Functional Expression of HGF and HGF Receptor/c-met in Adult HumanMesenchymal Stem Cells Suggests a Role in Cell Mobilization,

Tissue Repair, and Wound Healing

SABINE NEUSS,a,b* EVA BECHER,a,b* MICHAEL WÖLTJE,a LOTHAR TIETZE,b WILLI JAHNEN-DECHENTa

aInterdisciplinary Center for Clinical Research on Biomaterials, IZKF BIOMAT, Aachen, Germany;bInstitute of Pathology, University Hospital, Aachen, Germany

*These authors contributed equally.

Key Words. HGF · Mesenchymal stem cells · Cell migration · Mobilization

ABSTRACT

Human mesenchymal stem cells (hMSC) are adultstem cells with multipotent capacities. The ability of mes-enchymal stem cells to differentiate into many cell types,as well as their high ex vivo expansion potential, makesthese cells an attractive therapeutic tool for cell trans-plantation and tissue engineering. hMSC are thought tocontribute to tissue regeneration, but the signals govern-ing their mobilization, diapedesis into the bloodstream,and migration into the target tissue are largely unknown.Here we report that hepatocyte growth factor (HGF) and

the cognate receptor HGFR/c-met are expressed inhMSC, on both the RNA and the protein levels. Theexpression of HGF was downregulated by transforminggrowth factor beta. HGF stimulated chemotactic migra-tion but not proliferation of hMSC. Therefore theHGF/c-met signaling system may have an important rolein hMSC recruitment sites of tissue regeneration. The con-trolled regulation of HGF/c-met expression may be bene-ficial in tissue engineering and cell therapy employinghMSC. Stem Cells 2004;22:405-414

STEM CELLS 2004;22:405-414 www.StemCells.com

Correspondence: Willi Jahnen-Dechent, Ph.D., IZKF BIOMAT, University Hospital, Pauwelsstrasse 30, D-52074 Aachen,Germany. Telephone: 49-241-80-80163; Fax: 49-241-80-82573; e-mail: willi.jahnen@rwth-aachen.de Received October1, 2003; accepted for publication December 11, 2003. © AlphaMed Press 1066-5099/2004/$12.00/0

INTRODUCTION

A classic concept of tissue repair holds that inflamma-tory cells enter the damaged tissue and signal resident tis-sue-specific progenitor cells (e.g., parenchymal cells,fibroblasts) for mitosis. Several studies suggest that multi-potent (bone marrow) mesenchymal stem cells can alsocontribute to tissue repair after mobilization, migration, andengraftment of the damaged tissue [1]. In addition, circulat-ing immature cells seem to participate in regeneration ofmany different tissues [2, 3]. The role of mesenchymal stemcells in tissue remodeling was shown in different in vivo

models—for example, for hepatic regeneration [4, 5], mus-cle regeneration [6, 7], and infarcted myocardium [8, 9].Not surprisingly mesenchymal and circulating stem cellshave attracted increasing attention because they hold greattherapeutic potential for endogenous tissue repair and tissueengineering.

Since the original report from Friedenstein et al. [10], anumber of different protocols have been defined to isolatemultipotent adult mesenchymal stem cells from bone marrow specimen [11, 12]. Unlike hematopoietic stem cells,human mesenchymal stem cells (hMSC) adhere to cell culture

Stem Cells®

Original Article

Neuss, Becher, Wöltje et al. 406

plastic, which is exploited for their isolation [13, 14]. HMSCexpress CD105 and CD73 but not the lineage-specific surfaceantigens CD14, CD34, and CD45 [14]. Markers specific forhMSC are not known. Therefore, putative hMSC isolateshave to be verified by their capacity to differentiate at leastinto adipocytes, chondrocytes, and osteoblasts. In addition,bone marrow-derived mesenchymal stem cells can be differ-entiated in vitro into bone marrow stromal cells and intoendothelial, myogenic, hepatic, and neurogenic cells. Celltransplantation studies in human patients and in animals havedemonstrated that bone marrow-derived cells can colonizemost organs. The colonization was much enhanced by inflam-mation accompanying, for example, graft rejection or infarc-tion. Hence, successful engraftment of several organs wasenhanced by irradiation [15], chemical injury, and genetic dis-eases [5] or following infarction [8]. Despite major advancesin MSC biology, our knowledge of the signals required forMSC mobilization and migration to the injured tissue site lagsbehind the extensive experience with hematopoetic stemcells, which are in routine clinical use. Therefore we sought todetermine which factors may be responsible for mobilizationof hMSC. Hepatocyte growth factor/scatter factor (HGF/SF)is a multipotent growth factor that exerts a mitogenic, moto-genic, and morphogenic response on cells expressing c-met,the cellular HGF receptor. HGF/SF is essential in paracrinesignaling of mesenchymal and epithelial cells, particularlyduring embryogenesis, repair, and carcinogenesis [16]. Inmalignant and transfected cells autocrine stimulation has beendescribed [17]. In pathology, HGF/SF has been shown toinduce tumor cell invasion in tissues [18].

Studies on HGF and c-met expression in bone marrowcells were reported by Takai et al. [19]. These authorsdemonstrated that bone marrow stromal cells constitutivelyexpress HGF and promote hematopoiesis. In addition,expression of c-met by stromal cells suggested an autocrinestimulation of stromal cells by HGF. However, it was notdetermined if HGF and c-met were also expressed by hMSC.This study was undertaken to address this question, includingfunctional aspects of HGF and c-met-like cell migration andproliferation. We demonstrate that HGF and c-met are con-stitutively expressed by hMSC and that the expression ofHGF is downregulated by transforming growth factor-β(TGF-β). Furthermore, HGF exerted a strong chemotacticstimulus on hMSC, which may be further enhanced byautocrine signaling through the HGF c-met pathway.

MATERIALS AND METHODS

Cell CultureHMSC were isolated mechanically from femoral heads

of human orthopedic patients (with informed consent) and

by adherence to cell culture plastic according to protocolsof Haynesworth [13] and Pittenger [14]. Briefly, bone mar-row was rinsed thoroughly with stem cell medium(MSCBM Stem Cell BulletKit; CellSystems; St.Katharinen, Germany; http://www.cellsystems.com) sev-eral times, then bone marrow was removed and cell sus-pension was mixed and centrifuged for 10 min (500 g). Thecell pellet was resuspended in fresh medium and seeded ina T75 culture flask. Nonadherent cells were removed after24 hours by medium change. Cells were cultured in stemcell medium at 37°C in a 5% CO2 and 20% O2 humidifiedatmosphere. Medium was changed every 3-4 days. At con-fluency, cells were trypsinized (0.05% trypsin, 0.53 mMEDTA, 5 min) with stem cell trypsin (CellSystems) andseeded at a density of 5,000 cells/cm2 to expand cell culture.

Characterization of hMSCTo differentiate hMSC into adipocytes, chondrocytes,

and osteoblasts, protocols according to Pittenger et al. [14]were used. For adipogenic differentiation, cells were seededat a density of 8 × 104 cells/cm2 into stem cell medium. Atconfluency, the medium was changed every 3-4 days fromadipogenic induction to adipogenic maintenance mediumand back. This regimen was repeated twice. The adipogenicinduction medium consisted of Dulbecco’s modifiedEagle’s medium (DMEM) high glucose (PAA Labora-tories; Cölbe, Germany; http://www.paa.at) with 1 µM dex-amethasone, 0.2 mM indomethacin, 0.01 mg/ml insulin, 0.5mM 3-isobutyl-1-methyl-xanthine, and 10% fetal calfserum (FCS) (all Sigma; Steinheim, Germany; http://www.sigmaaldrich.com), while the adipogenic maintenancemedium was composed of DMEM high glucose (PAA)with 0.01 mg/ml insulin and 10% FCS. After three com-plete cycles of induction and maintenance, cells were fixedwith 10% formalin and stained for 10 min with Oil red Osolution (Sigma) to visualize lipids.

For chondrogenic differentiation, 0.5-ml cell suspension(5 × 105 cells/ml) was placed into a 15-ml polypropylenetube (Becton Dickinson; Heidelberg, Germany; http://www.bd.com) and centrifuged to obtain cell pellets. The pelletswere cultured in serum-free chondrocyte induction medium(DMEM high glucose; PAA), 100 nM dexamethasone, 0.17mM l-ascorbic acid 2-phosphate, 100 µg/ml sodium pyru-vate, 40 µg/ml proline (all Sigma), and 1% ITS-Plus (BectonDickinson). TGF-β3 (CellSystems) was added in a concen-tration of 10 ng/ml medium at each medium change. After 21days, pellets were formalin fixed and paraffin embedded.Thin sections were stained with Toluidin blue (Sigma).

For osteogenic differentiation, cells were seeded in adensity of 3.1 × 104 cells/cm2 and stimulated with osteo-genic induction medium after 24 hours. The medium

407 HGF/c-met in Mesenchymal Stem Cells

consisted of DMEM low glucose (PAA), 10% FCS, 100 nMdexamethasone, 10 mM sodium β-glycerophosphate, and0.05 mM L-ascorbic acid 2-phosphate (all Sigma) and wasreplaced every 3-4 days. After 16 days of differentiation,cells were fixed with 70% ethanol for 1 hour, washed withaqua bidest, and stained with Alizarin red solution (40 mM,pH 4.1, Sigma) for 10 min. Stained cells were washed threetimes with phosphate-buffered saline (PBS), and calciumdeposits were photographed.

FACS AnalysisAn aliquot of 2.5 × 105 cells was preincubated (4°C,

30 min) in PBS containing 1% bovine serum albumin (BSA).A 1-µg primary antibody was added to a 100-µ1 cell suspen-sion (4°C, 60 min). Cells were washed three times in 100 µ1PBS containing 0.1% BSA and resuspended in PBS contain-ing 1% BSA. The washing step was followed by incubationwith secondary antibody (1:20 dilution) in PBS/1% BSA(4°C, 45 min, in the dark). Cells were washed three times asdescribed previously and resuspended in 100-µ1 PBS contain-ing 1 µM propidium iodide. Monoclonal antibodies (CD4,CD14, CD34, CD49a, CD49c, CD49d, CD51, CD54, CD73,CD105, CD117) and corresponding secondary antibodies(conjugated with fluorescein isothiocyanate [FITC]) were pur-chased from DAKO (Glostrup, Denmark; http://www.dakocytomation.com). Species-matched immunoglobulin Gs(IgGs) served as negative control. Data of 10,000 stainedcells were collected and analyzed using a FACSCaliburinstrument and FACSCalibur software (Becton Dickinson).

REVERSE TRANSCRIPTASE POLYMERASE CHAIN

REACTION (RT-PCR)Total RNA was extracted using guanidinium thiocyanate

(RNeasy Kit; Qiagen; Hilden, Germany; http://www.qiagen.com). Reverse transcription was accomplished with 5 µg oftotal RNA using the “first-strand cDNA synthesis kit”(Amersham Pharmacia Biotech; Buckinghamshire, UK;http://www.amershambiosciences.com) with FPLCpure™

murine reverse transcriptase. PCR was carried out under thefollowing conditions: denaturation at 95°C for 1 min, anneal-ing at 54°C (HGF) or 60°C (c-met) for 1 min, extension at72°C for 1 min (30 cycles), and a final extension at 72°C for10 min. PCR amplicon size (266 bp for HGF and 440 bp forc-met) was analyzed by electrophoresis on a 2% agarose geland visualized with ethidium bromide. The oligonucletoideprimer and the GenBank/EMBL identifiers of templatesequences were as follows:

c-met (NM000245)forward (nt 1398-1423 [20]): 5′-AGAAATTCATCAGGCTGTGAAGCGCG-3′

reverse (nt 1814-1838 [20]): 5′-TTCCTCCGATCGCACACATTTGTCG-3′

HGF (XM168542)forward (nt 548-568): 5′-GGTAAAGGACGCAGCTACAAG-3′reverse (nt 794-814): 5′-ATAACTCTCCCCATTGCAGGT-3′

ImmunohistochemistrySubconfluent hMSCs were grown in chamber slides and

fixed for 30 min using 0.5% paraformaldehyde (in PBS).After washing with wash buffer (Dulbecco’s phosphate-buffered saline solution A [PBSSA], 0.5% BSA in PBS),unspecific protein-binding capacity was blocked for 15 minusing blocking buffer (PBS with 5% BSA). Cells were incu-bated for 45 min at RT with the first antibody, diluted 1:50in PBSSA-NP-40 (1:200 dilution of nonidet P-40 [NP-40] in PBSSA), washed three times for 10 min in PBSSA, fol-lowed by an incubation with the secondary antibody (1:250in PBSSA) for 30 min at RT and again three washing steps.FITC-conjugated streptavidin was added (1:250 in PBSSA)for 30 min, protected from light. Finally, cells were washedthree times with PBSSA for 10 min and mounted in 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI)-containingmounting medium.

Polyclonal rabbit c-met antibody (primary antibody)was purchased from Santa Cruz Biotechnology (SantaCruz, CA; http://www.scbt.com), and secondary antibody(biotin-conjugated goat anti-rabbit) and FITC-conjugatedstreptavidin from DAKO. As a negative control, cells wereincubated with the first antibody, which had been preincu-bated overnight at 4°C with a specific blocking peptide(Santa Cruz Biotechnology).

Western BlottingMesenchymal stem cells were lysed with insect cell

lysis buffer (PharMingen International; Hamburg,Germany; http://www.pharmingen.com), and protein con-centration was determined using the BCA-Kit (PierceBiotechnology; Rockford, IL; http://www.piercenet.com).Ten µg of protein lysate and protein marker (PerfectProtein AP WB Marker; Novagen; Darmstadt, Germany;http://www.emdbiosciences.com) were separated on 4%-12% SDS gels (NuPAGE; Karlsruhe, Germany) at 70 V for2.5 hours. Membranes were fixed with methanol, and afterelectroblotting (60 min, 150 mA) on polyvinylidene fluo-ride membrane (Bio-Rad; Munich, Germany; http://www.bio-rad.com), the membranes were blocked overnight withlow-fat milk at 4°C. Immunostaining was accomplished byincubation with rabbit polyclonal antibody against c-met(1:200, Santa Cruz Biotechnology) for 90 min at room

Neuss, Becher, Wöltje et al. 408

temperature. After 1 hour of incubation with alkaline phos-phatase (AP)-conjugated secondary antibody (1:5,000;Roche; Mannheim, Germany; http://www.roche.com) atroom temperature, staining was developed using Sigma Fast5-bromo,4-chloro,3-indoyl phosphate/nitroblue tetrazolium(BCIP/NBT) tablets (Sigma). As a negative control, the firstantibody was preincubated with a fivefold excess of blockingpeptide in a small volume of PBS at 4°C overnight.

HGF-ELISAHGF concentration in hMSC-conditioned media was

measured by Quantikine human HGF-ELISA (R&DSystems; Minneapolis, MN; http://www.rndsystems.com),which is based on a sandwich enzyme immunoassay tech-nique with a precoated HGF-specific antibody. To this end,cells were seeded in 96-well plates (20,000 cells/well) andstimulated for 24 hours with TGF-β3 (10, 1, and 0.1 ng/ml),interleukin-1β (IL-1β) (10, 1, and 0.1 ng/ml), and bFGF (10,1, and 0.1 ng/ml). Unstimulated cells served as a control.Supernatants were harvested and stored at -70°C until theywere analyzed.

Scratch AssayhMSC were grown to confluency in a six-well plate

(Becton Dickinson). A scratch in the cell layer was madewith a pipette tip over the total diameter of 34.5 mm. HGFwas added at 0, 25, 50, and 75 ng HGF/ml medium. Closureof this “wound” was documented photographically(Axiovert 25; Zeiss; Cologne, Germany; http://www.zeiss.com) after 24 hours, and cells in four segments of thescratched area, each of 320 µm × 320 µm, were counted.

Boyden-Chamber AssayFor analysis of cell motility, 1 × 105 hMSCs/ml were

seeded in the top compartment of a Boyden chamber(NeuroProbe; Gaithersburg, UK; http://www.neuroprobe.com). The bottom compartment contained different HGF con-centrations and was separated from the top compartment by apolycarbonate membrane with 8 µm pores (Corning;Düsseldorf, Germany; http://www.corning.com). Cells wereallowed to migrate for 16 hours at 37°C in a humidified atmos-phere. After removing cells from the upper side of the mem-brane with cotton swabs, membranes were fixed, stained withhematoxylin (Merck; Darmstadt, Germany; http://www.merck.com), and transferred onto glass slides. Cells on the bottom side of the membrane were counted in five differenthighpower fields. Each analysis was performed in triplicate.

Cell Proliferation AssayCells were seeded in 24-well plates (3,000 cells per

well) and stimulated for 24 hours with HGF (0, 25, and

50 ng HGF/ml medium) in low serum (2% FCS). Themedium containing 20% FCS was used as positive control.Proliferation was measured by detection of ATP content ofthe cells with a luciferase detection system (ViaLight HS;BioWhittaker; Verviers, Belgium; http://www.biowhittaker.be). This bioluminescent method uses luciferase,which catalyzes the formation of light from ATP andluciferin. The emitted light intensity is linearly related to theATP concentration [21]. ATP content was measured 1, 2, 3,4, and 7 days after stimulation. The medium with HGF wasrenewed after day 4.

RESULTS

Isolation and Characterization of hMSCIsolated MSC appeared as two distinct morphological

phenotypes—long spindle-shaped cells (Fig. 1A) and large,flat cells (Fig. 1B). This mixed appearance is typical ofhMSC [22]. As shown by flourescence-activated cell sorter(FACS) analysis (Fig. 1C), isolated cells did not express thehematopoietic markers CD4 (a), CD14 (b), and CD34 (c);the integrins CD49a (d) and CD49d (e); or the stem cell fac-tor receptor CD117 (c-kit, f). In contrast, integrin CD49c(g), CD51 (vitronectin receptor, h), and CD54 (intercellularadhesion molecule 1, i), as well as CD105 (Endoglin, k)and CD73 (1), were readily detected on the cell surface,confirming the results of Pittenger et al. [14].

Next we induced cell differentiation of our isolatedMSC, following published protocols [14]. Adipogenic dif-ferentiation resulted in lipid vacuole formation (Fig. 1D),chondrogenic differentiation led to the production of pro-teoglycans in the extracellular matrix (Fig. 1E), andosteogenic differentiation caused the production ofAlizarin Red-positive mineral deposits (Fig. 1F). Thus wehave shown that the isolated cell population can indeed be differentiated in vitro into adipocytes, chondrocytes,and osteoblasts. Phenotypical and functional characteriza-tion was repeated with each isolate and showed virtuallyidentical results.

hMSC Express HGF and c-met on RNA and Protein LevelWe analyzed HGF- and c-met-specific RNA transcripts

in hMSC of three different donors at several times of culture between passages one and six. Figure 2 shows thatboth HGF and c-met were constitutively expressed in cul-tured hMSC. To detect c-met on the protein level, weemployed immunoblotting and detected a 145-kDa bandrepresenting the c-met protein (Fig. 3). Upon preincubationof the antibody with a blocking peptide, the band at 145kDa was much reduced in intensity, demonstrating thespecificity of the antibody detection. c-met expression was

409 HGF/c-met in Mesenchymal Stem Cells

confirmed by immunohistochem-istry. Figure 4 illustrates thathMSC stained positive for c-met. Fluorescence labeling ofcells appeared uniform withoutapparent receptor clustering in flu-orescent “hot spots” (Fig. 4A, 4B).Consistently, more than 95% ofhMSC stained positive, and thispositive staining could be abro-gated in all cases by preincubationof the c-met antibody with a block-ing peptide, indicating that the anti-body indeed stained the cellularHGF receptor c-met.

Next we analyzed HGF pro-tein amounts in hMSC culturesupernatants using enzyme-linkedimmunosorbent assay. We deter-mined that the amount of HGFpresent in the medium after a 24-hour culture period of 2 × 104 cellsranged from 770 to 2,000 pg/ml.The amount varied slightly between cell donors. Figure 5illustrates the combined results obtained with MSC fromthree different donors. Compared to unstimulated cells,stimulation with TGF-β3 at 0.1, 1.0, and 10 ng/ml causeda downregulation of HGF expression by 33% ± 7%, 54% ±17%, and 50% ± 19%, respectively. The downregulation ofHGF expression was statistically significant in all cases,

when compared to the untreated control (p < 0.05), indi-cating that hMSCs are sensitive to TGF-β3 and that mod-erate amounts are sufficient to mediate downregulation ofHGF in these cells. IL-1β at 0.1, 1, and 10 ng/ml and bFGFat 0.1 and 1 ng/ml did not change HGF expression. bFGFat 10 ng/ml enhanced HGF expression to 180% ± 51% (p = 0.11).

Figure 1. Characterization of hMSCand demonstration of pluripotency. A) hMSC, spindle-shaped morphol-ogy. B) hMSC, flat morphology. C) FACS analysis of hMSC: a, CD4;b, CD14; c, CD34; d, CD49a; e,CD49d; f, CD117; g, CD49c; h,CD51; i, CD54; k, CD105;1, CD73.D) Adipogenic differentiation ofhMSC: stem cells were cultured for 21days alternately in adipogenic induc-tion and adipogenic maintenancemedium. Lipid vacuoles were visual-ized by Oil red O staining. E) Chon-drogenic differentiation of hMSC:pellet culture of hMSC after 21 daysin chondrogenic induction medium.Proteoglycans were stained withToluidine blue. F) Osteogenic differ-entiation of hMSC: stem cells werecultured for 16 days with osteogenicinduction medium, and calciumdeposits were visualized by alizarinred staining.

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HGF Enhances Cell Migration of Human Mesenchymal Stem Cells

To study potential biological consequences of c-metsignaling in hMSC, we analyzed cell migration using awell-established wounding model of a confluent cell mono-layer with and without HGF stimulation. In this model aconfluent cell layer is disrupted by scraping with a cellscraper of defined width, and the subsequent closure of this“wound” by migrating cells is studied histomorphometri-cally over time. Figure 6 illustrates that the “wound”afflicted to a confluent layer of about 150 hMSC/mm2

closed faster when HGF was added to the culture medium;this effect was concentration-dependent. At 0 ng/ml addedHGF (Fig. 6A), hardly any (1.07 cells/mm2) MSC hadmigrated into the wounded, cell-free area 24 hours afterwounding. Stimulation with 25 and 50 ng/ml HGFenhanced hMSC migrating into the wound to 1.6 and 3.2 cells/mm2, respectively (Fig. 6B, 6C). The addition of

Figure 3. Detection of c-met in hMSC on the protein level.Immunoblot analysis of hMSC extracts probed with an antibodydirected against the HGF receptor/c-met detected a protein band at145 kDa, which is characteristic of this receptor (lane 1, arrow).Preincubation of the antibody with a blocking peptide abolished bind-ing to this protein (lane 2). M, molecular weight marker protein. Thisfigure shows one representative result out of four.

Figure 4. HGF receptor c-met is detectable on the cell surface. hMSC were incu-bated with antibody directed against the HGF receptor/c-met (A, B) or with thesame antibody preincubated with a blocking peptide (C, D) followed by a sec-ondary FITC-labeled antibody. Consistently, over 95% of all cells stained positivefor c-met, albeit at variable levels. This figure shows typical views from one out offour experiments with one brightly staining cell (B) and one moderately bright yetdistinctly positive staining cell (A). Cell nuclei were counterstained with DAPI.

Figure 2. Human MSC expresses HGF and its receptor c-met onthe RNA-level. Amplicon of HGF gene (266 bp) and c-met gene (400bp); M, molecular weight marker protein, 100-bp ladder: 1) negativecontrol (without cDNA); 2) hMSC, HGF; 3) liver cells (positive con-trol for HGF); 4) hMSC, c-met; 5) mesothelial cells (positive controlfor c-met). This figure shows one representative result out of four.

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411 HGF/c-met in Mesenchymal Stem Cells

75 ng/ml HGF to the medium fur-ther enhanced cell migration to 8.8 cells/mm2, corresponding to an8-fold increase in cell migrationinto the wound (Fig. 6D).

HGF Is Chemotactic for hMSCTo investigate whether the

observed promigratory effect waschemotactic or chemokinetic, we performed Boyden chamberassays. The results of the Boyden chamber assays are shownin Figure 7. HGF at 37.5 ng/ml and 50 ng/ml added to the bot-tom compartment of the Boyden chamber caused a more thanthreefold increase of migrating cells (p < 0.005 and p < 0.001,respectively). When identical amounts of HGF were simulta-neously added to both the top and the bottom compartments,cell migration of hMSC remained unchanged. Collectively,these results demonstrate that HGF is strongly chemotacticfor hMSC.

HGF Inhibits Mesenchymal Stem Cell ProliferationTo examine the possibility that the observed promigratory

effects of HGF detected in the Boyden chamber and woundingassay were also associated with enhanced proliferation, we ana-lyzed the proliferation of hMSC with and without added HGF.Figure 8 illustrates the amount of ATP measured in hMSC cul-tures as a surrogate marker for the number of vital cells presentin the cells over a period of 7 days. The viability of HGF-stim-ulated and -unstimulated cells was assessed by trypan blueexclusion and was similar in all cells over the period of 7 days(data not shown). Therefore, changes in ATP content are ameasure of proliferation and not of cell death. Compared to thelow serum culture, serum stimulated ATP production andhence proliferation by approximately 160% ± 32% during the7-day incubation period. In contrast, the addition of HGF at 25 ng/ml and 50 ng/ml did not stimulate cell proliferation butinhibited cell proliferation in a dose-dependent fashion.Compared to the low serum culture, 25 ng/ml added HGFdecreased proliferation to 72% ± 21%, and 50 ng/ml HGFdecreased proliferation to 65% ± 18% of the low serum controlincubation. In summary, these results suggest that HGF/c-met

signaling may be involved in control of hMSC mobilizationby shutting off proliferation and augmenting cell migration.HGF may represent an important cue to attract migratoryhMSC to their site of final differentiation, which could be driven by local factors of the respective target organ.

DISCUSSION

Adult pluripotent stem cells are thought to reside inmany tissues. They can be isolated from skin [23] and adipose tissue [24], and a baboon study has shown thatinfused stem cells can colonize a wide range of tissues—forexample, gastrointestinal, kidney, lung, liver, thymus, andskin [25]. However, the amount of stem cells in most tis-sues is exceedingly low, which leads to the hypothesis that

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Figure 7. HGF is chemotactic for hMSC. Stem cells were seeded in the top com-partment of a Boyden chamber, and different amounts of HGF were added to thetop and bottom compartments (ng/ml medium). The cells were allowed to migratefor 16 hours at 37°C. The top and bottom compartments were separated by a poly-carbonate membrane with 8-µm pores. Data from one experiment representativeof three independent experiments are shown. *p < 0.005, **p ≤ 0.001.

Figure 6. HGF stimulates migration ofhMSC. Confluent cell monolayers ofhMSC were wounded by scratchingwith a pipette tip. After 24 hours of stim-ulation with HGF at 0 ng/ml cell culturemedium (A), 25 ng/ml (B), 50 ng/ml (C),and 75 ng/ml (D), wound closure wasdocumented photographically. This fig-ure shows one representative result outof five.

Neuss, Becher, Wöltje et al. 412

injured tissues produce appropriate cues for engraftmentand, furthermore, that the local microenvironment wouldstimulate the differentiation of engrafted (stem) cells intofunctional, specialized cells [15]. This begs the question ofwhich signals are necessary and sufficient for stem cellmobilization and recruitment. HGF is well known to aug-ment cell migration, scattering (i.e., “scatter factor”), andproliferation of many different cell types, predominantly ofepithelial cells. Furthermore, HGF is considered to be ahumoral mediator of organogenesis and morphogenesis ofvarious tissues and organs, as well as regeneration oforgans, tumor invasion, and metastasis [26].

In the bone marrow, HGF is known as an importanthematopoietic regulator [27, 28]. Synthesis and release ofHGF precursors could be part of an autocrine mechanism.Pre-HGF has to be activated by proteolytic cleavage—forexample, through serine proteases like uPA and tPA, whichare also released by hMSC (Neuss et al., unpublished obser-vations). Tissue repair and wound healing are regulated bysoluble mediators provided by inflammatory cells. Therefore,we investigated the effect of bFGF, IL-1, and TGF-β on theproduction of HGF. TGF-β, which is known to be growthinhibitory for epithelial cells, downregulates the production ofHGF by hMSC. This effect is not attributed to cell death afterTGF-β stimulation (data not shown). In contrast, basic fibrob-last growth factor, which stimulates blood vessel formationand angiogenesis [29], and the proinflammatory cytokine IL-1β have no significant influence on the expression of HGF

(determined by ELISA). Nevertheless, bFGF upregulates theproduction of the pre-HGF-activating serine protease tPAin hMSC (data not shown). Thus, production of matureHGF is regulated by the microenvironment, depending onprocesses like tissue damage or inflammation. Activatedmonocytes or macrophages, which are replete at damagedtissue sites, are known to produce HGF/c-met [30]. On amore general note, HGF concentration is increased at sitesof tissue damage [31-33]. After partial hepatectomy in therat, HGF levels are even measurably elevated in the blood[34]. Taken together, these increases in local and systemicHGF could provide a key chemotactic signal to mobilizeand attract hMSC for tissue repair.

Here we identified HGF/SF as a potent regulator ofhMSC function, regulating migration and proliferation invitro. Our study demonstrates the production of HGF andthe expression of the HGF receptor c-met by a defined stro-mal cell population, human mesenchymal stem cells. Thesecells were shown by FACS analysis to lack any lineage-specific surface marker expression, including CD4, CD14,CD34, and CD117. In addition, the cells were capable ofdifferentiation into adipocytes, chondrocytes, andosteoblasts. Accordingly, these cells represent the bonemarrow stromal cell population described by the groups ofPittenger [14] and Haynesworth [13] as MSC. Migration ofhMSC was increased by HGF in Boyden chamber assays(chemotactic effect), whereas proliferation of hMSC wasnegatively influenced. Furthermore, HGF promoted therepopulation of a cell-free “wound” in a cell monolayerwounding model. We attribute this repopulation mainly tothe exogenously added HGF, since hMSC did not upregu-late HGF production after the cell monolayer was wounded(data not shown).

Expression of HGF and c-met was also reported byTakai [19] in a bone marrow stromal cell population, but thispopulation was not further characterized. Bone marrow stro-mal cells are widely used as feeder layers for long-term cul-tures of hematopoietic stem cells. Due to different isolationand culture procedures, these cells are less homogenous thanthe MSC used here. It is possible, however, that hMSCs arealso contained in bone marrow stromal cells and that, in fact,hMSCs are the source of HGF required for hematopoiesis inthe coculture system. This conclusion is supported by the factthat our hMSC produced similar amounts of HGF as did thefeeder cells described by Takai et al. [19] and, furthermore,that neither the bone marrow stromal cells nor our hMSCshowed increased proliferation in response to HGF. Theamount of HGF expressed by hMSC (0.7-2 ng/ml) is higherthan the normal HGF serum concentration (0.24-0.33 ng/ml[35]). As mentioned, HGF concentration can be elevated in theblood after wounding occurs [34]. We suggest that increased

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Figure 8. HGF inhibits cell proliferation in hMSC. ATP content ofhMSC was measured as a surrogate marker of cell proliferation at 1,2, 3, 4, and 7 days after continuous stimulation with HGF at 0, 25,and 50 ng/ml. The medium containing 20% FCS served as a positivecontrol, while the stimulation medium was supplemented with only2% FCS. This figure shows mean values of three different donors,each in duplicate.

413 HGF/c-met in Mesenchymal Stem Cells

systemic HGF may mobilize bone marrow or tissue residenthMSC to colonize damaged target organs. According to ourin vitro results, the elevated HGF should also inhibit hMSCproliferation. We speculate that the local environment willultimately determine hMSC differentiation into mature tissuecells, as is the case in embryonic development.

HGF could possibly help to mobilize autologous hMSCand to direct possible allogeneic hMSC in future stem celltherapy. The role of HGF and its possible role in dysregu-lation of wound healing (like that described for TGF-β andother growth factors in diabetic mice, for instance) [36, 37]have to be further clarified. The observation that ischemicor traumatic rat brain extracts induced production of HGF

in hMSC [37, 38] also supports the idea of HGF as animportant factor for tissue repair by (transplanted or autolo-gous) hMSC. The notion that HGF/c-met signaling may beinvolved in hMSC mobilization and recruitment to damagedtissues is entirely compatible with previous reports that thisimportant regulator of cell motion and differentiation is crit-ically involved in normal development of epithelial tissues[39], as well as the metastatic spread of tumor cells [40].

ACKNOWLEDGMENTS

We thank the Department of Orthopedic Surgery,University Hospital Aachen, and their patients for providingbone spongiosa.

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