Mesenchymal stem cells for treatment and prevention of graft-versus-host disease after allogeneic hematopoietic cell transplantation.
ABSTRACT Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective therapy for hematological malignancies and inherited diseases. However, acute graft-versus-host-disease (aGVHD) is a major life-threatening complication after allo-HCT and there are few therapeutic options for severe steroid-refractory aGVHD. Preliminary studies on co-transplantation of mesenchymal stem cells (MSCs) have shown an improvement in or resolution of severe aGVHD. However, the mechanism underlying this immunosuppressive effect has not been elucidated. Most of the data suggest that the immunosuppressive effect involves soluble factors such as IL-6 or TGF-beta as well as cell-cell contact dependence. MSCs interact either directly with T cells or indirectly via other immune cells such as dendritic cells and NK cells. Here we review the immunomodulatory function of MSCs in allo-HCT and their potential usefulness in the treatment or prevention of severe acute GVHD.
- Transplantation 04/1968; 6(2):230-47. · 3.78 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The hematopoietic system of vertebrates can be completely reconstituted with hematopoietic stem cells derived from the bone marrow, fetal liver, or cord blood, or even from peripheral-blood-derived cells. A cellular marker to identify those cells is the proteoglycan CD34, although we have shown that the earliest identifiable hematopoietic stem cell is a CD34− fibroblast-like cell which can differentiate into CD34+ hematopoietic precursors. Peripheral blood mononuclear cells were isolated from the heparinized blood of a dog and incubated in tissue culture in the presence of interleukin 6. After 10-14 days, an adherent layer of fibroblast-like cells had developed and cells were immortalized using the SV-40 large T antigen. Cells were cloned and subcloned by measures of limiting dilution, and various fibroblast-like clones were established. These fibroblast-like cells either do not express the CD34 antigen or express CD34 on a low level, although transcribing CD34. The CD34−/low cells express osteocalcin as a mesenchymal cell marker. The fibroblast-like cells eventually differentiate spontaneously in vitro into CD34+ precursors and show colony formation. Prior to autologous stem cell transplantation, one clone of choice (IIIG7) was transfected with a retroviral construct containing the green-fluorescence protein (GFP). The recipient dog was totally irradiated with 300 cGy and received a stem cell transplant with GFP-containing, immortalized, fibroblast-like monoclonal autologous stem cells (0.5 × 108/kg dog). No additional growth factors were applied. The peripheral blood counts recovered after 23 days (WBC >500; platelets >10,000). A peripheral blood smear showed some dim but definite, although timely, limited expression of the GFP protein in nucleated peripheral blood cells just five weeks after transplantation. A bone marrow biopsy showed GFP-positive cells in the marrow cavity predominantly as “bone-lining cells.”Stem Cells 06/2000; 18(4):252 - 260. · 7.70 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Human mesenchymal stem/progenitor cells (MSCs) have been identified in adult bone marrow, but little is known about their presence during fetal life. MSCs were isolated and characterized in first-trimester fetal blood, liver, and bone marrow. When 10(6) fetal blood nucleated cells (median gestational age, 10(+2) weeks [10 weeks, 2 days]) were cultured in 10% fetal bovine serum, the mean number (+/- SEM) of adherent fibroblastlike colonies was 8.2 +/- 0.6/10(6) nucleated cells (69.6 +/- 10/microL fetal blood). Frequency declined with advancing gestation. Fetal blood MSCs could be expanded for at least 20 passages with a mean cumulative population doubling of 50.3 +/- 4.5. In their undifferentiated state, fetal blood MSCs were CD29(+), CD44(+), SH2(+), SH3(+), and SH4(+); produced prolyl-4-hydroxylase, alpha-smooth muscle actin, fibronectin, laminin, and vimentin; and were CD45(-), CD34(-), CD14(-), CD68(-), vWF(-), and HLA-DR(-). Fetal blood MSCs cultured in adipogenic, osteogenic, or chondrogenic media differentiated, respectively, into adipocytes, osteocytes, and chondrocytes. Fetal blood MSCs supported the proliferation and differentiation of cord blood CD34(+) cells in long-term culture. MSCs were also detected in first-trimester fetal liver (11.3 +/- 2.0/10(6) nucleated cells) and bone marrow (12.6 +/- 3.6/10(6) nucleated cells). Their morphology, growth kinetics, and immunophenotype were comparable to those of fetal blood-derived MSCs and similarly differentiated along adipogenic, osteogenic, and chondrogenic lineages, even after sorting and expansion of a single mesenchymal cell. MSCs similar to those derived from adult bone marrow, fetal liver, and fetal bone marrow circulate in first-trimester human blood and may provide novel targets for in utero cellular and gene therapy.Blood 11/2001; 98(8):2396-402. · 9.06 Impact Factor
252 Current Stem Cell Research & Therapy, 2009, 4, 252-259
1574-888X/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.
Mesenchymal Stem Cells for Treatment and Prevention of Graft-Versus-
Host Disease After Allogeneic Hematopoietic Cell Transplantation
Tomomi Toubai*,1,2, Sophie Paczesny1, Yusuke Shono2, Junji Tanaka2, Kathleen P. Lowler1,
Chelsea T. Malter1, Masaharu Kasai3 and Masahiro Imamura2
1Blood and Marrow Transplantation Program, Department of Internal Medicine, Division of Hematology/Oncology,
University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA; 2Department of Hematology and Onco-
logy, Hokkido University Graduate School of Medicine, Sapporo, Japan; 3Department of Hematology, Sapporo Hokuyu
Hospital, Sapporo, Japan
Abstract: Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective therapy for hematological malignan-
cies and inherited diseases. However, acute graft-versus-host-disease (aGVHD) is a major life-threatening complication
after allo-HCT and there are few therapeutic options for severe steroid-refractory aGVHD. Preliminary studies on co-
transplantation of mesenchymal stem cells (MSCs) have shown an improvement in or resolution of severe aGVHD. How-
ever, the underlying mechanism this immunosuppressive effect has not been elucidated. Most of the data suggest that this
immunosuppressive effect involves soluble factors such as IL-6 or TGF-? as well as cell-cell contact dependence.
MSCs interact either directly with T cells or indirectly via other immune cells such as dendritic cells and NK cells. Here
we review the immunomodulatory function of MSCs in allo-HCT and their potential usefulness in the treatment or pre-
vention of severe acute GVHD.
Keywords: Mesenchymal stem cells (MSCs), acute graft-versus-host disease (aGVHD), allogeneic hematopoietic cell trans-
plantation (Allo-HCT), steroid-refractory acute GVHD.
Fridenstein in the late 1960’s as the source of osteoblastic,
adipogenic, and chondrogenic cells in adult bone marrow
. Since then, MSCs have been found in other human tis-
sues, including peripheral blood , liver , fetal lung 
and umbilical cord blood . Subsequently, it has been
demonstrated that MSCs are present in all human tissues [6,
7]. Possible avenues of MSC research have recently been
multiplied in an allogeneic hematopoietic cell transplantation
(allo-HCT) setting because MSCs have the ability not only
to support hematopoiesis but also to regulate immune re-
sponses. In the latter case, Le Blanc et al. reported in 2004
successful treatment by haploidentical third party MSCs in a
patient with steroid-resistant acute GVHD . Subsequently,
a clinical trial was initiated in Europe and encouraging re-
sults have recently been published . The relationship be-
tween MSCs and immunomodulatory functions has been
described in detail in several recent reviews [10-17]. Herein,
we review the major immunomodulatory effects of MSCs
and focus on the narrow field of treatment and prevention of
acute GVHD with MSCs.
Mesenchymal stem cells (MSCs) were first identified by
IMMUNOSUPPRESSIVE EFFECTS OF MSCs
1. MSC Phenotype
human and mouse MSC studies by surface expression of
*Address correspondence to this author at the Blood and Marrow Trans-
plantation Program, University of Michigan Comprehensive Cancer Cen-
ter 1500 E. Medical Center Drive, 6411 CGC Ann Arbor, Michigan 48109-
0942, USA; Tel: (734) 615-7128; Fax: (734) 647-9271;
E-mail: firstname.lastname@example.org and email@example.com
The phenotype of MSCs has been characterized in both
CD29, CD44, CD58, CD71, CD73, CD90, CD105, CD166,
CD271, MHC class I and recently CD146 at the surface of a
sub-endothelial subset and by the absence of expression of
CD34, CD80, CD86, CD40 and HLA class II expression [18,
19]. The variability in expression of these markers may de-
pend on species differences, tissue source and culture condi-
2. MSC Immune Targets
fect of MSCs suggested direct interaction between MSCs
and T lymphocytes. MSCs directly suppress the proliferation
of both naïve and memory lymphocytes T cells via cell-cell
contact or mitogenic stimuli [20-22]. This suppression is not
MHC-restricted. MSCs can decrease IFN-? producing T cells
and skew the T cell population towards Th2 cells producing
Regulatory T Cells
Generation of CD4+CD25+ regulatory T cells (Tregs) in
the presence of MSCs is still controversial. In early studies,
no association with Tregs was observed [20-22]. Later stud-
ies suggested that Tregs are directly generated in the pres-
ence of MSCs [24-26]. However, using a Foxp3sf mouse
model, Parekkadan et al. found that the immunosuppressive
function of MSCs is not associated with Tregs . In addi-
tion, CD8+ regulatory T cells have been shown to play a role
in MSC-mediated immunosuppressive effects . MSCs
have been reported to trigger the generation of Tregs indi-
rectly, often through the intervention of tolerogenic antigen-
presenting cells as described below. Several studies have
shown an interaction between MSC and antigen-presenting
Early studies demonstrating the immunosuppressive ef-
GVHD Treatment by MSCs Current Stem Cell Research & Therapy, 2009, Vol. 4, No. 4 253
tremely potent antigen-presenting cells (APCs) that play a
major role in the processing and presentation of antigens to
different immune cells and have the unique capacity to prime
naïve T lymphocytes [29, 30]. MSCs were first shown to
strongly inhibit alloantigen-induced DC1 differentiation and
indirectly induce CD4+CD25+ Tregs . MSCs were subse-
quently shown to inhibit the differentiation, function and
migration of both monocyte-derived DCs [23,31-34] and
CD34+-derived DCs [35, 36] by inhibiting the expression of
their co-stimulatory molecules, CD40, CD80, CD83 and
CD86, decreasing secretion of IL-12 and inhibiting mixed
lymphocyte reaction (MLR). The mechanism of the inhibi-
tory effect of MSCs on DCs function is controversial. MSCs
have been reported to increase IL-10 production by mature
DC2  or to inhibit the differentiation to DC1 with an
enhancement of differentiation of CD4+CD25+ Tregs .
More recently, Li et al. reported that human DCs generated
in co-culture with MSC have immunosuppressive effects
mediated through activation of the Notch pathway,  and
Zhang et al. reported that mouse bone marrow-derived
MSCs induce the differentiation of DCs into Jagged-2-
dependent regulatory DCs . Regarding inhibitory effects
on migration, English et al. showed that MSCs decreased
CCR7 surface expression on DCs and subsequently their
functional migration to CCL19 . However, details of the
molecular interactions between MSCs and DCs remain un-
clear. Macrophages might also be a target of MSCs, since
Nemeth et al. have recently shown that administration of
cultured human bone marrow-derived MSCs reduced mortal-
ity and improved organ function of septic mice, through in-
creases in PGE2 and IL-10 by macrophages .
Dendritic cells (DCs) represent a rare population of ex-
[23, 40-44]. MSCs can inhibit IL-2-induced NK cell prolif-
eration [43, 44] via activation of indoleamine 2,3-
dioxygenase (IDO) and prostaglandin E2 (PGE2) . MSCs
also inhibit the cytolytic function and IFN-? production of
NK cells [23, 42]. Downregulation of NK receptors (NKp30,
NKp44 and NKG2D) has been suggested to be responsible
for the decreased NK cell functions .
Effects of MSCs on NK cells have also been reported
experimental model for human systemic lupus erythematous,
showed inhibition of the proliferation, activation and IgG
secretion of B cells in the presence of allogeneic MSCs .
Corcione et al. have further shown that MSCs not only in-
hibit B cell proliferation and differentiation to antibody-
producing cells but also chemotaxis in vitro . MSCs may
play this role through STAT3 inactivation and PAX5 induc-
tion . These recent findings contradict results of previous
studies suggesting that MSCs induce the proliferation of
memory B cells and their differentiation into plasma cells
[48, 49]. Further analyses are therefore required.
3. Mechanisms of MSC-Mediated Immunosuppressive
Results of a study by Deng et al. using BXSM mice, an
into two types: soluble factor-mediated effects and cell-cell
MSC-mediated immunosuppressive effects are classified
TGF-?, hepatocyte growth factor (HGF), IDO, PGE2 and
heme oxygenase-1 (HO-1), have been implicated in the im-
munosuppressive effects of MSCs, though the exact mecha-
nism is still unclear [22,23,32,50-53]. For instance, human
MSCs decreased tumor necrosis factor ? (TNF?) secretion
from mature DC1 and IFN-? secretion from NK cells, in-
creased IL-10 secretion from mature DC2 and induced T cell
polarization from Th1 to Th2, which are supposedly induced
by elevated PGE2 production from MSCs . MSCs resid-
ing in the lung also have an immunosuppressive effect that is
associated with increased secretion of PGE2 in response to
IL-1? . The role of IDO in the immunosuppressive effect
of MSCs is controversial. Prior studies have shown a posi-
tive association between IDO and the immunosuppressive
effects of MSCs, particularly NK cells inhibition [43, 53]
whereas two recent studies have shown a negative associa-
tion [54, 55]. Min et al. examined the role of IL-10 in the
immunosuppressive effects of MSCs using IL-10-transduced
MSCs in a GVHD mouse model and showed that co-
injection of IL-10-transduced MSCs reduced GVHD severity
and prolonged survival . However, a significant effect of
IL-10 was not found in other studies [35, 54]. Djouad et al.
suggested that IL-6 is involved because murine MSCs se-
creted an appreciable amount of IL-6 and its neutralization
resolved this immunosuppressive effect . Recently, two
studies have shown a correlation between nitric oxide (NO)
and MSC-mediated immunosuppression [54, 55]. NO is pro-
duced by NO synthases (NOSs) and has three subtypes: in-
ducible NOS (iNOS), endothelial NOS, and neuronal NOS.
Sato et al. reported that NO produced by MSCs plays a ma-
jor role in T cell suppression through the suppression of
Stat5 phosphorylation . Ren et al. showed in both in
vitro and in vivo models of GVHD that the immunosuppres-
sive function of MSCs was elicited by iNOS and chemoki-
nes induced by INF-? with concomitant presence of either
TNF-?, IL-1? or IL-1? . Tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) has no effect on prolif-
eration or differentiation of MSCs but does promote the mi-
gration of human MSCs .
Cell-cell contact-mediated effects were first suggested by
the finding that addition of few MSCs in co-culture did not
inhibit an MLR, whereas addition of larger numbers of
MSCs resulted in strong suppression of the MLR . In
addition, Di Nicola et al. clearly showed that MSCs directly
suppress the proliferation of T cells via cell-cell contact .
The cell-cell contact between MSCs and targets cells by en-
gagement of PD1 and its ligand is also a mechanism for in-
hibition of T cell proliferation . Toll-like receptors have
recently been shown to be associated with inhibition of T
cell function by MSCs [61-64]. Selmani et al. found that
secretion by MSCs of HLA-G5, a tolerogenic non-classical
MHC class?Ib molecule, was critical for reducing allogeneic
T cell proliferation, inhibition of NK cell function, and ex-
pansion of Tregs .
Soluble factors, such as Th2 cytokines (IL-10 and IL-4),
ated with cell cycle arrest of T cells, inducing their anergy.
Glennie et al. found that the lack of early activation of T
cells is due to down-regulation of cyclin D2 expression and
increased levels of p27Kip1 (cyclin-dependent kinase inhibi-
tor). Cyclin D2 is a marker of early G1 phase and inhibition
of its expression is associated with T cell arrest at this stage
On the other hand, MSCs have been shown to be associ-
254 Current Stem Cell Research & Therapy, 2009, Vol. 4, No. 4 Toubai et al.
. Ramasamy et al. have shown that MSCs blocked DCs
from entering into G1 phase, causing an accumulation of
cells in G0 phase .
4. In Vivo Prevention and Treatment of GVHD Using
MSCs differ from in vitro studies in several ways. Although
MSCs have an immunosuppressive function in vitro, there
are not many in vivo experimental data to support this. Table
1 summarizes studies using mouse models. The first study
using a mouse model was by Chung et al. in 2004, and their
study showed that co-transplantation of MSCs prevented
lethal GVHD in a major histocompatibility complex (MHC)-
mismatched model . Later, Yanez et al. reported that
adipose derived-MSCs could also prevent GVHD . The
mechanisms involved are IFN-? activation of MSCs  and
increase of CD4+CD25+ T cells . On the other hand, Su-
dres et al. did not detect any significant difference between
the treated group and control group in a study using an
MHC-mismatched model in which MSCs were injected be-
fore BMT (one injection) . Tisato et al. demonstrated
prevention of GVHD in their xenogenic model using 4
weekly MSC injections instead of a single injection, but
when MSCs were injected at the onset of GVHD, they could
not find any significant effect . Badillo et al. also re-
ported the failure of prevention of GVHD by treatment with
MSCs in their haploidentical mouse model,  although the
experimental procedure they used was the same as that used
by Yanez et al. One possible reason for this discrepancy in
results may be the difference in tissue of origin: adipose tis-
sues versus bone marrow. The latter authors have also re-
ported that allogeneic MSCs could not induce tolerance in a
skin graft . Possible explanations for the variability of
these results are: (i) the timing of injection possibly due to
the milieu of cytokines present as this time, (ii) the dose of
MSCs used, (iii) MSC tissue of origin, (iv) strain differences
between donor and host and (v) possible rejection of MSCs
In vivo studies on the immunosuppressive function of
shortly after injection (by an unknown mechanism). Further
discernment of the critical parameters will require additional
in vivo studies.
5. Clinical Application of MSCs After Allo-HCT
MSCs that were expanded ex vivo was a safe treatment for
15 patients with hematological malignancies in complete
remission . Other clinical studies using MSCs were per-
formed in advanced breast cancer patients for promoting
rapid hematopoietic recovery after autologous stem cell
transplantation . Lazarus et al. also reported the results
of an open-label, multicenter study on safety and feasibility
of co-transplantation of HLA-identical expanded MSCs in
46 patients with hematological malignancies after a myelo-
ablative conditioning regimen. However, the engraftment of
neutrophils and platelets was similar to historical reports (at
days 14 and 20, respectively) . In contrast, Le Blanc et
al. reported in 2007 that co-transplantation of MSCs en-
hanced the engraftment of hematopoietic stem cells after
allo-HCT in 7 patients in comparison to their historical con-
trols . Co-transplantation of ex vivo-expanded MSCs for
prevention of graft failure in haploidentical allo-HCT
showed no graft failure in the treated group versus 14.9%
graft failure in the historical group and faster recovery of
total leukocyte count and NK cells . Macmillan et al.
have recently confirmed the safety of transplantation of ex-
vivo culture-expanded parental haploidentical MSCs in pedi-
atric recipients of unrelated donor umbilical cord blood with
good engraftment parameters .
Lazarus et al. demonstrated that infusion of autologous
9-year-old boy with steroid-refractory grade IV aGVHD
involving the gut and liver was successfully treated with
MSCs derived from a haplo-identical donor . His symp-
toms dramatically improved after administration of the first
dose. This case report was followed by a pilot study using
MSCs to treat steroid-refractory GVHD after allo-HCT .
Nine patients (8 patients with steroid refractory acute GVHD
Regarding GVHD, Le Blank et al. reported in 2004 that a
The Prevention and Prophylaxis of Acute GVHD in Murine Models
Authors Source of MSCs Model of Mice Conditioning Dose of MSCs Results
Chung et al.
1x105 GVHD prevented
Yanez et al.
GVHD prevented by administration at day
0, 7, and 14
Sudres et al.
T cells 5x105
5x105, 3x106, 4x106 No prevention of GVHD
Tisato et al.
PBMC 2x107 3x106 Single dose, no GVHD prevention,
4 times, GVHD prevention
After onset GVHD, no efficacy
Badillo et al.
1.5x105- 1x106 (once)
5x104 (3 times)
GVHD prevention; no efficacy
GVHD prevention; no efficacy
GVHD treatment; no efficacy
Polchert et al.
Success by administration at day 2 and day
20, but the BMT from IFN-?-/- donor was
BM, bone marrow; PBMC, peripheral blood mono nuclear cells.
GVHD Treatment by MSCs Current Stem Cell Research & Therapy, 2009, Vol. 4, No. 4 255
and 1 patient with chronic GVHD) were treated with MSCs
derived from an HLA identical sibling (n=2), haploidentical
donor (n=5) and HLA-mismatched donor (n=4), resulting in
the disappearance of acute GVHD in six of the eight pa-
tients; two died soon after MSC administration with no re-
sponse. Overall survival (OS) was significantly better in the
MSC-treated group than in the control group, although 2 of
the 6 patients with complete responses developed pneumonia
and bronchiolitis. Fang et al. also reported some success
using human adipose tissue-derived MSCs for treatment of
steroid-refractory GVHD [82-84]. Muller et al. reported re-
sults of 11 infusions of expanded MSCs to treat acute
GVHD, chronic GVHD, hemophagocytosis and graft rejec-
tion after allo-HCT  showing a moderate response in
patients with acute and chronic GVHD. Le Blanc et al. re-
ported the results of a phase II?multicenter study for treat-
ment of steroid-refractory severe acute GVHD with ex-
panded MSCs . A total of 55 patients received treatment
(20 patients receiving a single infusion, 22 receiving 2 infu-
sions, and 6 receiving 3-5 infusions). Thirty patients showed
a complete response and nine patients showed partial re-
sponse, an overall response rate of 71%. There was a signifi-
cant improvement in 2-year OS in the complete response
group (53%) and a significant decrease in 1-year transplant-
related mortality (TRM) (37%). These encouraging results
could not be reproduced in a study by von Bonin et al. that
included 13 patients with steroid-refractory aGVHD treated
with third-party MSCs; only 2 patients (15%) responded and
did not require further escalation of immunosuppressive
therapy . The donor source of MSCs might account for
these differences, and studies addressing this question are
required. Regarding GVL effects, three patients had recur-
rent malignant disease and one developed de novo acute
myeloid leukemia of recipient origin. As for infections, 16
patients (29%) developed various infections, including as-
pergillosis, cytomegalovirus, and septicemia (caused by En-
terococci, Klebsiella sp, Escherichia coli, and unidentified
pathogens), and three patients developed infection with Ep-
stein-Barr virus (EBV), one of which developed post-
transplantation lymphoproliferative disease related to the
prophylaxis of GVHD are summarized in Table 2. However,
since other immunosuppressants such as steroids were used
in all of those studies, we are unable to distinguish between
the effects induced by MSCs and those induced by the im-
The results of several clinical studies on treatment and
6. Controversy About the Use of MSCs After Allo-HCT
steroid-refractory GVHD. However, some controversy has
resulted this approach; some may be resolved, some will
require further analysis.
a. Absence of MHC-Restriction and MSC Donor Source
MSCs might be an attractive option for the treatment of
MHC recognition, suggesting that MSCs lack specificity,
creating the problem of a global immunosuppression. Fur-
thermore, results obtained using different donor sources of
MSCs (haploidentical MSCs, third party MSCs or identical
Immunosuppression via MSCs is not mediated through
MSCs) are not clear. This question should be answered by
future in vivo studies and trials.
b. Absence of In Vivo Detection
ministered in vivo, preventing any biological measurement of
the efficacy of treatment. The only human study which could
successfully detect gene marked MSCs dealt with engraft-
ment in children with osteogenesis imperfecta .
c. Transformation and Facilitation of Primary Disease
MSCs have been almost impossible to detect when ad-
culture, murine bone marrow-derived MSCs transformed
into malignant fibrosarcoma with chromosomal abnormali-
ties, elevated telomerase activity, and increased expression
of c-myc . Tolar et al. reported the development of sar-
coma in mice after injection of cultured MSCs . In con-
trast, Bernardo et al. demonstrated that human bone marrow-
derived MSCs did not have a risk for malignant transforma-
tion, even after several passages . However, long-term
follow-up observations for the development of secondary
malignancies after co-transplantation of MSCs are needed.
MSCs also have the potential to promote tumor growth in
vivo . Since GVHD is closely associated with GVL, we
must confirm whether the GVL effect is decreased after in-
fusion of MSCs, particularly in the high relapse risk patient
group. In fact, Ning et al. reported significantly higher rates
of relapse which developed faster in the group co-
transplanted with MSCs than in the untreated group, al-
though GVHD was decreased . However, in this study
(i) the population was small, (ii) few MSCs were infused so
the correlation is not clear, and (iii) the disease status at
transplant is unknown. In addition, according to Le Blanc’s
finding, only 3 of 55 patients had recurrent malignancies at
the 2 years follow-up . Therefore, we must monitor pri-
mary malignant disease closely, especially in patients with a
high risk of relapse, and confirm the safety and efficacy of
treatment using large-scale randomized studies.
d. Global Immunosuppression and Infections
Miura et al. demonstrated that after several passages in
severe infections [9,75,78,79,81-85,92,93]. However, some
have found a correlation between MSC treatment and the
occurrence of infections. Kang et al. demonstrated that
MSCs suppress approximately 50% of virus-induced CTLs,
 and Sundin et al. examined whether MSCs can be in-
fected with cytomegarovirus, HSV-1, HSV-2, EBV, or
varicella-zoster virus (VZV). Although herpes family viruses
were not detected by PCR in ex vivo-expanded MSCs de-
rived from seropositive donors, MSCs could be infected in
vitro with CMV and HSV-1, but not EBV . There was a
suppression of the lymphocyte proliferative response to
HSV, candida, and bacteria infection . Conversely,
Karlsson et al. demonstrated that MSCs did not affect on
EBV and CMV T cell responses in vitro and in vivo, sup-
pressing only the alloantigen response . Persistent par-
vovirus B19 in MSCs can also infect hematopoietic stem
cells . However, recently, septic mice had improved or-
gan failure and mortality rates after administration of MSCs
cultured from mouse bone marrow . Thus, the associa-
tion between increased incidence of infection and MSCs
Previous clinical studies have shown no increased risk of
256 Current Stem Cell Research & Therapy, 2009, Vol. 4, No. 4 Toubai et al.
remains controversial, and we should remember this uncer-
tainty in treating steroid-refractory GVHD.
e. Efficacy Without Other Immunosuppressants
other immunosuppressants such as tacrolimus, cyclosporine,
mycophenolate mofetil, and sirolimus. Therefore, the effi-
cacy of MSCs should be assessed accordingly. For instance,
in a heart transplant model, Hoogduijn et al. found that
therapeutic doses of mycophenolate mofetil and sirolimus
inhibited MSC proliferation, and high doses of tacrolimus
In many clinical situations, MSCs are administered with
significantly reduced the number of viable MSCs . Con-
versely, MSCs had an adverse effect on the immunosuppres-
sive efficacy of tacrolimus and sirolimus. This might be dif-
ferent if MSCs are isolated from bone marrow. In conclu-
sion, we need to be cautious when using a combination of
MSCs and other immunosuppressants.
oid-refractory GVHD patients. Although steroid administra-
MSCs are an attractive alternative for treatment of ster-
The Clinical Study for Treatment and Prophylaxis of GVHD in Allo-HSCT
Authors Source MSC donor
Number of patients
Conditioning Dose of MSCs Effect on GVHD
et al. 
1 (9-year-old boy) /
Grade4?improved? Reccurence? improved
et al. 
9 (12 times infu-
acute GVHD 8,
Complete response 6, Response of GVHD 1,
Slight effect 1, No response 4
Fang, et al.
2 / steroid-refractory
1 / chronic hepatic
6 / steroid-refractory
5/6 complete response
Muller, et al.
7 (11 times infu-
/ aGVHD n=2,
0.4-3x106/kg aGVHD, 1/2 alive and well
cGVHD, 1/3 slightly improved
Hemophagocytosis, good response
Graft rejection prophylaxis, alive and well
et al. 
55 (92 infusions
/ Grade 2 n=5,
Grade 3 n=25,
Grade 4 n=25
Children: complete response 17/25, partial
Adult: complete response 13/30, partial re-
Total: complete response 30/55 (54.5%),
partial response 9/55 (16.3%)
One year transplantation related mortality
37% (complete response) vs 72% (partial or
Overall survival at 2 years 53% (complete
response) vs 16% (partial or no response)
et al. 
13 (32 infusions)/
Grade 3 n=2, Grade
2 patients (15%) responded within a week
after first MSC infusion.
5/11 (45%) patients showed a response after
received additional salvage immunosuppres-
sive therapy with MSC infusion
BM, bone marrow: RIC, reduced-intensity conditioning.
GVHD Treatment by MSCs Current Stem Cell Research & Therapy, 2009, Vol. 4, No. 4 257
tion is the mainstay treatment for aGVHD, there are many
adverse side-effects. Therefore, treatment using MSCs with
new compounds has become the hope of many clinicians.
Although the European clinical study results are encourag-
ing, they need to be confirmed by a phase III trial. Since
mouse models are controversial and depend on experimental
conditions, the biologic mechanisms induced by MSCs in
vivo should be further evaluated. The acceptance and promo-
tion of this treatment will depend on (i) more basic research
conducted under clinically relevant conditions, (ii) the stan-
dardization of MSC culture protocol, and (iii) validation by
several large-scale multi-institutional clinical trials.
Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP. Het-
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Received: September 29, 2008
Revised: February 04, 2009 Accepted: March 11, 2009