Rho Inhibition Induces Migration of Mesenchymal Stromal Cells

Abstract

Although mesenchymal stromal cells (MSCs) are being increasingly used as cell therapeutics in clinical trials, the mechanisms that regulate their chemotactic migration behavior are incompletely understood. We aimed to better define the ability of the GTPase regulator of cytoskeletal activation, Rho, to modulate migration induction in MSCs in a transwell chemotaxis assay. We found that culture-expanded MSCs migrate poorly toward exogenous phospholipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) in transwell assays. Moreover, plasma-induced chemotactic migration of MSCs was even inhibited after pretreatment with LPA. LPA treatment activated intracellular Rho and increased actin stress fibers in resident MSCs. Very similar cytoskeletal changes were observed after microinjection of a cDNA encoding constitutively active RhoA (RhoAV14) in MSCs. In contrast, microinjection of cDNA encoding Rho inhibitor C3 transferase led to resolution of actin stress fibers, appearance of a looser actin meshwork, and increased numbers of cytoplasmic extensions in the MSCs. Surprisingly, in LPA-pretreated MSCs migrating toward plasma, simultaneous addition of Rho inhibitor C2I-C3 reversed LPA-induced migration suppression and led to improved migration. Moreover, addition of Rho inhibitor C2I-C3 resulted in an approximately 3- to 10-fold enhancement of chemotactic migration toward LPA, S1P, as well as platelet-derived growth factor or hepatocyte growth factor. Thus, inhibition of Rho induces rearrangement of actin cytoskeleton in MSCs and renders them susceptible to induction of migration by physiological stimuli.
Disclosure of potential conflicts of interest is found at the end of this article.

Full text

Rho Inhibition Induces Migration of Mesenchymal Stromal Cells
BITHIAH GRACE JAGANATHAN,
a
BRIGITTE RUESTER,
a
LARS DRESSEL,
a
STEFAN STEIN,
b
MANUEL GREZ,
b
ERHARD SEIFRIED,
a
REINHARD HENSCHLER
a
a
Institute of Transfusion Medicine and Immune Hematology, German Red Cross Blood Donor Service,
University of Frankfurt, Frankfurt am Main, Germany;
b
Chemotherapeutic Research Institute Georg Speyer Haus,
Frankfurt, Germany
Key Words. Rho GTPases • Actin • Chemotaxis • Migration • Mesenchymal stem cells
ABSTRACT
Although mesenchymal stromal cells (MSCs) are being in-
creasingly used as cell therapeutics in clinical trials, the
mechanisms that regulate their chemotactic migration be-
havior are incompletely understood. We aimed to better
define the ability of the GTPase regulator of cytoskeletal
activation, Rho, to modulate migration induction in MSCs in
a transwell chemotaxis assay. We found that culture-ex-
panded MSCs migrate poorly toward exogenous phospho-
lipids lysophosphatidic acid (LPA) and sphingosine-1-phos-
phate (S1P) in transwell assays. Moreover, plasma-induced
chemotactic migration of MSCs was even inhibited after
pretreatment with LPA. LPA treatment activated intracel-
lular Rho and increased actin stress fibers in resident MSCs.
Very similar cytoskeletal changes were observed after mi-
croinjection of a cDNA encoding constitutively active RhoA
(RhoAV14) in MSCs. In contrast, microinjection of cDNA
encoding Rho inhibitor C3 transferase led to resolution of
actin stress fibers, appearance of a looser actin meshwork,
and increased numbers of cytoplasmic extensions in the
MSCs. Surprisingly, in LPA-pretreated MSCs migrating
toward plasma, simultaneous addition of Rho inhibitor
C2I-C3 reversed LPA-induced migration suppression and
led to improved migration. Moreover, addition of Rho in-
hibitor C2I-C3 resulted in an approximately 3- to 10-fold
enhancement of chemotactic migration toward LPA, S1P, as
well as platelet-derived growth factor or hepatocyte growth
factor. Thus, inhibition of Rho induces rearrangement of
actin cytoskeleton in MSCs and renders them susceptible
to induction of migration by physiological stimuli. S
TEM
CELLS 2007;25:1966 –1974
Disclosure of potential conflicts of interest is found at the end of this article.
I
NTRODUCTION
Increasing evidence suggests that mesenchymal stromal cells
(MSCs) are a promising cell source for tissue engineering, tissue
regeneration, and gene therapy applications. MSCs isolated
from human bone marrow can differentiate into adipocytes,
chondrocytes, and osteocytes [1] but can also acquire markers of
other cell types such as myocytes, endothelial cells, cardiomy-
ocytes, and neuron-like cells [2, 3]. These cells could be cul-
tured in an undifferentiated state up to 20 – 40 population dou-
blings [2]. Apart from bone marrow, MSCs have been isolated
from adipose tissue, peripheral blood, cord blood [4 – 6], umbil-
ical cord, muscle, and pancreas [7, 8]. In addition, MSCs have
been identified in the hematopoietic microenvironment and have
been shown to mediate accelerated hematopoietic recovery after
hematopoietic stem cell transplantation [9] or to confer immu-
nosuppressive properties [10].
The defective migration potential of MSCs has been discussed
to hamper their effective use for gene therapy or tissue regenera-
tion. For example, after i.v. injection, it has been difficult to trace
the transplanted MSCs in different organs [11]. Data from other
groups as well as our own show that i.v. injected MSCs may be
physically trapped in the lungs [12, 13]. However, preclinical
models of MSC transplantation have also shown engraftment into
various tissues [11, 14]. It has been postulated that for egress from
the bloodstream, i.v. injected MSCs are capable of performing at
least principally some of the steps that have been recognized to
regulate the extravasation of leukocytes as defined by Springer et
al. [15, 16]. These imply, in addition to selectin-dependent inter-
actions, also integrin-mediated binding and its enhancement
through chemokines [13, 17].
Rho family GTPases have been described as important
signaling molecules to the cytoskeleton, regulating the coordi-
nated assembly and activation of actin with actin-binding pro-
teins such as paxillin and
␣
-actinin [18, 19]. Gu et al. have
implicated a crucial role of the activation status of Rac GTPases
in the proliferation and migration of hematopoietic stem and
progenitor cells [20]. We have recently demonstrated that treat-
ment of hematopoietic progenitor cells with stromal cell-derived
factor (SDF)-1
␣
results in an intracellular signal that requires
intact Rho GTPase signaling [21, 22] and that Rho itself is a
regulator of migration responses in hematopoietic progenitor
cells [23]. We therefore hypothesized that signaling through
Rho family GTPases would influence the migration response in
MSCs.
We show here that modulation of Rho GTPase can increase
the responsiveness of human MSCs to physiological stimuli
such as stimulation by lysophospholipids. This involves a
change in their actin cytoskeletal activation status and can turn
a signal that negatively regulates MSC chemotaxis into a signal
that effectively induces chemotactic migration.
Correspondence: Reinhard Henschler, M.D., Institute of Transfusion Medicine and Immune Hematology, University Hospital Frank-
furt, Sandhofstrasse 1, D 60528 Frankfurt, Germany. Telephone: ⫹49 69 6782 191; Fax: ⫹49 69 6782 258; e-mail:
rhenschler@web.de Received March 8, 2007; accepted for publication May 3, 2007; first published online in S
TEM CELLS EXPRESS May
17, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0167
T
HE
S
TEM
C
ELL
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ICHE
STEM CELLS 2007;25:1966 –1974 www.StemCells.com

M
ATERIALS AND
M
ETHODS
Chemicals and Reagents
Isobutylmethylxanthine,
␤
-glycerophosphate, dexamethasone, ascorbic
acid, indomethacin, insulin, sphingosine-1-phosphate, lysophosphatidic
acid, paraformaldehyde, human laminin, and cell culture tested bovine
serum albumin (BSA) were purchased from (Sigma-Aldrich, St. Louis,
http://www.sigmaaldrich.com). Rho kinase inhibitor Y27632 was from
(Calbiochem, San Diego, http://www.emdbiosciences.com). Trans-
forming growth factor (TGF)-
␤
1, basic fibroblast growth factor
(bFGF), platelet-derived growth factor (PDGF)-bb, and hepatocyte
growth factor (HGF), as well as fluorescence-conjugated monoclonal
antibodies for CD45 (phycoerythrin-Cy5), CD73 (phycoerythrin),
CD90 (fluorescein isothiocyanate), and CD105 (phycoerythrin), were
from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com).
Trypsin was from Invitrogen (Carlsbad, CA, http://www.invitrogen.
com). Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) and
anti-pan-cadherin antibody (CH-19) were from Sigma. Anti-Rho anti-
body was from Pierce (Rockford, IL, http://www.piercenet.com) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Ab-
cam (Cambridge, U.K., http://www.abcam.com).
Isolation of MSCs from Bone Marrow
MSCs were isolated from bone marrow samples of patients under-
going hip surgery after informed consent by a procedure approved
by the local ethics committee. The bone marrow cells were sus-
pended in Iscove’s modified Dulbecco’s medium supplemented
with 500 U/ml heparin. Mononuclear cells were separated by den-
sity gradient centrifugation at 300g for 20 minutes. The cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM) low glu-
cose with 20% (vol/vol) fetal bovine serum (FBS) supplemented
with bFGF (25 ng/ml) at a density of 5 ⫻ 10
6
cells per milliliter.
The medium was changed every 2–3 days. After 2–3 weeks, a layer
of spindle-shaped cells had formed (MSC). The cells were passaged
1:3 at 80% confluence.
Differentiation of MSCs
To assess their differentiation potential, freshly trypsinized MSCs
were seeded into differentiation medium. Briefly, MSCs at passages
3, 6, and 9 were seeded at 1 ⫻ 10
4
per cm
2
on tissue culture plastic
in the presence of 10 mM
␤
-glycerophosphate, 0.1
␮
M dexameth-
asone, and 60
␮
M ascorbic acid-2-phosphate in DMEM/10% FBS
for induction of osteogenic differentiation; 1
␮
M dexamethasone,
0.2 mM indomethacine, 0.5 mM isobutylmethylxanthine, and 10
␮
g/ml insulin in DMEM/10% FBS for adipocytic differentiation; or
at 2 ⫻ 10
5
cells per milliliter in a 15-ml polypropylene tube (Becton,
Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) in
0.1
␮
M dexamethasone, 50
␮
g/ml ascorbic acid, 6.25 ng/ml selenic
acid, 6.25
␮
g/ml linoleic acid, 6.25
␮
g/ml insulin, and 10 ng/ml
TGF-
␤
in DMEM without FBS in a micromass culture for chon-
drogenic differentiation. After 3 weeks, differentiation of the cells
was assessed after fixation with methanol and subsequent staining
with oil red O, alkaline phosphatase, or addition of safranin O for
chondrogenic cells. All three, adipogenic, osteogenic, and chondro-
genic differentiation, yielded more than 50% of differentiated cells
at the different tested passages.
Chemotaxis Assay
The transwell migration assay for human MSCs has been described
previously [24]. Briefly, MSCs between passages 3 and 10 were
trypsinized, washed once by centrifugation in DMEM/10% FBS
(Gibco, Grand Island, NY, http://www.invitrogen.com), and kept in
suspension in DMEM with 0.1% BSA at 37°C at a density of 1–5 ⫻
10
5
cells per milliliter in 15-ml polypropylene tubes in a cell culture
incubator for up to 1 hour. To the lower wells of 48-well chambers
(Neuro Probe, Gaithersburg, MD, http://www.neuroprobe.com), mi-
gration medium (DMEM/1% BSA) and migration-inducing sub-
stances were added. Upper wells were filled with migration medium
with cells (approximately 32
␮
l, 20,000 cells per well), in some
cases also containing test substances. Filter pore size was 8
␮
m. The
mounting steps were performed within 15 minutes, and the chamber
was placed into a humidified incubator. After 6 hours, assays were
stopped by removal of the medium from the upper wells and careful
removal of the filters. Filters were fixed with methanol by brief
submersion and were subsequently wiped on the upper side using
the Neuro Probe wiper. Filters were stained with May-Gru¨nwald-
Giemsa (Merck & Co., Whitehouse Station, NY, http://www.merck.
com) solution for 5 (May-Gru¨nwald) and 15 (Giemsa) minutes
each. Evaluation of completed transmigration was performed under
a light microscope, and random fields were scanned (2– 4 per filter)
for the presence of cells at the lower membrane side only. Absolute
numbers of migrated cells were calculated by counting migrated
cells in parts of the entire filter using scaled grids. Results of the
migration experiments were expressed as percentage of input
MSCs, calculated by dividing the absolute numbers of migrated
cells by input number of MSCs added.
Immunohistochemistry
For analysis of cytoskeleton-associated proteins, trypsinized MSCs
were seeded on plastic slides precoated with fibronectin (10
␮
g/ml)
(Becton Dickinson), allowed to adhere overnight, rinsed carefully
two times with phosphate-buffered saline (PBS), and fixed for 10
minutes in PBS containing 4% paraformaldehyde. Subsequently,
cells were permeabilized by incubation with 0.5% Triton X-100 in
PBS for 10 minutes. For immunolabeling, the cells were incubated
with PBS, 3% BSA, and 0.5% Triton X-100 containing phalloidin-
TRITC for 2 hours at room temperature and analyzed using a
fluorescence microscope (Carl Zeiss, Jena, Germany, http://www.
zeiss.com). Images were taken on a PC using a 1.3 megapixel CCD
camera and Axion Software (Zeiss).
Microinjection of cDNA
cDNAs encoding RhoA V14 (kindly provided by Dr. M. Ruthardt,
University of Frankfurt, Germany) or C3 transferase from Clostrid-
ium limosum (kindly provided by Dr. H. Barth, University of
Freiburg, Germany) [23, 25] were subcloned into the PINCO
gamma retroviral vector, which is derived from Moloney murine
leukemia virus [26]. Plasmids, including an empty PINCO control
vector, were dissolved in sterile water at 0.2–1
␮
g/ml and were
microinjected into MSCs that were seeded on sterile glass cover
slides precoated with human laminin (100 ng/ml) using an Eppen-
dorf InjectMan NI2 fitted with a microinjection needle and Femto-
Jet mounted to a Zeiss microscope Axiovert 135. Cells were incu-
bated at 37°C, 5% CO
2
for another 6 –12 hours before fluorescent
microscopic analysis.
Real-Time Polymerase Chain Reaction
Total cellular RNA was isolated from MSCs in TRIzol RNA ex-
traction buffer according to manufacturer’s instructions (Invitro-
gen). Quantification of RNA was performed by measuring the
absorbance at 260 nm (NanoDrop, Wilmington, DE, http://www.
nanodrop.com). cDNA was produced from 1
␮
g of RNA by reverse
transcription in a 50-
␮
l reaction using SuperScript II (Invitrogen)
and oligo(dT) primers at 55°C for 1 hour. The conditions for
subsequent real-time polymerase chain reaction (PCR) for Edg
receptors were 35 cycles at a denaturation temperature of 94°C
for 15 seconds, annealing at 60°C for 45 seconds, and extension at
72°C for 30 seconds using a SYBR Green PCR mix. Primers for
Edg-2, -4, -5, and -7 were designed using Primer3 Software (http://
fokker.wi.mit.edu/primers). The primers used for these were: Edg-2,
sense 5⬘-TGTCTCGGCATAGTTCTGGA-3⬘ antisense 5⬘-TTCTT-
TGTCGCGGTAGGAGT-3⬘; Edg-4, sense 5⬘-AGGCTGTGAGTC-
CTGCAATGT-3⬘ antisense 5⬘-TCTCAGCATCTCGGCAAGAGT-
3⬘; Edg-5, sense 5⬘-GGCCTAGCCAGTTCTGAAA-3⬘ antisense
5⬘-GCAATGAGCACCAGAAGGTT-3⬘; Edg-7, sense 5⬘-GCA-
TACAAGTGGGTCCATCA-3⬘ antisense 5⬘-TCACGACGGAGT-
TGAGCA-3⬘; and GAPDH, sense 5⬘-GGGAAGGTGAAGGTCG-
GAGT-3⬘ antisense 5⬘-GGGTCATTGATGGCAACAATA-3⬘. PCR
fragments were analyzed on a 2% agarose gel stained with ethidium
bromide.
1967Jaganathan, Ruester, Dressel et al.
www.StemCells.com

Subcellular Fractionation and Detection of
Membrane-Bound Rho
MSCs were pretreated with lysophosphatidic acid (LPA) (25
␮
M)
or C2I-C3 (1
␮
g/ml) for 2 hours. The cells were briefly washed by
centrifugation with ice-cold PBS twice. The membrane and cyto-
solic proteins were separated using Qproteome Cell Compartment
Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) according
to manufacturer’s instructions. Protein content was quantified using
a protein detection kit (Bio-Rad, Hercules, CA, http://www.bio-
rad.com). The membrane (30
␮
g/lane) and cytosolic fractions (40
␮
g/lane) were run on a 12% polyacrylamide gel. The gels were
blotted onto a nitrocellulose membrane and detected with either
anti-Rho antibody or anti-pan-cadherin and GAPDH followed by
horseradish peroxidase-conjugated anti-mouse IgG antibody (Sigma).
The immunoblotted proteins were visualized with the enhanced chemi-
luminescent reagents (Amersham Biosciences, Piscataway, NJ, http://
www.amersham.com).
Lentiviral Production, Titration, and Infection of
Human MSCs
The packaging plasmid pCMV⌬R8.91 encodes the human immu-
nodeficiency virus-1 regulatory proteins tat and rev as well as
⌬plasmid pCMV gag and pol precursors [27]. Plasmid pMD.G
expresses vesicular stomatitis virus glycoprotein G. Pseudotyped
lentiviruses were produced by transient calcium-phosphate transfec-
tion of 293T cells with pCMV⌬R8.91, pMD.G, and the lentiviral
transfer vectors (SIEW) [28] into which cDNAs encoding RhoV14
or C3 transferase (kindly provided by Dr. Holger Barth, University
of Freiburg, Germany) were subcloned. Viral supernatants were
collected 48 –72 hours after transfection. Viral titers were deter-
mined on 293T cells as described previously and amounted to
0.1–1 ⫻ 10
8
titer units/ml [29]. Lentiviral transduction of human
MSCs was performed by seeding the cells in a 6-well plate (1 ⫻ 10
5
cells per well) in DMEM/20% FBS and bFGF (5 ng/ml). The cells
were allowed to attach for 24 hours. Viral supernatants of different
vectors containing multiplicity of infection 1–5 were added to the
cells in the presence of polybrene (4
␮
g/ml). After 24 hours, the
cells were washed and incubated with fresh medium. The cells
were split 72 hours later and expanded. Subsequently, transduced
cells were identified by the presence of green fluorescent protein
reporter gene by flow cytometry.
Flow Cytometry
For flow cytometric analysis, cells were harvested by trypsinization,
washed once with PBS, and resuspended in PBS containing 2%
FBS. The cells were incubated with the conjugated antibodies for 30
minutes on ice. The cells were washed by centrifugation and ana-
lyzed in a flow cytometer (Becton, Dickinson).
Statistical Evaluation
Analysis was performed through Microsoft (Redmond, WA, http://
www.microsoft.com) Excel statistics modules.
R
ESULTS
To characterize the regulation of chemotactic migration in
MSCs by the GTPase Rho in conjunction with several physio-
logical stimuli, culture-expanded MSCs were established from
the bone marrow of 10 different donors by adherence selection.
As shown in Figure 1A and 1B, MSCs could be expanded up to
six log scales and were found to be negative for the hematopoi-
etic marker CD45 and positive for the mesenchymal markers
CD73, CD90, and CD105 by flow cytometric analysis. The
isolated MSCs could be differentiated into adipocytes, osteo-
cytes, or chondrocytes using specific induction medium as
shown by the positive staining using oil red O for adipocytes,
which stain the lipid globules, alkaline phosphatase, for osteo-
blastic cells or safranin O, indicating induction of chondrocytic
differentiation (Fig. 1C). We next investigated the chemotaxis
of MSCs toward various chemoattractants in the transwell assay.
In a positive control, human plasma added to the lower wells
resulted in a bell-shaped dose-response curve with mean 4.5%
of total MSCs transmigrated, as observed previously (Fig. 1D)
[24]. PDGF and bFGF have previously been reported to induce
chemotactic migration in MSCs [24, 30, 31]. Similar to our
previous study, where PDGF induced chemotactic migration of
MSCs with variable efficiency and with on average lower effi-
ciency than plasma [24], we observed a comparatively weaker
transmigration of the tested MSCs as measured with plasma
(Fig. 1D). SDF-1
␣
, which induced chemotaxis in hematopoietic
stem cells, failed to cause detectable chemotactic migration of
MSCs (data not shown). Also, the lysophospholipids LPA or
sphingosine-1-phosphate (S1P) did not induce MSC migration
(Fig. 1D).
MSCs Express LPA Receptors, but LPA Inhibits
Their Plasma-Induced Chemotaxis and Stimulates
Actin Stress Fiber Formation
Stimulation with phospholipid mediators LPA and S1P has been
reported to regulate migration and adhesion events in several
cell types [32–37]. We therefore investigated whether MSCs
would express receptors for these substances. Figure 2 shows
the results of semiquantitative real-time PCR reactions from
MSC preparations from three different donors. We found three
of the LPA receptors (Edg-2, -4, -7) and one S1P receptor
(Edg-5) expressed. The expression of these receptors was con-
sistently found in different donors, comparable with human
umbilical vein endothelial cells that were included as control.
We next pretreated MSCs with LPA and tested them in plasma-
induced transwell migration. This resulted in clear inhibition of
MSC transmigration (Fig. 2B). Similar data were also obtained
with S1P (not shown). These findings led us to investigate
whether LPA would also induce alterations in the actin cytoskel-
eton. Staining of actin with fluorescence-tagged phalloidin re-
vealed the presence of actin stress fibers in untreated control
MSCs, which became more prominent after exposure of MSC to
LPA (Fig. 3A, 3B). Moreover, at lower magnification, a more
intense actin staining was observed after LPA treatment com-
pared with controls. The intensity of the overall actin staining
signals between the cells remained relatively homogeneous un-
der each of the conditions (Fig. 3C, 3D).
Activation of Rho Regulates Actin Cytoskeleton
in MSCs
Since Rho has been shown to control cytoskeletal activation in
other adherent cell types such as endothelial cells, we investi-
gated whether LPA would induce Rho activation also in MSCs.
The Rho activation assay shown in Figure 2C revealed induction
of membrane-bound Rho, corresponding to Rho activation, after
exposure of MSCs to LPA. Involvement of Rho in induction of
actin stress fiber formation in MSCs was also observed after
microinjection of cDNA encoding the dominant active isoform
of Rho, RhoAV14. Actin stress fibers became more prominent
compared with vector transfected control cells (Fig. 3E, 3F).
The data prompted us to examine the possibility that inac-
tivation of Rho, on the opposite, might suppress stress fiber
formation in MSCs. We therefore microinjected cDNA encod-
ing C3 transferase, a known specific inhibitor of Rho, into
MSCs. Stress fibers in MSCs almost disappeared, and actin
staining revealed much thinner, more flexible-appearing fiber
structures (Fig. 3G). These appeared as shorter in length and
were included in relatively smaller meshwork-like structures
that extended toward the cytoplasmic membrane in more fila-
mentous extensions (Fig. 3G). To investigate whether Rho could
1968 MSC Migration and Rho Activation

Figure 1. Characterization of mesenchymal stromal cells (MSCs) used. (A): Growth curve of MSCs isolated from human bone marrow of six of
the donors used in this study. Values represent means ⫾ SD of three determinations of cell concentrations at each time point. Cumulative cell numbers
were calculated. (B): Flow cytometric analysis of cell surface markers on MSCs using antibodies against CD45, CD73, CD90, and CD105. (C):
Differentiation of MSCs into adipocytes, osteocytes, and chondrocytes. MSCs were treated with specific induction medium and cultured for 14 days.
The cultures were stained with oil red O staining for adipocytes and alkaline phosphatase for osteocytes. MSCs were grown in a micromass culture
for chondrogenic differentiation, and differentiation into chondroblasts was revealed by safranin O staining; magnification ⫻100. (D): Migration of
MSCs induced by various stimuli present in the lower wells of transwell chambers. Twenty thousand MSCs were seeded into the upper wells of
chemotaxis chambers and allowed to migrate for 6 hours toward various indicated factors at the indicated range of concentrations, and absolute
numbers of transmigrated MSCs were determined. The responses varied in percentage among different donors, and representative results are shown.
Values are means of four replicate determinations. MSCs were from passages 4 and 6 (B), passage 6 (C), or passage 4 (D). Abbreviations: LPA,
lysophosphatidic acid; No., number; PDGF, platelet-derived growth factor; S1P, sphingosine-1-phosphate.
1969Jaganathan, Ruester, Dressel et al.
www.StemCells.com

be inactivated by the cell-permeable soluble Rho inhibitor C2I-
C3, we assessed the degree of intracellular Rho activation by
determination of cytoplasmic and membrane-bound Rho. A
decrease in membrane-bound Rho was seen after treatment of
MSCs with C2I-C3 (Fig. 2C). Taken together, modulation of
Rho activity is associated with changes in cytoskeletal activa-
tion as revealed by the presence of actin stress fibers in MSCs.
Inhibition of Rho Influences Transwell Migration
of MSCs
From the previous findings, we hypothesized that modification
of the actin cytoskeleton might alter the migration behavior of
MSCs. We therefore investigated the transwell migration of
MSCs after Rho inhibition. In a first set of experiments, we
analyzed the transwell migration induced by human plasma.
Inhibition of Rho with C2I-C3 transferase or Y27632 resulted in
significantly increased migration toward plasma (Fig. 4A, 4B).
Both treatment of MSCs during the migration period as well as
pretreatment and subsequent washout of C2I-C3 before the
assay were equally effective in inducing chemotactic migration
(data not shown). A different behavior was observed in MSCs
under serum-deprived conditions after treatment with LPA; al-
though compared with untreated MSCs, pretreatment with LPA
inhibited migration toward plasma (Fig. 4C), migration of LPA
pretreated MSCs was enhanced when Rho was inhibited (Fig.
4C). Therefore, the inactivation of Rho can reverse the migra-
tory response of MSCs to the same signal.
Since the effects of treatment with soluble inhibitors are
expected to be transient in the cells, we performed lentiviral
transduction of MSCs with cDNA encoding dominant active
Rho (RhoV14) or C3 transferase. Due to the relatively high
serum requirements of the transduced MSCs, the transwell mi-
gration assays had to be performed in the presence of 10%
plasma, yielding a relatively high spontaneous migration com-
pared with the previous serum-deprived conditions (Fig. 5).
Consistent with the previous results after short-term modulation
of Rho, C3 transferase transduced MSCs, however, showed
increased migration capacity compared with the mock trans-
duced controls, whereas MSCs transduced with constitutively
active RhoA migrated less efficiently under these conditions
(Fig. 5), confirming the data obtained with soluble Rho
inhibitor.
In addition to plasma-induced MSC migration, we also
tested the chemotactic response of MSCs to phospholipids or
growth factors in the absence and presence of Rho inhibition but
in the complete absence of serum. Whereas MSCs showed to be
nonresponsive or only weakly responsive to PDGF, HGF, LPA,
and S1P present in the lower wells in the absence of serum,
C2I-C3 treated MSCs showed 3- to 10-fold increased chemo-
taxis toward PDGF, HGF, LPA, and S1P (Fig. 6).
In conclusion, our results demonstrate that the inactivation
of Rho GTPase by exogenous C2I-C3 or transfection with C3
transferase cDNA alters the actin activation status in MSCs,
reducing stress fiber formation and appearance of a more fila-
mentous actin fiber type. In parallel, induction of MSC migra-
tion toward plasma is enhanced, and MSCs can now be induced
to migrate to a substantial degree toward single physiological
stimuli such as LPA, S1P, PDGF, or HGF.
D
ISCUSSION
Migration of MSCs to sites of tissue injury is a necessary feature
for tissue reconstitution, a process which was found to be guided
by specific soluble factors such as growth factors and/or che-
mokines. Our results show a pivotal role for Rho activation in
actin cytoskeletal modification and in the induction of a migra-
tory response in MSCs by soluble factors.
Role of Rho in Chemotactic Migration of
Hematopoietic and Mesenchymal Progenitor Cells
Rho GTPases have been shown to be critical regulators of the
migration of several cell types, including hematopoietic progen-
itor cells (HPCs). Absence of the Rac1 and -2 GTPases results
in severe migration defects in hematopoietic progenitor cells
and impaired engraftment after transplantation [20]. In contrast,
inhibition of Rho using C2I-C3 or transfection with C3 trans-
ferase cDNA led to increased in vitro migration of HPCs [23].
Ghiaur et al. [38] have furthermore demonstrated that overex-
Figure 2. MSCs express Edg receptors, and the Edg-2, -4, and -7
cognate ligand LPA modulates chemotaxis and the activation status of
Rho in MSCs. (A): Real-time polymerase chain reaction analysis of Edg
receptors for LPA (Edg-2, Edg-4, Edg-7) and S1P (Edg-5) in MSCs with
HUVECs as positive control. (B): Transwell migration of MSCs against
plasma (0.5%) prestimulated with LPA. MSCs were pretreated or not
with LPA (25
␮
M) and allowed to migrate for 6 hours in a transwell
chamber. Values are means ⫾ SD (n ⫽ 4) of total migrated cells. (C):
Analysis of Rho activation status in MSCs. MSCs were treated with
LPA (25
␮
M) or C2I-C3 (1
␮
g/ml) for 2 hours. Membrane and cyto-
plasmic fractions were isolated, and equal amounts of protein were
analyzed using a Western blot for all three treatments in each cell
compartment type with Rho-specific antibody. Staining with anti-pan-
cadherin antibody was included to control the membrane fraction, and
anti-GAPDH antibody served as loading control; ⴱⴱⴱ p ⱕ .0001. MSCs
were from passages 4 or 5. Abbreviations: GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; HUVEC, human umbilical vein endothelial
cell; LPA, lysophosphatidic acid; MSC, mesenchymal stromal cell; NC,
negative control (H
2
O).
1970 MSC Migration and Rho Activation

pression of the dominant negative RhoA N19 mutant enhances
hematopoietic engraftment in a murine bone marrow transplan-
tation model. The results of this study demonstrate that, in
MSCs, a cell type which shows much stronger activation of
F-actin than HPCs, inhibition of Rho will not only result in a
quantitative difference of migration but instead reverse induc-
tion of adhesion into induction of migration.
In HPCs, modification of SDF-1
␣
and its receptor CXCR4
has been postulated to be of importance also for Rac GTPase
mediated migration induction [20]. Although we and others
have demonstrated the presence of the SDF-1
␣
receptor CXCR4
also on human MSCs [17, 24, 39], SDF-1
␣
did not induce
chemotactic migration of MSCs in our hands even after pre-
treatment with Rho inhibitor C2I-C3. Therefore, the SDF-1
␣
signaling pathway seems not to be significantly involved in
migration responses of MSCs. Yet, our in vitro data suggest that
inhibition of Rho in MSCs and their subsequently increased
migratory function might lead to improved in vivo functions of
MSCs (e.g., to a more efficient passage through lung capillaries
and to increased responsiveness to the physiological signals that
induce their extravasation and deposition in target tissues).
Alterations in Actin Cytoskeleton Morphology
Induced After Inhibition of Rho
Previous studies have indicated the involvement of Rho
GTPases in the cytoskeletal activation in fibroblasts, with over-
expression of Rho resulting in formation of stress fibers and of
focal adhesion complexes [40, 41]. The cytoskeletal changes
brought about by Rho GTPases have been shown to correlate
with changes in cell morphology and with altered cell migration
[19]. We have shown here the functionality of the cell-perme-
able C3 transferase C2I-C3 to inhibit Rho and to induce similar
changes in actin cytoskeleton in MSCs as seen in fibroblasts
after transfection with cDNAs encoding Rho isoforms. Clearly,
finer actin structures within the MSCs were associated with an
induced chemotactic migration response. We also showed that
an opposite effect, that is, induction of a nonmigratory pheno-
type, was induced by overexpression of an activating mutant of
RhoA in MSCs. MSCs are therefore susceptible to modulation
of their migratory response through modifications of their Rho
GTPase activation.
Activation of Actin Cytoskeleton and Migration in
MSCs by Lysophospholipids
S1P and LPA have been described previously as inducers of
Rho GTPases that modulate the migration response of various
cell types [32, 36, 37, 42]. Meriane et al. have recently impli-
cated RhoA and Rho kinase in the induction of migration in
MSCs [43]. In a collagen gel migration assay implying activa-
tion of matrix metalloproteinases, the authors demonstrate that
S1P leads to an increase in serum-induced MSC migration,
which is decreased after treatment with Rho kinase inhibitor.
Interestingly, in the migration model of Meriane et al., ma-
trix metalloproteinase-mediated migration stimulated by S1P
Figure 3. Modification of the actin cytoskeleton by Rho GTPase. Fluorescent microscopic analysis of mesenchymal stromal cells (MSCs)
immobilized on fibronectin and 2 hours after treatment with phosphate-buffered saline (A, C) and lysophosphatidic acid (25
␮
M [B, D]) or 6 hours
after microinjection of immobilized MSCs with mock vector (E), RhoAV14 (F), or C3 transferase (G) cDNAs. Actin filaments were stained with
phalloidin-tetramethylrhodamine B isothiocyanate and visualized through an inverted microscope. Magnification: (A, B), ⫻100; (C, D), ⫻20; (E–G),
⫻40. MSCs were from passage 5 or 6.
1971Jaganathan, Ruester, Dressel et al.
www.StemCells.com

through gelatin-precoated filters was associated with an increase
in actin stress fibers in MSCs, whereas in our transwell model,
induction of chemotactic migration was found to be enhanced
when Rho activity is downregulated and when stress fibers are
decreased. This may be explained by the fact that, during
chemotaxis, presence of S1P acting as a chemoattractant in the
lower well might not necessarily increase stress fiber formation
in the migrating MSCs. In contrast, actin stress fiber formation
is required in the migration situation described by Meriane et al.,
which involves matrix degradation. Therefore, a different acti-
vation status of the actin cytoskeleton may be optimal at differ-
ent stages of migration activation in MSCs, that is, gradient
sensing, polarization, and orientation versus subsequent migra-
tion through more solid matrix, as reflected by the observations
in the two studies.
Our results demonstrate that MSCs express receptors for
both S1P and LPA, although the results do not necessarily imply
that these receptors are functional. We demonstrated that pre-
treatment with LPA can induce divergent migration responses
depending on the activation status of Rho. Decreased transwell
migration responses were observed when RhoA activation was
induced in the presence of LPA, but this was reversed to an
induction of migration by LPA when C2I-C3 is present. Thus,
MSCs can respond bidirectionally to LPA, dependent on
whether RhoA can be activated or not.
Taken together, our results suggest that the regulation of
chemotactic migration in MSCs is dependent on the activation
status of Rho. Substances such as phospholipids LPA and S1P
can thus either act to induce the formation of a more flexible
Figure 4. Migration of mesenchymal stromal cells (MSCs) induced by
0.3% plasma present in the lower well of transwell chambers. (A–C):
MSCs were pretreated with C2I-C3 (1
␮
g/ml unless indicated other-
wise), Y27632 (10
␮
M), and/or LPA (25
␮
M) for 2 hours and seeded at
20,000 into upper wells of transwell chambers. Lower wells contained
0.3% human plasma. Cells were allowed to migrate for 6 hours, and
absolute cell numbers were determined. (A): Dose-response curve for
C2I-C3 pretreatment; (B): comparison of Rho inhibitor C2I-C3 and Rho
kinase inhibitor Y27632 pretreatment; and (C): effect of the simulta-
neous presence of Rho inhibitor C2I-C3 on migration of LPA pretreated
MSCs. Values are means ⫾ SD; n ⫽ 4; ⴱ p ⱕ .01, ⴱⴱ p ⱕ .001, ⴱⴱⴱ p ⱕ
.0001. MSCs were from passage 4 (A), passage 5 (B), or passage 6 (C).
Abbreviation: LPA, lysophosphatidic acid.
Figure 5. Transwell migration of lentivirally transduced mesenchymal
stromal cells (MSCs). MSCs were transduced using cDNAs encoding
vector only, RhoV14, or C3 transferase. Thirty-six hours after transduc-
tion, 20,000 MSCs were seeded into upper wells of transwell chambers
containing Dulbecco’s modified Eagle’s medium/10% plasma and al-
lowed to migrate for 6 hours, and absolute numbers of migrated cells
were determined. Values are means ⫾ SD; n ⫽ 4; ⴱ p ⱕ .01, ⴱⴱ p ⱕ
.001. MSCs were from passage 5.
Figure 6. Transmigration of mesenchymal stromal cells (MSCs) in
serum-free conditions induced by different stimuli present in the lower
well. MSCs were pretreated without or with C2I-C3 (1
␮
g/ml), washed
by centrifugation, seeded into chemotaxis chambers that contained the
indicated substances in the lower wells, and allowed to migrate for 6
hours. Absolute numbers of transmigrated cells were determined; values
are means ⫾ SD; n ⫽ 4. MSCs were from passages 4– 6. Abbreviations:
HGF, hepatocyte growth factor; LPA, lysophosphatidic acid; PDGF,
platelet-derived growth factor; S1P, sphingosine-1-phosphate.
1972 MSC Migration and Rho Activation

actin cytoskeleton or to increase stress fiber formation, depen-
dent on the activation status of Rho in MSCs. These results will
be of importance to further decipher the steps that lead to the
directional migration activation of mesenchymal progenitor
cells, and they may allow us to better influence or engineer
MSCs as improved cellular therapeutics for tissue regeneration.
A
CKNOWLEDGMENTS
We thank Heike Nu¨rnberger and Dr. Martin Ruthardt for pro-
viding us with the PINCO vectors encoding C3 transferase and
RhoA V14, Sabrina Boehme for technical support with migra-
tion experiments, and Kristine Eschedor for secretarial assis-
tance. This work was financially supported by Grants 0312625
and 05GN0525 from the German Ministry of Health and Re-
search (BMBF). B.G.J. is currently affiliated with the Hemato-
poietic Stem Cell Lab, Cancer Research UK, London, U.K.
D
ISCLOSURE OF
P
OTENTIAL
C
ONFLICTS
OF
I
NTEREST
The authors indicate no potential conflicts of interest.
R
EFERENCES
1 Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of
adult human mesenchymal stem cells. Science 1999;284:143–147.
2 Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of
postnatal human marrow mesodermal progenitor cells. Blood 2001;98:
2615–2625.
3 Sato Y, Araki H, Kato J et al. Human mesenchymal stem cells xe-
nografted directly to rat liver are differentiated into human hepatocytes
without fusion. Blood 2005;106:756 –763.
4 Bieback K, Kern S, Kluter H et al. Critical parameters for the isolation
of mesenchymal stem cells from umbilical cord blood. S
TEM CELLS
2004;22:625– 634.
5 He Q, Wan C, Li G. Multi-potent mesenchymal stromal cells in blood.
S
TEM CELLS 2007;25:69 –77.
6 Lin Y, Liu L, Li Z et al. Pluripotency potential of human adipose-derived
stem cells marked with exogenous green fluorescent protein. Mol Cell
Biochem 2006;291:1–10.
7 da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem
cells reside in virtually all post-natal organs and tissues. J Cell Sci
2006;119:2204 –2213.
8 Karahuseyinoglu S, Cinar O, Kilic E et al. Biology of stem cells in
human umbilical cord stroma: In situ and in vitro surveys. S
TEM CELLS
2007;25:319 –331.
9 Muguruma Y, Yahata T, Miyatake H et al. Reconstitution of the func-
tional human hematopoietic microenvironment derived from human mes-
enchymal stem cells in the murine bone marrow compartment. Blood
2006;107:1878 –1887.
10 Bocelli-Tyndall C, Bracci L, Spagnoli G et al. Bone marrow mesenchymal
stromal cells (BM-MSCs) from healthy donors and auto-immune disease
patients reduce the proliferation of autologous- and allogeneic-stimulated
lymphocytes in vitro. Rheumatology (Oxford) 2007;46:403– 408.
11 Horwitz EM, Gordon PL, Koo WK et al. Isolated allogeneic bone
marrow-derived mesenchymal cells engraft and stimulate growth in
children with osteogenesis imperfecta: Implications for cell therapy of
bone. Proc Natl Acad SciUSA2002;99:8932– 8937.
12 Anjos-Afonso F, Siapati EK, Bonnet D. In vivo contribution of murine
mesenchymal stem cells into multiple cell-types under minimal damage
conditions. J Cell Sci 2004;117:5655–5664.
13 Ruster B, Gottig S, Ludwig RJ et al. Mesenchymal stem cells display
coordinated rolling and adhesion behavior on endothelial cells. Blood
2006;108:3938 –3944.
14 Bartholomew A, Sturgeon C, Siatskas M et al. Mesenchymal stem cells
suppress lymphocyte proliferation in vitro and prolong skin graft survival
in vivo. Exp Hematol 2002;30:42– 48.
15 Springer TA. Traffic signals for lymphocyte recirculation and leukocyte
emigration: The multistep paradigm. Cell 1994;76:301–314.
16 Springer TA. Traffic signals on endothelium for lymphocyte recircula-
tion and leukocyte emigration. Annu Rev Physiol 1995;57:827– 872.
17 Honczarenko M, Le Y, Swierkowski M et al. Human bone marrow
stromal cells express a distinct set of biologically functional chemokine
receptors. S
TEM CELLS 2006;24:1030 –1041.
18 Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature
2002;420:629 – 635.
19 Hall A, Nobes CD. Rho GTPases: Molecular switches that control the
organization and dynamics of the actin cytoskeleton. Philos Trans R Soc
Lond B Biol Sci 2000;355:965–970.
20 Gu Y, Filippi MD, Cancelas JA et al. Hematopoietic cell regulation by
Rac1 and Rac2 guanosine triphosphatases. Science 2003;302:445– 449.
21 Bug G, Rossmanith T, Henschler R et al. Rho family small GTPases
control migration of hematopoietic progenitor cells into multicellular
spheroids of bone marrow stroma cells. J Leukoc Biol 2002;72:837– 845.
22 Henschler R, Piiper A, Bistrian R et al. SDF-1alpha-induced intracellular
calcium transient involves Rho GTPase signalling and is required for
migration of hematopoietic progenitor cells. Biochem Biophys Res Com-
mun 2003;311:1067–1071.
23 Gottig S, Mobest D, Ruster B et al. Role of the monomeric GTPase Rho
in hematopoietic progenitor cell migration and transplantation. Eur J Im-
munol 2006;36:180 –189.
24 Ruster B, Grace B, Seitz O et al. Induction and detection of human
mesenchymal stem cell migration in the 48-well reusable transwell assay.
Stem Cells Dev 2005;14:231–235.
25 Barth H, Preiss JC, Hofmann F et al. Characterization of the catalytic site
of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-
directed mutagenesis. J Biol Chem 1998;273:29506 –29511.
26 Zheng X, Beissert T, Kukoc-Zivojnov N et al. Gamma-catenin contrib-
utes to leukemogenesis induced by AML-associated translocation prod-
ucts by increasing the self-renewal of very primitive progenitor cells.
Blood 2004;103:3535–3543.
27 Zufferey R, Nagy D, Mandel RJ et al. Multiply attenuated lentiviral
vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997;15:
871– 875.
28 Brocke-Heidrich K, Kretzschmar AK, Pfeifer G et al. Interleukin-6-
dependent gene expression profiles in multiple myeloma INA-6 cells
reveal a Bcl-2 family-independent survival pathway closely associated
with Stat3 activation. Blood 2004;103:242–251.
29 Demaison C, Parsley K, Brouns G et al. High-level transduction and gene
expression in hematopoietic repopulating cells using a human immuno-
deficiency [correction of imunodeficiency] virus type 1-based lentiviral
vector containing an internal spleen focus forming virus promoter. Hum
Gene Ther 2002;13:803– 813.
30 Fiedler J, Etzel N, Brenner RE. To go or not to go: Migration of human
mesenchymal progenitor cells stimulated by isoforms of PDGF. J Cell
Biochem 2004;93:990 –998.
31 Schmidt A, Ladage D, Schinkothe T et al. Basic fibroblast growth factor
controls migration in human mesenchymal stem cells. S
TEM CELLS
2006;24:1750 –1758.
32 Allende ML, Dreier JL, Mandala S et al. Expression of the sphingosine
1-phosphate receptor, S1P1, on T-cells controls thymic emigration.
J Biol Chem 2004;279:15396 –15401.
33 Matsuyuki H, Maeda Y, Yano K et al. Involvement of sphingosine
1-phosphate (S1P) receptor type 1 and type 4 in migratory response of
mouse T cells toward S1P. Cell Mol Immunol 2006;3:429 – 437.
34 Ryu Y, Takuwa N, Sugimoto N et al. Sphingosine-1-phosphate, a plate-
let-derived lysophospholipid mediator, negatively regulates cellular Rac
activity and cell migration in vascular smooth muscle cells. Circ Res
2002;90:325–332.
35 Sengupta S, Kim KS, Berk MP et al. Lysophosphatidic acid downregu-
lates tissue inhibitor of metalloproteinases, which are negatively in-
volved in lysophosphatidic acid-induced cell invasion. Oncogene 2007;
26:2894 –2901.
36 Spiegel S, English D, Milstien S. Sphingosine 1-phosphate signaling: Pro-
viding cells with a sense of direction. Trends Cell Biol 2002;12:236 –242.
37 Tanski W, Roztocil E, Davies MG. Sphingosine-1-phosphate induces
G(alphai)-coupled, PI3K/ras-dependent smooth muscle cell migration.
J Surg Res 2002;108:98 –106.
38 Ghiaur G, Lee A, Bailey J et al. Inhibition of RhoA GTPase activity
enhances hematopoietic stem and progenitor cell proliferation and en-
graftment. Blood 2006;108:2087–2094.
39 Wynn RF, Hart CA, Corradi-Perini C et al. A small proportion of
mesenchymal stem cells strongly expresses functionally active CXCR4
receptor capable of promoting migration to bone marrow. Blood 2004;
104:2643–2645.
40 Nobes CD, Hall A. Rho, rac and cdc42 GTPases: Regulators of actin
structures, cell adhesion and motility. Biochem Soc Trans 1995;23:
456 – 459.
1973Jaganathan, Ruester, Dressel et al.
www.StemCells.com

41 Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly
of multimolecular focal complexes associated with actin stress fibers,
lamellipodia, and filopodia. Cell 1995;81:53– 62.
42 Vouret-Craviari V, Bourcier C, Boulter E et al. Distinct signals via Rho
GTPases and Src drive shape changes by thrombin and sphingosine-1-
phosphate in endothelial cells. J Cell Sci 2002;115:2475–2484.
43 Meriane M, Duhamel S, Lejeune L et al. Cooperation of matrix
metalloproteinases with the RhoA/Rho kinase and mitogen-activated
protein kinase kinase-1/extracellular signal-regulated kinase signaling
pathways is required for the sphingosine-1-phosphate-induced mobi-
lization of marrow-derived stromal cells. S
TEM CELLS 2006;24:
2557–2565.
1974 MSC Migration and Rho Activation