methods

The investigation conforms to the principles of lab-oratory animal care according to the Guide for theCare and Use of Laboratory Animals (Department ofHealth and Human Services, NIH Publication No. 86-23, revised 1996).

j Animals

Adult C57BL/6 mice (age 6–10 weeks, body wt 20–25 g) were used. Mice were bred and maintained at

Indiana University according to protocols approvedby the laboratory animal research facility (LARC) ofthe Indiana University School of Medicine. Eachexperimental group of donor C57BL/6 mice was ob-tained from the same litter.

j Isolation of skeletal muscle-derived primitive cells

Cells were isolated according to previously publishedmethods [35]. Briefly, following euthanasia, the hindlimbs were removed and placed in Dulbecco’s Modi-fied Eagle Medium (DMEM, Invitrogen). Adiposetissue and large vasculature were removed carefullywith fine forceps and discarded. Skeletal muscle(sartorius, quadriceps, adductors, soleus, and gas-trocnemius muscles) was carefully dissected, excisedfrom tendons, and placed in 15 ml of fresh medium.Muscle tissue was triturated by repeated gentlepipetting and centrifuged at 500 rpm for 10 min. Thepelleted muscle tissue was digested in collagenase I(220 U/ml, Worthington Biochemical) and Dispase(33 U/ml, BD Biosciences) in 50 ml of DMEM for45 min at 37�C with gentle agitation [35]. Cells werethen filtered through a 40-lm cell strainer into asterile 50-ml conical tube. Viability was assessed byTrypan blue (Sigma) staining. Approximately 4 · 106

cells were obtained from each donor mouse.

j Flow cytometric cell sorting

Freshly isolated cells were pelleted in PBS containing1% FBS (HyClone), and FITC-conjugated monoclonalantimouse Sca-1 (E13–161.7, BD Pharmingen), allo-phycocyanin (APC)-conjugated monoclonal anti-mouse CD45 and c-kit (30-F11 and 2B8, respectively,BD Pharmingen), and biotin-conjugated antimouseThy-1.2 (53-2.1, BD Pharmingen) primary antibodieswere added simultaneously. PE-conjugated streptavi-din (Caltag Laboratories) was added subsequently. Allstaining was performed at 4�C for 20 min, and cellswere washed with PBS supplemented with 1% FBS afterstaining. Flow cytometric cell sorting was performedusing a MoFlo (Dako) and a FACS Vantage SE [35]. Thesorting strategy is summarized in Fig. 1. Sorted cellswere collected into tubes containing 2 ml DMEM with10% FBS. Isolated cells were reanalyzed immediatelyafter sorting to determine purity of sorting. The via-bility of sorted cells always exceeded 90%.

j Cell culture

Freshly isolated SM+ and SM) cells were seeded at5,000 cells/plate in gelatin-coated 22-mm diameterplates. The basic medium (BM) consisted of DMEM

A. Abdel-Latif et al. 515TGF-b1 and cardiac differentiation

supplemented with 10% FBS, IGF-1 (25 nM [2]; R&DResearch), and dynorphin B (1 lM [32]; NeoMPS).IGF-1 and dynorphin B, both of which have beenshown to induce cardiac differentiation [2, 32], wereadded to the BM to synergistically augment theoverall cardiomyogenic effects of these media. Todetermine the impact of specific supplements oncardiomyogenic differentiation, cells were cultured inBM alone and BM supplemented with insulin (10 lg/ml [3]; Sigma), oxytocin (0.1 lM [22]; Becham Cal-ifornia), bFGF (20 ng/ml [23, 30]; R&D Research), orTGF-b1 (2.5 ng/ml [6]; R&D Research). These con-centrations were based on our careful review of theliterature. We specifically avoided using a higherdose of TGF-b1 to obviate the induction of osteo-blastic features [14]. Half of the medium was chan-ged twice a week and cells were allowed todifferentiate for 30 days.

j Phenotypic analysis

Cells in each of 10 groups were evaluated on a dailybasis under light microscopy and images were ac-quired using a digital camera (MRC5, Carl Zeiss, Inc.,Thornwood, NY) and the Axiovision software (CarlZeiss) to document changes in cellular morphology.

j Assessment of mRNA expression by real-timeqRT-PCR

For the analysis of mRNA expression of markers ofcardiac differentiation (Nkx2.5, GATA-4, and Myl2),total mRNA was isolated from freshly isolated cellsSM+ and SM) cells and after 4 and 9 days of culture(RNeasy, Qiagen), and reverse-transcribed (TaqMan,Applied Biosystems). Quantitative assessment of

mRNA expression of all of the genes of interest andb2-microglobulin was performed by real-time qRT-PCR using an ABI PRISM� 7000 System [35].

All of the primer sequences are provided in sup-plemental Table 1. Primers were designed with thePrimer Express software. A 25-ll reaction mixturecontaining 12.5 ll of SYBR Green PCR Master Mixand 10 ng of forward and reverse primers was used.The threshold cycle (Ct), i.e., the cycle number atwhich the amount of amplified gene of interestreached a fixed threshold, was subsequently deter-mined. Relative quantitation of mRNA expression wascalculated with the comparative Ct method. The rel-ative quantitative value of target, normalized to anendogenous control b2-microglobulin gene and rela-tive to a calibrator, was expressed as 2)DDCt (-folddifference), where DCt = (Ct of target genes [Nkx2.5,GATA-4, and Myl2]) ) (Ct of endogenous controlgene [b2-microglobulin]), and DDCt = (DCt of sam-ples for target gene) ) (DCt of calibrator for the targetgene). To avoid the possibility of amplifying con-taminating DNA (1) all of the primers for real-timeRT-PCR were designed to contain an intron sequencefor specific cDNA amplification; (2) reactions wereperformed with appropriate negative controls (tem-plate-free controls); (3) a uniform amplification of theproducts was rechecked by analyzing the meltingcurves of the amplified products (dissociationgraphs); and (4) the melting temperature (Tm) was57–60�C with the probe Tm at least 10�C higher thanthe primer Tm [35]. Three independent experimentswere performed for each set of genes.

j Immunocytochemistry

The expression of cardiac-specific transcription fac-tors (cTFs) and structural proteins (cSPs) was exam-

R531.0%

R450.7%

R214.0%

R34.3%

Thy-1.2-PE

CD45

/c-k

it-AP

C

R1

FSC

SSC

R76.5%

R619.2%

Coun

ts

Sca-1-FITC

256 104

103

102

101

100

100 101 102 103 104 100 101 102 103 104

192

64

128

00 64 128 192 256

105

78

52

26

0

Fig. 1 Representative dot-plots show the expression (in percentage) of Sca-1(FITC), CD45 (APC), c-kit (APC), and Thy-1.2 (PE) in skeletal muscle-derived cells.Blue lines indicate regions included into the sorting logic. From region 1 (R1),CD45-/c-kit- cells positive for Thy-1.2 are identified in region 5 (R5). Based on

Sca-1 expression, cells from R5 are divided into Sca-1+/CD45-/c-kit-/Thy-1+(SM+) cells in region 7 (R7) and Sca-1-/CD45-/c-kit-/Thy-1+ (SM-) cells inregion 6 (R6). The percentages in R6 and R7 relate to the total number of cellsin R1. FSC forward scatter characteristics, SSC side scatter characteristics

516 Basic Research in Cardiology, Vol. 103, No. 6 (2008)� Steinkopff Verlag 2008

ined by immunocytochemical staining after 30 daysof differentiation [35]. Briefly, cells were fixed (4%paraformaldehyde), permeabilized (0.2% Triton X-100), blocked (10% donkey serum) and incubatedwith primary antibodies against Nkx2.5 (Santa Cruz),GATA-4 (Santa Cruz), cardiac myosin heavy chain(cMyHC, Novus Biologicals), a-sarcomeric actin(Sigma), and a panel of primary antibodies againstspecific markers of noncardiac lineages (as describedbelow) at a concentration of 1:100–1:200 for 16 h at4�C. Following washing with PBS, appropriate fluo-rochrome-conjugated secondary antibodies (JacksonImmunoresearch) were added at a concentration of1:100. Following staining with DAPI, cells were wa-shed with PBS and a coverslip was applied formicroscopic examination. Appropriate negative andspecific positive controls were employed for eachantigen to ensure the specificity of staining.

j Quantitative assessment of cardiomyocyticdifferentiation

All images were acquired using a LSM 510 (Zeiss)confocal microscope. For quantitative assessment of theexpression of cardiac-specific antigens, 500 cells/plateon average were systematically evaluated in multiplefields from all four quarters of the plate [35]. As previ-ously reported [12, 33, 35], cardiac-specific transcrip-tion factors (Nkx2.5 [12, 35] and GATA-4 [33, 35]) werenoted both in cytoplasm and in the nucleus. Cells withnuclear staining with or without cytoplasmic stainingwere counted as positives. For the purpose of exclusion,we also stained cells for markers specific for severalnoncardiac lineages (skeletal muscle [myogenin andMyf5]; neuron [nestin and GFAP]; fibroblast [PDGFR-aand -b]; chondrocyte [collagens I and II]; epithelial cell[cytokeratin 17]; macrophage [CD68]; and others) [35].This rigorous approach enabled us to specificallyidentify cells with a cardiomyocytic phenotype. Cellsthat were positive for cardiac-specific markers andnegative for noncardiac lineage markers were countedas positive, and the total number of cells in each fieldwas calculated based on nuclear staining with DAPI.The percentage of cells differentiating into cardiomyo-cytes was calculated as the number of positive cells di-vided by the total number of nuclei in the field [35].These experiments were performed in triplicate.

j Statistical analysis

Data are mean ± SEM. The percentage of cells with acardiac phenotype and quantitative mRNA data (-foldchanges in mRNA levels) for cardiac-specific markerswas analyzed with one-way or two-way (time and

group) ANOVA. If the ANOVA showed an overalldifference, post hoc contrasts were performed withStudent’s t tests for unpaired data, and the resultingprobability values were adjusted according to theBonferroni correction (SPSS). A P < 0.05 was con-sidered statistically significant.

Results

j Isolated skeletal muscle-derived primitive cells

Skeletal muscle-derived primitive cells were isolatedby FACS. Based on the expression of Sca-1, CD45,c-kit, and Thy-1, cells were sorted into Sca-1+/CD45-/c-kit-/Thy-1+ (SM+) and Sca-1-/CD45-/c-kit-/Thy-1+(SM-) populations according to the protocol sum-marized in Fig. 1.

j Impact of media composition on cell morphologyduring culture

Within the first 48 h after plating, both SM+ and SM-cells adhered to the gelatin-coated surface. Duringculture, SM+ cells cultured in all five different mediaexhibited greater morphological heterogeneity com-pared with SM- cells (Fig. 2a–e). During the 2nd–4thweeks, SM+ cells cultured in BM alone graduallydifferentiated into a combination of large flat cellswith epithelial morphology and positive immuno-staining for cytokeratin 17 (Fig. 2f), isolated spindle-shaped cells, oval cells with endothelial morphology,and scattered cellular aggregates with an adipocyticphenotype (Fig. 2a). Addition of insulin (Fig. 2b) oroxytocin (Fig. 2c) to the BM resulted in markedlygreater differentiation into adipocytes. AlthoughBM+bFGF (Fig. 2d) and BM+TGF-b1 (Fig. 2e)induced differentiation into varied phenotypes, thefrequency of spindle-shaped cells was less comparedwith SM- cells in respective media. In addition, SM+cells cultured in BM+bFGF (Fig. 2d) andBM+TGF)b1 (Fig. 2e) exhibited greater predisposi-tion to differentiate into myotubes (Fig. 2g–j).Overall, SM+ cells exhibited greater propensity todifferentiate into an adipose phenotype in all media.

Compared with SM+ cells, SM) cells cultured in allfive media combinations exhibited greater morpho-logical homogeneity (Fig. 3a–e). SM- cells cultured inBM alone differentiated into cells with epithelial andendothelial phenotypes with isolated spindle-shapedcells (Fig. 3a). SM- cells in BM+insulin (Fig. 3b) andBM+oxytocin (Fig. 3c) differentiated into similarcombinatorial phenotypes with predominant epithe-lial cells. In contrast, a greater fraction of SM- cells


cultured in BM+bFGF (Fig. 3d) and BM+TGF)b1(Fig. 3e) showed predominantly spindle-shapedmorphology. However, a fraction of these SM- cellscultured in BM+bFGF gradually acquired the pheno-type characteristic of myotubes, while the mononu-cleate spindle-shaped SM- cells in BM+TGF)b1predominantly remained isolated with formation ofoccasional small clusters. Spontaneous rhythmiccontractions were observed in both single cells andclusters of small cells in BM+TGF-b1 medium.

j Analysis of mRNA expression of cardiac markers

The expression of Nkx2.5 was greater in all media at4 days of culture as compared with 9 days, and greaterin SM- cells compared with SM+ cells (Fig. 4). In SM-cells, the expression of Nkx2.5 was greatest in cellscultured in BM+TGF)b1, and to a lesser extent in BMalone and BM+bFGF (Fig. 4). The expression ofGATA-4 followed a similar pattern except that thegreatest expression was in SM- cells cultured in

Fig. 2 Representative light microscopic images of SM+ cells after 30 days ofdifferentiation demonstrating the effects of culture media composition oncellular morphology. Panels a-c show the heterogeneous (epithelial,endothelial, adipose, and others) morphology of SM+ cells cultured in basicmedium (BM) containing IGF-1 and dynorphin B (a), and BM supplementedwith insulin (b), or oxytocin (c). Exposure of SM+ cells to insulin or oxytocinincreased differentiation into an adipose phenotype. Supplementation of BMwith bFGF (d) or TGF-b1 (e) resulted in enhanced myotube formation fromSM+ cells. Morphological observations were confirmed via immunostaining (f-j). Panel f shows differentiation of SM+ cells cultured in BM supplemented with

insulin into epithelial cells positive for the epithelial cytoskeletal markercytokeratin 17 (red). Panels g–j show representative images of SM+ cellsstained for the cardiac-specific marker cardiac myosin heavy chain (i,j, red), andskeletal muscle-specific transcription factor myogenin (h-j, green). Nuclei arestained with DAPI (f,g,i,j, blue). Myogenin expression was localized to nuclei(H,I, exemplified by arrowheads). Nuclei expressing myogenin (h-j, exemplifiedby arrowheads) belonged to cells negative for cardiac marker in redfluorescence (h,j) and exhibited morphology characteristic of myotubes (j).Scale bar = 40 lm (f) or 20 lm (g-j)


BM+bFGF, followed by BM+TGF-b1, and BM alone(Fig. 4). Compared with SM+ cells, Myl2 expressionwas uniformly greater in SM- cells, particularly inpresence of bFGF and TGF-b1. Importantly, Myl2expression was greater after 9 days compared withlevels after 4 days of culture. These observations areconsistent with the paradigm that the expression oftranscription factors would logically precede theexpression of structural proteins during lineage com-mitment. Although SM+ cells expressed low levels ofMyl2 (a structural protein) at 4 and 9 days, theexpression of Nkx2.5 and GATA-4 (transcription fac-tors) was clearly detectable in SM+ cells at these time-

points (Fig. 4). Cell type-specific differences in thetime-course of antigen expression may be responsiblefor the differential expression levels of Myl2 inSM- and SM+ cells at these earlier time-points.

j Impact of media composition on cardiomyogenicdifferentiation

Freshly isolated SM+ and SM- cells were allowed todifferentiate in five different media combinations.After 30 days, cells were immunostained for cardiac-specific transcription factors (Nkx2.5 and GATA-4)

Fig. 3 Representative light microscopic images of SM- cells after 30 days ofdifferentiation demonstrating the effects of culture media composition oncellular morphology. Panels a–c show the predominant epithelial morphologyof SM-cells cultured in basic medium (BM) containing IGF-1 and dynorphin B(a), and BM supplemented with insulin (b), or oxytocin (c). Scattered cells withadipose morphology were noted throughout the culture plates. Panels d and eshow the increased frequency of spindle-shaped cells when SM- cells wereexposed to BM supplemented with bFGF (d) or TGF-b1 (e). Cardiomyogenicdifferentiation was verified via immunostaining. Panels f-i show representative

confocal microscopic images of SM- cells after 30 days of differentiation in BMsupplemented with TGF-b1. Mononucleate spindle-shaped cells are identifiedin the transmission image (f). Cardiac differentiation is evidenced by positivityfor cardiac-specific transcription factor (Nkx2.5, h–i, green) and structuralprotein (cardiac myosin heavy chain, g–i, red). Panels h–i identify cells positivefor both cardiac-specific transcription factor (in green) and structural protein (inred) resulting in yellow fluorescence. Nuclei are stained with DAPI (f,g,i, blue).Scale bar = 20 lm


and cardiac-specific structural proteins (cMyHC anda-sarcomeric actin). Fig. 3f–i demonstrates cardi-omyocytic differentiation of SM- cells cultured inmedium containing BM+TGF-b1. After 30 days, BMalone, BM+insulin, BM+oxytocin, BM+bFGF, andBM+TGF-b1 induced cardiac differentiation in11.1 ± 1.0%, 6.2 ± 0.4%, 7.1 ± 1.0%, 9.7 ± 0.8%, and13.1 ± 1.5% of SM+ cells, respectively (Fig. 5). In cul-tured SM- cells, BM alone, BM+insulin, BM+oxytocin,BM+bFGF, and BM+TGF-b1 induced cardiac differen-tiation in 14.0 ± 1.3%, 9.3 ± 1.0%, 10.1 ± 0.2%,14.0 ± 1.1% and 21.6 ± 1.6% cells, respectively (Fig. 5).Addition of insulin or oxytocin to BM significantlyattenuated cardiac differentiation. BM+TGF-b1 wasmost effective in inducing cardiomyogenic differentia-tion of SM- cells. Compared with insulin, oxytocin, andbFGF, addition of TGF-b1 also resulted in greater cardiacdifferentiation of SM+ cells. These findings are consis-tent with data from mRNA analysis, which showed thataddition of TGF-b1 enhanced the expression of bothcardiac-specific transcription factors and structuralproteins. In addition, we observed spontaneous rhyth-mic contractions in SM- cell-derived cardiomyocyticcells (supplemental Video 1) confirming their cardi-omyocytic nature. Overall, compared with SM+ cells,

SM- cells exhibited greater predisposition to undergocardiomyocytic differentiation (Fig. 5). The myotubedifferentiation rate was not determined because cellswere cultured in cardiomyogenic media, and becausethese myotubes frequently fused to form syntitia,thereby precluding an accurate cell count.

Discussion

In order to improve the outcomes of cell therapy forcardiac repair, it is imperative to critically evaluatethe ability of the candidate cell to differentiate intocardiac lineages in vitro. However, little consensusexists regarding the most effective medium compo-sition for inducing cardiac differentiation in vitro.Our results demonstrate that: (1) the addition ofTGF-b1 to medium containing IGF-1 and dynorphinB significantly enhances cardiomyogenic differentia-tion of adult skeletal muscle-derived primitive cells;(2) the addition of insulin or oxytocin reduces cardiacdifferentiation and promotes adipocytic differentia-tion; and (3) SM- cells exhibit greater predispositionto differentiate into a cardiomyocytic phenotype,indicating that this specific cell type is inherently

0

1

2

3

4

5

6

7

8

9

10

mRN

A (-

fold

diff

eren

ce)

A

Nkx2.5

0

2

4

6

8

10

12

14

16

18

20

22

mRN

A (-

fold

diff

eren

ce)

GATA-4

0

5

10

15

20

25

30

35

40

mRN

A (-

fold

diff

eren

ce)

Myl2

E : BM + TGF-β1

A : BM

D : BM + bFGF

C : BM + oxytocin

B : BM + insulin

*

* *

4 days

* P<0.05 vs. respective BM groups

# P<0.05 vs. respective SM groups

*

##

#

#

* #

* #* #

* #* #

#

SM-

B C D E A B C D E

A B C D E A B C D E

SM-

SM-

9 days

SM+

+

SM+ SM+

Fig. 4 Quantitative assessment of mRNAexpression by qRT-PCR of cardiac-specificmarkers in SM+ and SM- cells after 4 and9 days of culture in 5 different media.mRNA level is expressed as -fold changecompared with freshly isolatedunfractionated skeletal muscle-derivedcells. Three independent experimentswere performed. Data are mean ± SEM


more amenable to cardiac differentiation. These re-sults have considerable implications for the selectionof media for in vitro experiments aimed at identifyingthe most suitable cell population for cardiac repair invivo.

Members of the TGF-b superfamily play impor-tant roles in cardiac development during embryo-genesis [27, 31] as well as in various cardiacpathologies [24, 34]. TGF-b1 has been shown toinduce cardiac differentiation in vitro in embryonicexplants and stem cells [6, 21] as well as in adultbone marrow-derived cells [14]. Our results showthat compared with other combinations, theBM+TGF-b1 combination is most effective ininducing cardiomyocytic differentiation of SM- cellsin vitro, an effect that was verified by morphologicalexamination as well as expression of cardiac-specifictranscription factors and structural proteins at themRNA and protein levels. TGF-b1 enhanced theexpression of Nkx2.5 and GATA-4 at 4 days of cul-ture, which was followed by increased expression ofstructural proteins (Myl2 at day 9, and cMyHC anda-sarcomeric actin at later stages). Following TGF-b1receptor binding, the receptor-regulated Smads (R-Smads: Smad2 and Smad3) are phosphorylated andform a complex with the common-mediator Smad(Co-Smad: Smad4) that migrates to the nucleus and

binds to specific DNA sequences leading to tran-scriptional activation [31]. Importantly, the regula-tory region of the Nkx2.5 gene, an early [17] andessential [19] transcription factor for cardiomyo-genesis, contains several Smad [16] as well as GATA[7] binding sites. Notwithstanding the complex andpoorly understood interaction between Nkx2.5,GATA-4, and other factors during cardiac develop-ment, it is plausible that TGF-b1-induced cardiacdifferentiation is triggered via Smad activation ofNkx2.5 and GATA-4, which is followed by theexpression of cardiac structural proteins.

In a recent study by Li et al. [15], TGF-b1 induceddifferentiation of bone marrow-derived CD117+ cellsinto immature cardiomyocytes in vitro. Importantly,in a previous study [14], transplantation of TGF-b1-primed CD117+ cells into infarcted hearts resulted inmyocyte regeneration and improvement in LV func-tion. In our study, while a rigorous assessment of thecardiomyocyte phenotype via a combinatorial ap-proach (morphology, transcription factor, structuralprotein, spontaneous contraction) enabled an accu-rate assessment of the comparative efficacy of differ-ent growth factors in cardiac differentiationinduction, whether these cardiomyocytic cellsachieved the status of mature cardiomyocytes in vitroremained unclear. Therefore, to address this issue of

0

5

10

15

20

25

30

35

Expression of cardiac-specific markers

Transcription factors (cTFs)

Structural proteins (cSPs)

cTFs and cSPs

Posi

tive

cells

(%)

**

#

SM-

* P<0.05 vs. respective BM groups# P<0.05 vs. respective SM groups

*#

BM +

Insu

lin

BM +

OT

BM +

bFGFBM

BM +

TGF-

β1

*#

*#

BM +

Insu

lin

BM +

OT

BM +

bFGFBM

BM +

TGF-

β1

SM+

+

Fig. 5 Percentage of cells positive for cardiac-specificmarkers (Y axis), demonstrating the effects of differentmedia (X axis) on cardiomyogenic differentiation ofSM+ and SM) cells. Cells positive for cardiactranscription factors (cTFs) and/or structural proteins(cSPs) were counted and expressed as a percentage oftotal cells. The percentage of cells positive for cTFs isrepresented by solid bars, cSPs by white bars, and cellspositive for both by cross-hatched bars. Cardiomyocyticdifferentiation was noted in both SM+ and SM)populations. However, compared with SM+ cells, SM-cells exhibited greater cardiomyogenic potential in allmedia. Supplementation of BM with TGF-b1 resultedin cardiomyocytic differentiation in the greatestfraction of cells in both groups


maturity from a translational standpoint, futurestudies will be necessary to investigate whether TGF-b1-pre-differentiated SM- cells can improve LVstructure and function in vivo following transplanta-tion into the infarcted heart.

bFGF (FGF-2) also induced expression of Nkx2.5and GATA-4; however, immunocytochemical quanti-tation revealed significantly less cardiac differentia-tion. Although bFGF is expressed in the developingheart [28], its role on cardiac specification vs. prolif-eration is unclear [21, 29]. In the study by Hidai et al.[10], FGF-2 suppressed the expression of retinoicacid-induced GATA-4 expression, and promotedskeletal muscle differentiation in embryonal P19 cells.Interestingly, inhibition of FGFR-1, which serves as areceptor for both FGF-1 and FGF-2, was able to blockthe expression of GATA-4 without affecting a-MyHCexpression. In the study by Barron et al. [4], transientexposure to FGF-2 with continuous BMP-2 presencewas able to induce a contractile phenotype in non-precardiac mesoderm. However, longer exposure toFGF-2 was necessary to achieve Nkx2.5 induction. Ourresults are consistent with this dichotomy between theinduction of cardiac transcription factors vs. struc-tural proteins by bFGF, and its role in skeletal myo-genesis. Further studies will be necessary to determinethe signaling machinery activated by bFGF in adultprimitive cells.

IGF-1 receptor is expressed in the developing heart[5] and IGF-1 has been shown to induce cardiac dif-ferentiation of transplanted embryonic stem cells[13]. Similarly, the P19 embryonal cells expressprodynorphin, and dynorphin B can induce cardiacdifferentiation in these cells [32]. In our study, al-though BM (containing IGF-1 and dynorphin B) aloneupregulated the expression of Nkx2.5 and GATA-4 inSM- cells, cardiomyocytic differentiation was consid-erably less than that achieved with the addition ofTGF-b1. These differences from previous reportscould be accounted for by the differences in sub-strates, embryonic cells vs. adult primitive cells.

The addition of insulin to BM negatively im-pacted the expression of cardiac markers. In addi-tion, insulin induced marked adipocyticdifferentiation. In previous studies, insulin has beenshown to induce contractility in embryonic explants[29] and promote cardiac differentiation in embry-onic neural precursors [3]. Similar observationshave been made in adult bone marrow-derivedmesenchymal stem cells [26]. It is plausible thatadding insulin to a medium containing IGF-1 anddynorphin B negatively impacted cardiac differenti-ation. Similarly, the addition of oxytocin also re-duced cardiac differentiation compared with BM

alone and promoted an adipocytic phenotype.Oxytocin is expressed in the developing heart [11],and the evidence supporting a role of oxytocin incardiac differentiation comes primarily from studiesthat used the embryonal P19 cells [8, 22]. Interest-ingly, amongst adult cells, the cardiomyogenicproperties of oxytocin has been documented in Sca-1+ cardiac progenitors [20]. However, in our study,the effects of oxytocin in Sca-1+ (SM+) and Sca-1-(SM-) cells were similar. Thus, the role of oxytocinin cardiac differentiation of non-cardiac adultprimitive cells remains to be established.

The current results also expand our previousobservations regarding the impact of Sca-1 expressionon cardiac differentiation of CD45-/c-kit-/Thy-1+adult skeletal muscle-derived cells [35]. Comparedwith SM- cells, SM+ cells consistently exhibited lowerlevels of cardiac-specific gene and protein expressionin response to the various growth factors used, indi-cating that the negative influence of Sca-1+ expres-sion on cardiac differentiation of skeletal muscle-derived CD45-/c-kit-/Thy-1+ primitive cells [35] isindependent of the culture medium. The greater car-diomyocytic differentiation of SM- cells vs. SM+ cellswith the same growth factors implies cell type-specificdifferences. Therefore, these results also underscorethe importance of carefully selecting the candidate cellfor cardiac repair.

Our study has several limitations. First, we did notdemonstrate calcium transients in differentiated cells.Instead, we documented spontaneous rhythmic con-tractions in cardiomyocytic cells in culture (supple-mental Video 1). Second, the cardiomyogenic effectsof insulin and oxytocin might have been influenced bythe presence of IGF-1 and dynorphin B in the BM.However, both IGF-1 and dynorphin B are known toinduce cardiomyogenesis [2, 32], and their presencewas therefore more likely to synergistically augmentthe cardiomyogenic effects in all arms. Since thecomposition of BM was constant in all groups, ourcomparison remains valid. Finally, we did not fullyassess whether these SM- cell-derived cardiomyocytesin vitro attained features consistent with matureadult cardiomyocytes, a question that may perhapsbe addressed better in a more conducive milieu invivo following transplantation into the infarctedheart.

In conclusion, our results indicate that TGF-b1enhances cardiomyogenic differentiation of skeletalmuscle-derived adult primitive cells. This influencewas achieved via the upregulation of Nkx2.5 andGATA-4 expression. These results may have consid-erable implications for the formulation of optimalmedium for the induction of cardiac differentiation in


vitro, and selecting the candidate cell population fortherapeutic cardiac repair in vivo.

j Acknowledgments We gratefully acknowledge Barbara Turgeonfor expert secretarial assistance. This study was supported in partby NIH grants R01 HL-72410, HL-55757, HL-68088, HL-70897, HL-76794, HL-78825, and R21 HL-89737.

References

1. Abdel-Latif A, Bolli R, Tleyjeh IM,Montori VM, Perin EC, Hornung CA,Zuba-Surma EK, Al-Mallah M, Dawn B(2007) Adult bone marrow-derived cellsfor cardiac repair: a systematic reviewand meta-analysis. Arch Intern Med167:989–997

2. Antin PB, Yatskievych T, DominguezJL, Chieffi P (1996) Regulation of avianprecardiac mesoderm development byinsulin and insulin-like growth factors.J Cell Physiol 168:42–50

3. Bani-Yaghoub M, Kendall SE, MooreDP, Bellum S, Cowling RA, NikopoulosGN, Kubu CJ, Vary C, Verdi JM (2004)Insulin acts as a myogenic differentia-tion signal for neural stem cells withmultilineage differentiation potential.Development 131:4287–4298

4. Barron M, Gao M, Lough J (2000)Requirement for BMP and FGF signal-ing during cardiogenic induction innon-precardiac mesoderm is specific,transient, and cooperative. Dev Dyn218:383–393

5. Bassas L, Lesniak MA, Serrano J, Roth J,de Pablo F (1988) Developmental regu-lation of insulin and type I insulin-likegrowth factor receptors and absence oftype II receptors in chicken embryotissues. Diabetes 37:637–644

6. Behfar A, Zingman LV, Hodgson DM,Rauzier JM, Kane GC, Terzic A, PuceatM (2002) Stem cell differentiation re-quires a paracrine pathway in the heart.Faseb J 16:1558–1566

7. Brown CO 3rd, Chi X, Garcia-Gras E,Shirai M, Feng XH, Schwartz RJ (2004)The cardiac determination factor, Nkx2-5, is activated by mutual cofactorsGATA-4 and Smad1/4 via a novel up-stream enhancer. J Biol Chem279:10659–10669

8. Danalache BA, Paquin J, Donghao W,Grygorczyk R, Moore JC, Mummery CL,Gutkowska J, Jankowski M (2007) Nitricoxide signaling in oxytocin-mediatedcardiomyogenesis. Stem Cells 25:679–688

9. Dawn B, Bolli R (2005) Adult bonemarrow-derived cells: regenerative po-tential, plasticity, and tissue commit-ment. Basic Res Cardiol 100:494–503

10. Hidai C, Masako O, Ikeda H, Nagashi-ma H, Matsuoka R, Quertermous T,Kasanuki H, Kokubun S, Kawana M(2003) FGF-1 enhanced cardiogenesis indifferentiating embryonal carcinomacell cultures, which was opposite to theeffect of FGF-2. J Mol Cell Cardiol35:421–425

11. Jankowski M, Danalache B, Wang D,Bhat P, Hajjar F, Marcinkiewicz M, Pa-quin J, McCann SM, Gutkowska J (2004)Oxytocin in cardiac ontogeny. Proc NatlAcad Sci USA 101:13074–13079

12. Kodama H, Hirotani T, Suzuki Y, Oga-wa S, Yamazaki K (2002) Cardiomyo-genic differentiation in cardiac myxomaexpressing lineage-specific transcrip-tion factors. Am J Pathol 161:381–389

13. Kofidis T, de Bruin JL, Yamane T, Bal-sam LB, Lebl DR, Swijnenburg RJ, Ta-naka M, Weissman IL, Robbins RC(2004) Insulin-like growth factor pro-motes engraftment, differentiation, andfunctional improvement after transferof embryonic stem cells for myocardialrestoration. Stem Cells 22:1239–1245

14. Li TS, Hayashi M, Ito H, Furutani A,Murata T, Matsuzaki M, Hamano K(2005) Regeneration of infarcted myo-cardium by intramyocardial implanta-tion of ex vivo transforming growthfactor-beta-preprogrammed bone mar-row stem cells. Circulation 111:2438–2445

15. Li TS, Komota T, Ohshima M, Qin SL,Kubo M, Ueda K, Hamano K (2008)TGF-b induces the differentiation ofbone marrow stem cells into immaturecardiomyocytes. Biochem Biophys ResCommun 366:1074–1080

16. Liberatore CM, Searcy-Schrick RD,Vincent EB, Yutzey KE (2002) Nkx-2.5gene induction in mice is mediated by aSmad consensus regulatory region. DevBiol 244:243–256

17. Lints TJ, Parsons LM, Hartley L, LyonsI, Harvey RP (1993) Nkx-2.5: a novelmurine homeobox gene expressed inearly heart progenitor cells and theirmyogenic descendants. Development119:419–431

18. Lyngbaek S, Schneider M, Hansen JL,Sheikh SP (2007) Cardiac regenerationby resident stem and progenitor cells inthe adult heart. Basic Res Cardiol102:101–114

19. Lyons I, Parsons LM, Hartley L, Li R,Andrews JE, Robb L, Harvey RP (1995)Myogenic and morphogenetic defects inthe heart tubes of murine embryoslacking the homeo box gene Nkx2-5.Genes Dev 9:1654–1666

20. Matsuura K, Nagai T, Nishigaki N, Oy-ama T, Nishi J, Wada H, Sano M, TokoH, Akazawa H, Sato T, Nakaya H,Kasanuki H, Komuro I (2004) Adultcardiac Sca-1-positive cells differentiateinto beating cardiomyocytes. J BiolChem 279:11384–11391

21. Muslin AJ, Williams LT (1991) Well-defined growth factors promote cardiacdevelopment in axolotl mesodermalexplants. Development 112:1095–1101

22. Paquin J, Danalache BA, Jankowski M,McCann SM, Gutkowska J (2002) Oxy-tocin induces differentiation of P19embryonic stem cells to cardiomyo-cytes. Proc Natl Acad Sci USA 99:9550–9555

23. Rosenblatt-Velin N, Lepore MG, Car-toni C, Beermann F, Pedrazzini T (2005)FGF-2 controls the differentiation ofresident cardiac precursors into func-tional cardiomyocytes. J Clin Invest115:1724–1733

24. Sakata Y, Chancey AL, Divakaran VG,Sekiguchi K, Sivasubramanian N, MannDL (2008) Transforming growth factor-beta receptor antagonism attenuatesmyocardial fibrosis in mice with car-diac-restricted overexpression of tumornecrosis factor. Basic Res Cardiol103:60–68

25. Segers VF, Lee RT (2008) Stem-celltherapy for cardiac disease. Nature451:937–942

26. Shim WS, Jiang S, Wong P, Tan J, ChuaYL, Tan YS, Sin YK, Lim CH, Chua T,Teh M, Liu TC, Sim E (2004) Ex vivodifferentiation of human adult bonemarrow stem cells into cardiomyocyte-like cells. Biochem Biophys Res Com-mun 324:481–488

27. Solloway MJ, Harvey RP (2003) Molec-ular pathways in myocardial develop-ment: a stem cell perspective.Cardiovasc Res 58:264–277

28. Spirito P, Fu YM, Yu ZX, Epstein SE,Casscells W (1991) Immunohistochem-ical localization of basic and acidicfibroblast growth factors in the devel-oping rat heart. Circulation 84:322–332


29. Sugi Y, Lough J (1995) Activin-A andFGF-2 mimic the inductive effects ofanterior endoderm on terminal cardiacmyogenesis in vitro. Dev Biol 168:567–574

30. Ulloa-Montoya F, Verfaillie CM, Hu WS(2005) Culture systems for pluripotentstem cells. J Biosci Bioeng 100:12–27

31. Valdimarsdottir G, Mummery C (2005)Functions of the TGF-b superfamily inhuman embryonic stem cells. Apmis113:773–789

32. Ventura C, Maioli M (2000) Opioidpeptide gene expression primes cardi-ogenesis in embryonal pluripotent stemcells. Circ Res 87:189–194

33. Winitsky SO, Gopal TV, Hassanzadeh S,Takahashi H, Gryder D, Rogawski MA,Takeda K, Yu ZX, Xu YH, Epstein ND(2005) Adult murine skeletal musclecontains cells that can differentiate intobeating cardiomyocytes in vitro. PLoSBiol 3:e87

34. Wollert KC (2007) Growth-differentia-tion factor-15 in cardiovascular disease:from bench to bedside, and back. BasicRes Cardiol 102:412–415

35. Zuba-Surma EK, Abdel-Latif A, Case J,Tiwari S, Hunt G, Kucia M, Vincent RJ,Ranjan S, Ratajczak MZ, Srour EF, BolliR, Dawn B (2006) Sca-1 expression isassociated with decreased cardiomyo-genic differentiation potential of skele-tal muscle-derived adult primitive cells.J Mol Cell Cardiol 41:650–660


Documents

TGF-β1 enhances cardiomyogenic differentiation of skeletal muscle-derived adult primitive cells