Cell Transplantation, Vol. 22, pp. 2041–2051, 2013
Printed in the USA. All rights reserved.
Copyright 2013 Cognizant Comm. Corp.
0963-6897/13 $90.00 + .00
Received November 1, 2011; final acceptance September 23, 2012. Online prepub date: February 26, 2013.
Address correspondence to Yu-Hua Chao, M.D., Department of Pediatrics, Chung Shan Medical University Hospital, No. 110, Sec. 1, Chien-Kuo N.
Road, Taichung 402, Taiwan. Tel: +886-4-24739595 ext. 21728; Fax: +886-4-24710934; E-mail: firstname.lastname@example.org
Human Application of Ex Vivo Expanded Umbilical Cord-Derived
Mesenchymal Stem Cells: Enhance Hematopoiesis After
Cord Blood Transplantation
Kang-Hsi Wu,*† Chris Tsai,‡ Han-Ping Wu,§ Martin Sieber,‡ Ching-Tien Peng,†¶ and Yu-Hua Chao#**††
*School of Chinese Medicine, China Medical University, Taichung, Taiwan
†Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan
‡Bionet Corp, Taipei, Taiwan
§Department of Pediatrics, Buddhist Tzu-Chi General Hospital, Taichung Branch, Taichung, Taiwan
¶Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan
#Department of Pediatrics, Chung Shan Medical University Hospital, Taichung, Taiwan
**Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
††School of Medicine, Chung Shan Medical University, Taichung, Taiwan
Delayed hematopoietic reconstitution after cord blood (CB) transplantation (CBT) needs to be overcome. Bone
marrow-derived mesenchymal stem cells (BMMSCs) have been found to enhance engraftment after hematopoi-
etic stem cell transplantation. However, getting BMMSCs involves an invasive procedure. In this study, umbili-
cal cord-derived mesenchymal stem cells (UCMSCs) were isolated from Wharton’s jelly and cryopreserved in
the UCMSCs bank. Compared with BMMSCs, we found that UCMSCs had superior proliferative potential. We
found that NOD/SCID mice cotransplanted with CB and UCMSCs demonstrated significant human CD45+ cell
engraftment compared with those transplanted with CB alone. Then, 20 patients with high-risk leukemia were
prospectively randomized to either receive cotransplantation of CB and ex vivo expanded banked UCMSCs
or to receive CBT alone. No serious adverse events were observed in the patients receiving UCMSC infusion.
The time to undergo neutrophil engraftment and platelet engraftment was significantly shorter in the eight
patients receiving cotransplantation than that in the 12 patients receiving CBT alone (p = 0.003 and p = 0.004,
respectively). Thus, application of ex vivo expanded banked UCMSCs in humans appears to be feasible and
safe. UCMSCs can enhance engraftment after CBT, but further studies are warranted.
Key words: Mesenchymal stem cells (MSCs); Umbilical cord; Cord blood transplantation (CBT)
Mesenchymal stem cells (MSCs) can have clinical
applications in regenerative medicine and tissue repair in
adverse health events (9) such as heart diseases (14), neu-
rological problems (13), lung diseases, liver diseases, renal
diseases (5), diabetes, and bone problems. The immuno-
modulatory properties of MSCs make them clinically
valuable in immune disorders (8,15,25,31,35). In most
previous reports, MSCs in human clinical applications
were derived from bone marrow (BM) (4,16,18–22,27,29).
However, harvesting BM-derived MSCs (BMMSCs)
involves an invasive and painful procedure. MSCs also
can be isolated without an invasive procedure from fetal
tissues, such as amniotic fluid (2), amniotic membrane
(23), placenta, cord blood (CB) (5), and umbilical cord
(UC) (12,32). The UC is rich with MSCs, and UC-derived
MSCs (UCMSCs) have been shown to be easy to isolate
and culture (32). Therefore, the UC can be considered as
an alternative source of MSCs for clinical applications.
Hematopoietic stem cell transplantation (HSCT) is an
effective therapeutic modality for a variety of disorders.
CB is a source of hematopoietic stem cells (HSCs), and
there are some advantages in CB transplantation (CBT),
including fast availability, tolerance of one to two human
leukocyte antigen (HLA) mismatch, low incidence and
severity of acute graft-versus-host disease (GVHD),
low risk of transmitting infections, and lack of risks for
donors (11). However, because of the limited number of
HSCs in CB, patients receiving CBT have been found to
have delayed hematopoietic engraftment, which might
result in more complications, such as infections and hem-
orrhage, and prolonged hospitalization (11,30).
2042 WU ET AL.
Previous studies have documented that cotransplantation
of HSCs and BMMSCs can enhance hematopoiesis, reduce
the incidence of GVHD, accelerate lymphocyte recovery,
and reduce the risk of graft failure (4,16,18,21,22,27).
BMMSCs were also reported to promote hematopoietic
engraftment after CBT (22). Our previous studies found
that no severe adverse effects were noted in patients receiv-
ing UCMSCs. We also found that UCMSCs could enhance
engraftment after HSCT in patients with severe aplastic
anemia and effectively treat severe acute GVHD (7,34).
In the present study, after ex vivo and animal studies, we
prospectively randomized subjects to investigate the safety
and effect of cotransplantation of CB and UCMSCs in
patients with high-risk leukemia.
PATIENTS AND METHODS
Isolation and Culture of BMMSCs
BM cells of healthy individuals were obtained from iliac
crest aspirates. The institutional review board (IRB) of the
China Medical University Hospital approved this protocol,
and written informed consents were obtained. Mononuclear
cells were isolated from BM aspirates by Ficoll-Paque den-
sity centrifugation (1.077 g/ml; Amersham Biosciences,
Uppsala, Sweden) and then seeded in minimum essential
medium with a modification (a-MEM; Invitrogen, Carls-
bad, CA, USA) supplemented with 10% fetal bovine serum
(Invitrogen) and penicillin– streptomycin (Invitrogen). Cells
were incubated at 37°C with 5% CO2 in a humidified
atmosphere. After 48 h, the medium with suspension of
nonadherent cells was discarded, and fresh medium was
added. Thereafter, the medium was replaced twice a week.
When reaching 80–90% confluence, cells were detached
with 0.25% trypsin-EDTA (Invitrogen) and replated at a
concentration of 8.5 × 103/cm2 in 10-cm dishes.
Collection and Cryopreservation of UCMSCs
IRB approval and informed consent was obtained from
all eligible UC donors who tested negative for human immu-
nodeficiency virus (HIV)-I, HIV-II, human T-lymphotropic
virus (HTLV)-I, HTLV-II, hepatitis B and C, cytomegalovi-
rus (CMV), and syphilis and were free of any active infec-
tions during UCMSC harvesting. The UC was collected
immediately after parturition and express-shipped to the
UCMSC bank for MSC processing. UCMSCs were har-
vested from Wharton’s jelly as previously described (7,34).
Briefly, the main vessels were carefully removed, and the
UC was sectioned to acquire Wharton’s jelly. The jelly
was digested using 1 mg/ml collagenase (Sigma, St. Louis,
MO, USA) and plated in media (as above) at 5% CO2 and
37°C in a humidified atmosphere. Forty-eight hours later,
the suspended nonadherent cell-containing medium was
discarded and replaced with fresh medium. The cells were
detached with 0.25% trypsin-EDTA at 80–90% confluence
and subcultured for further expansion. Once the cells had
been proven negative for bacteria, fungi, mycoplasma, and
endotoxins and UCMSCs were identified, the cells were
cryopreserved in 10% dimethyl sulfoxide (DMSO; WAK-
Chemie Medical GmbH, Steinbach, Germany) using a
controlled-rate freezer and maintained in liquid nitrogen
at the UCMSCs bank. All procedures for UCMSC pro-
cessing complied with the current good tissue practice
Identification of MSCs
BMMSCs and UCMSCs were immunolabeled with
the following mouse anti-human antibodies: cluster of
differentiation 14 (CD14), CD34, CD45, CD13, CD29,
CD31, CD44, CD90, and HLA-DR (BD Biosciences, San
Jose, CA, USA); CD105 (AbD Serotec, Oxford, UK);
and CD73 (BD Pharmingen, San Diego, CA, USA). The
cells were incubated with a secondary antibody, anti-
mouse IgG-fluorescein isothiocyanate (FITC) or IgG-
phycoerythrin (PE) (BD Biosciences) and analyzed by
flow cytometry (BD Biosciences). The MSCs then under-
went osteogenic, adipogenic, and chondrogenic differen-
tiation under specific induction conditions (6,26).
Proliferative Potential Assay
The population doublings of the cultured UCMSCs
(n = 10) and BMMSCs (n = 10) were calculated according
to the equation: population doubling = log2 (the number
of viable cells at harvest/the number of seeded cells) (6).
The population doubling time was derived from the time
interval divided by the cumulative population doubling
from passages 2 to 6.
Cotransplantation of NOD/SCID Mice With CB
The male nonobese diabetic/severe combined immuno-
deficient (NOD/SCID) mice were obtained from the National
Science Council (Taipei, Taiwan) and maintained at the
Animal Center of China Medical University. There were
two groups of 10 mice each (age 6–8 weeks) sublethally
irradiated with 350 cGy (Varian, Palo Alto, CA, USA)
and then transplanted with the following cells: (a) 106
total nucleated cells (TNCs) in the CB together with 106
UCMSCs and (b) 106 TNCs in the CB alone. The cells
in these two groups were suspended in 250 ml of
phosphate-buffered saline (Invitrogen) and injected into
the lateral tail veins. These mice were sacrificed 6 weeks
after transplantation. Peripheral blood samples were col-
lected by cardiac puncture, and BM was harvested by flush-
ing femurs and tibias with phosphate-buffered saline. To
document hematopoietic cells engraftment, these cells
from peripheral blood and BM were stained with a FITC-
conjugated antibody to human CD45 (BD Pharmingen),
UCMSCs ENHANCE CORD BLOOD TRANSPLANTATION 2043
and the percentages of human CD45+ cells were measured
by flow cytometry.
In addition, in order to compare the time of achieving
complete chimerism between NOD/SCID mice cotrans-
planted with CB and UCMSCs and those transplanted
with CB alone, we analyzed the percentages of human
CD45+ cells in the peripheral blood collected from the tail
vein every week in another two groups of 10 mice each
(age 6–8 weeks) sublethally irradiated with 350 cGy and
then transplanted. We defined the end point when com-
plete chimerism (100% human CD45+ cells in peripheral
blood) in mice was achieved.
After the IRB at the China Medical University Hospital
and the Department of Health in Taiwan approved this study,
children with high-risk leukemia but lacking HLA-matched
siblings or unrelated donors were enrolled in this study at
the China Medical University Hospital. The high-risk acute
lymphoblastic leukemia (ALL) was defined as ALL with
cytogenetic abnormalities indicating poor prognosis or in
second remission. The high-risk acute myeloid leukemia
(AML) was defined as AML with cytogenetic abnormali-
ties indicating poor prognosis, in second remission, or with
myelodysplastic syndrome. We prospectively randomized
patients to receive cotransplantation of CB and UCMSCs or
CBT alone. Parents or legal guardians of patients provided
written informed consent for inclusion in this study. Written
informed consent in accordance with the Declaration of
Helsinki was also obtained from patients receiving UCMSC
infusion by an independent physician to explain risks asso-
ciated with UCMSC infusion. Patients who received CBT
alone were enrolled as the control group.
Expansion, Thawing, and Infusion of UCMSCs
After we recruited suitable patients for the study (see
Table 1 for demographics), we selected a unit of banked
UCMSCs matching HLA based on antigen-level HLA-A,
HLA-B, and HLA-DR typing with the recipients. UCMSC
units and recipients should match at least three HLAs. The
selected UCMSCs were thawed and subcultured in a-MEM
supplemented with 10% fetal bovine serum at 37°C in a
humidified atmosphere with 5% CO2. The medium was
replaced twice a week. When reaching 80–90% conflu-
ence, cells were detached with 0.25% trypsin-EDTA and
replated. When adequate cell dosages were obtained, the
expanded UCMSCs were then cryopreserved again. After
UCMSC expansion and before recryopreservation, karyo-
typing and screening for bacteria, fungi, mycoplasma, and
endotoxins tests were performed. After the cells were shown
to have a normal karyotype and were negative for infectious
contamination, these cells were shipped from the UCMSC
bank to our hospital. On the day of CBT, the UCMSCs
were thawed, washed (16), and intravenously infused into
the recipient through a central venous line, followed by CB
infusion 4 h afterward. Some residual UCMSCs and suspen-
sion were tested again to rule out infectious contamination.
All cotransplanted patients received one unit of CB and one
unit of expanded UCMSCs. The collection, cryopreserva-
tion, expansion, thawing, and infusion of UCMSCs are sum-
marized in Figure 1.
Toxicity Assessment About UCMSCs
Vital signs were monitored at 15 and 30 min, 1, 2, 4, 6,
10, and 14 h after UCMSC infusion. Patients were moni-
tored continuously for UCMSC infusion-related toxici-
ties defined as alterations in vital organ functions, which
cannot be explained by other intercurrent complications
such as infection or chemoradiotherapy within 28 days of
UCMSC infusion. To assess the presence of ectopic tis-
sue formation, patients were evaluated by magnetic reso-
nance imaging (MRI; GE Medical Systems, Milwaukee,
WI, USA) and positron emission tomography (PET; GE
Medical Systems) every 3 months after UCMSC infusion.
Searches for unrelated CB donors were processed
through the CB bank approved by the Department of
Health in Taiwan. The compatibility of HLA-A, -B, and
-DRB1 was assessed by high-resolution typing. The CB
units were selected according to the TNCs in the CB units
and the HLA match between recipients and the CB units.
The selected CB units had to contain at least 3 × 107 TNCs/
kg of the recipients (prethaw). A minimum of four HLA
antigens shared with the recipients was required, and only
one HLA-DRB1 single-locus mismatch was allowed. All
patients enrolled in this study received one unit of CB,
whether cotransplanted with UCMSCs or transplanted
alone. Any patient receiving two or more units of CB was
Conditioning Regimens, GVHD Prophylaxis, and
Conditioning regimens were different according to ALL
or AML. Patients with ALL received cyclophosphamide
(Baxter Oncology GmbH, Halle, Germany) 60 mg/kg/day
intravenously for 2 days, etoposide (Bristol-Myers Squibb,
Princeton, NJ, USA) 30 mg/kg intravenously for 1 day,
and total body irradiation 200 cGy twice a day for 3 days.
Patients with AML were treated with intravenous busulfan
(Otsuka, Tokyo, Japan) 1 mg/kg intravenously every 6 h
for 4 days, and cyclophosphamide 60 mg/kg/day intrave-
nously for 2 days. GVHD prevention consisted of intrave-
nous or oral cyclosporine (Novartis, Basle, Switzerland)
and intravenous methylprednisolone (Pfizer, NY, USA). All
patients received rabbit thymoglobulin (Genzyme, Marcy
2044 WU ET AL.
Table 1. Characteristics of Patients Receiving Cotransplantation of CB and UCMSCs
Age at transplant (years)
AML in 2nd
AML with MDS AML with MDS ALL with Ph
AML in 2nd
ALL with Ph
ALL in 2nd
Characteristics of CB
TNC dose (×107/kg)
CD34+ cell dose (×105/kg)
Gender of CB donor
Characteristics of UCMSCs
UCMSC dose (×106/kg)
Duration of expansion to sufficient
Gender of UCMSCs donor
ANC > 0.5 × 109/L (days)
Platelet count > 20 × 109/L (days)
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANC, absolute neutrophil count; CB, cord blood; F, female; GVHD, graft-versus-host disease; HLA, human leukocyte antigen;
M, male; MDS, myelodysplastic syndrome; Ph, Philadelphia chromosome; TNCs, total nucleated cells; UCMSCs, umbilical cord-derived mesenchymal stem cells.
UCMSCs ENHANCE CORD BLOOD TRANSPLANTATION 2045
L’Etoile, France) 2.5 mg/kg/day intravenously for 4 days.
Patients received antimicrobial prophylaxis with oral
ciprofloxacin (Bayer, Leverkusen, Germany), oral voricon-
azole (Pfizer, Illertissen, Germany), oral trimethoprim-
sulfamethoxazole (Shionogi, Osaka, Japan), and intravenous
acyclovir (GlaxoSmithKline, Torrile, Italy). CMV pp65
antigen and pp65 genes were tested from peripheral blood
samples every week for CMV infection (24). Within 4 h
of specimen collection, the CMV pp65 antigen assay was
carried out using the Light Diagnostics™ CMV pp65
Antigenemia Immunofluorescence Assay (Chemicon
International, Temecula, CA, USA) according to the
manufacturer’s instructions. The result was expressed as
the number of CMV antigen-positive cells per 2 × 105 leu-
kocytes in the slide. For detection of CMV pp65 genes,
total nucleic acids were extracted from 350 μl of EDTA-
anticoagulant whole blood and eluted in 200 μl of elu-
tion buffer using the automatic Bio Robot Ez1 extractor
(Qiagen, Valencia, CA, USA). According to the manufac-
turer’s protocol, CMV DNA was quantified using a com-
mercially available real-time PCR test (TaqMan Q-CMV
Rt Complete KIT; Cepheid, Torino, Italy). Once CMV
was detected, ganciclovir (Roche, Basel, Switzerland) was
administered intravenously. After transplantation, com-
plete blood count was checked every day, and granulocyte
colony- stimulating factor (G-CSF; Kirin, Tokyo, Japan;
Figure 1. The flow chart of collection, cryopreservation, expansion, thawing, and infusion of umbilical cord-derived mesenchymal
stem cells (UCMSCs). HLA, human leukocyte antigen.
2046 WU ET AL.
10 μg/kg/day) was administrated subcutaneously until
absolute neutrophil count (ANC) > 1.0 × 109/L. The ANC
is calculated by multiplying the total leukocyte count by
the percentage of segmented neutrophils and bands. Short
tandem repeat analysis was used for assessment of engraft-
ment after CBT (17). For short tandem repeat analysis,
DNA was prepared from mononuclear cells obtained from
the CB and peripheral blood of the recipient before CBT.
Specific primers designed to flank repeated units of the
apolipoprotein B (APO-B) and D1S80 gene regions were
used for PCR amplification. Transplant-related mortality
was defined as patients expired due to CBT.
A boxplot was made to compare the human CD45+
cells between NOD/SCID mice cotransplanted with CB
and UCMSCs and those transplanted with CB alone. A
Mann–Whitney test and Fisher’s exact probability test
were used to assess differences between patients receiv-
ing cotransplantation of CB and UCMSCs and patients
receiving CBT alone. The data are presented as median and
range. A value of p < 0.05 was considered significant.
Comparisons Between UCMSCs and BMMSCs
UCMSCs showed the same uniform spindle-shaped
morphology as BMMSCs, and they were both positive for
CD13, CD29, CD44, CD73, CD90, and CD105 but nega-
tive for CD14, CD31, CD34, CD45, and HLA-DR. Under
specific induction conditions, UCMSCs and BMMSCs
possessed the capacity for adipogenic, osteogenic, and
chondrogenic differentiation (6,26). A significant increase
in cumulative population doubling from passages 2 to 6 was
observed between UCMSCs (median, 6.3; range, 4.6–7.8)
and BMMSCs (median, 2.9; range, 1.6–4.4) (p = 0.004).
The population doubling time of UCMSCs (median, 1.4
days; range, 1.1–1.9 days) was significantly shorter than
that of BMMSCs (median, 9.6 days; range, 11.7–7.2 days)
(p = 0.003).
Comparisons Between NOD/SCID Mice Cotransplanted
With CB and UCMSCs and Those Transplanted With
As shown in Figure 2, we found that NOD/SCID mice
transplanted with 106 TNCs in the CB together with 106
UCMSCs demonstrated a significantly higher median
28.2% (range, 24.6–33.1%) number of human CD45+ cells
engrafting in the peripheral blood than those transplanted
with 106 TNCs in the CB alone (median, 5.3%; range,
4.2–6.5%; human CD45+ cell engraftment in the peripheral
blood) (p = 0.001). NOD/SCID mice transplanted with 106
TNCs in the CB together with 106 UCMSCs demonstrated
a significantly greater median 6.9% (range, 5.9–7.3%)
number of human CD45+ cells engrafting in the BM than
those transplanted with 106 TNCs in the CB alone (median,
1.7%; range, 1.5–2.3%; human CD45+ cell engraftment in
the BM) (p = 0.015).
Of the 10 mice receiving cotransplantation of CB
and UCMSCs, complete chimerism was observed after
a median time of 9 weeks (range, 8–11 weeks). Of the 10
mice receiving CBT alone, complete chimerism was not
observed until after a median time of 16 weeks (range,
14–20 weeks). By defining complete chimerism in mice
as the end point, we found that faster engraftment was
Figure 2. Cotransplantation of umbilical cord-derived mesenchymal stem cells (UCMSCs) enhanced engraftment of human CD45+
cells in NOD/SCID mice. The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice that were transplanted with
cord blood (CB) together with 106 UCMSCs demonstrated a significantly higher human CD45+ cell engraftment in the peripheral blood
and bone marrow compared to those transplanted with CB alone.
UCMSCs ENHANCE CORD BLOOD TRANSPLANTATION 2047
achieved in mice receiving cotransplantion with CB and
UCMSCs in comparison to those receiving CBT alone
(p = 0.007). Based on the results of animal experimen-
tal investigation, we found that the cotransplantation of
UCMSCs did enhance engraftment of CBT.
Characteristics of Patients and CB
The characteristics of eight patients with a median age
of 9.8 years (range, 3.2–12.1 years) receiving cotrans-
plantation of CB and UCMSCs are summarized in Table 1.
Of the 8 units of CB cotransplanted with UCMSCs, three
CB donors were female, and five were male. Of the eight
patients, three patients had ALL, and five patients had
AML. Twelve patients with a median age of 8.5 years
(range, 3.6–13.1 years) received CBT alone. Of these 12
patients, five patients had ALL, and seven patients had
AML. The characteristics of the 12 patients are summa-
rized in Table 2. The age at transplant, gender, leukemia
type, TNCs in CB, and CD34 cells in CB between patients
receiving cotransplantation and those receiving CBT
alone were not significantly different (Table 3).
Characteristics of UCMSCs
Of the eight patients receiving cotransplantation of CB
and UCMSCs, the range of HLA match between UCMSCs
and recipients was 3/6 to 5/6. The median cell dose (×106/
kg) and passage of UCMSCs were 7.19 (range, 2.44–10.12)
and 3 (range, 2–4), respectively. The median duration of
expansion to sufficient UCMSCs was 7 days (range, 5–8
days). Of the 8 units of UCMSCs, five UCMSC donors
were female, and three were male. UCMSCs infused to
the eight patients were shown to have a normal karyo-
type and the absence of pathogenic contamination after
ex vivo expansion. The characteristics of UCMSCs are
summarized in Table 1.
Safety of UCMSCs
The ex vivo expanded banked UCMSCs were infused
during 30 min through a central venous line. The infusion
of the UCMSCs was well tolerated by all recipients. There
were no infusion-related toxicities such as allergic reac-
tions, respiratory distress, or hypertensive responses. Of
the eight patients receiving cotransplantation of CB and
UCMSCs, no ectopic tissue was noted after MRI and PET
survey in the interval of 3 months after UCMSC infusion.
The median duration of follow-up after cotransplantation
was 16.5 months (range, 8–27 months).
Engraftment, GVHD, and Outcome
All of the 20 patients, including the patients receiving
cotransplantation and CBT only, achieved 100% donor
chimerism assessed with the method of short tandem repeat
on the 60th day after transplantation. Of the eight patients
receiving cotransplantation of CB and UCMSCs, an
ANC > 0.5 × 109/L was seen after a median time of 12 days
(range, 8–16 days) (Table 1). Platelets count > 20 × 109/L
was achieved at a median time of 30 days (range, 20–45
days) (Table 1) after cotransplantation. Of the 12 patients
receiving CBT alone, an ANC > 0.5 × 109/L was seen after
a median time of 21 days (range, 17–43 days). Platelet
count > 20 × 109/L was achieved at a median time of 73
days (range, 42–135 days) after CBT. Patients receiving
cotransplantion had faster recovery of ANC (p = 0.003)
and platelets (p = 0.004) in comparison to those receiving
CBT alone (Table 3).
None of the eight patients receiving cotransplantation
encountered severe infection, including CMV disease, sep-
tic shock, or fungal infection. Of the eight patients receiv-
ing cotransplantation, four patients developed localized
skin rashes (grades I–II acute GVHD), which responded
to steroids. One of these four patients with acute GVHD
developed limited chronic GVHD, which responded to
long-term use of cyclosporine and steroids. Of the eight
patients receiving cotransplantation, two patients expired
due to leukemia relapse, and the other six patients remained
alive and disease free with a median follow-up time of
16.5 months (range, 11–27 months). Of the 12 patients
receiving CBT alone, one patient died from bacteremia
due to Pseudomonas aeruginosa, one died from invasive
pulmonary aspergillosis, and two patients died from leu-
kemia relapse. The other eight patients remained alive and
disease-free with a median follow-up time of 18.5 months
(range, 12–31 months). The comparison between patients
receiving cotransplantation and those receiving CBT alone
is summarized in Table 3.
In humans, BMMSCs are safe to infuse with no severe
adverse events and no formation of ectopic tissue in
clinical application (4,16,18–22,27,29). However, all
the experiences about MSC safety are from BMMSCs.
Amariglio et al. reported that a donor-derived brain
tumor following neural tissue that included neural stem
cells derived from fetal tissue was transplanted in an
ataxia telangiectasis patient with immune deficiency (1).
UCMSCs, one kind of fetal tissue-derived MSCs, are
more primitive than BMMSCs. Although fetal tissue-
derived MSCs are easier to obtain than BMMSCs and
cause no suffering to the donor, the safety of these cells in
human clinical application is still unknown but is likely
to be very important. Few human clinical experiences of
fetal tissue-derived MSCs have been reported until now
(7,12,34). In the present study, after we infused banked
UCMSCs from third-party HLA-mismatched donors to
patients who were severely immunocompromised in the
course of CBT, there was no infusion toxicity and no ecto-
pic tissues detected by MRI and PET after 8–27 months
follow-up. Therefore, we speculate that UCMCSs may
2048 WU ET AL.
Table 2. Characteristics of Patients Receiving CBT Alone
Characteristics of CB
ANC > 0.5 × 109/L
Count > 20 × 109/L
ALL in 2nd
AML with MDS
ALL in 2nd
ALL with Ph
AML in 2nd
AML in 1st
ALL with Ph
ALL in 2nd
Expired due to
AML with MDS
AML in 1st
Extensive Expired due
AML in 2nd
AML in 2nd
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANC, absolute neutrophil count; CB, cord blood; CBT, cord blood transplantation; F, female; GVHD, graft-versus-host disease; HLA, human leukocyte antigen; M, male; MDS, myelodysplastic syndrome; Ph, Philadelphia chromosome; TNCs, total nucleated cells.
UCMSCs ENHANCE CORD BLOOD TRANSPLANTATION 2049
be as safe as BMMSCs for clinical use, even in severe
MSCs have low immunogenicity and do not express
HLA class II (25). Le Blanc et al. found that the response
rates of severe acute GVHD after BMMSC treatment
were not related to the HLA match between recipients and
BMMSCs (19). In the present study, after we infused 3/6
to 5/6 HLA-matched UCMSCs in the patients, no severe
adverse effects were noted, and these cells enhanced
hematopoiesis after CBT. Therefore, we speculate that the
restriction of MSC matches between donors and recipi-
ents is less than other cells, such as HSCs. Besides, we
found that UCMSCs proliferated faster than BMMSCs
(34), as previously reported (3). In the present study, we
could get an adequate cell dose of UCMSCs for recipi-
ents within 8 days, which is shorter than the time to get
an adequate cell dose of BMMSCs for clinical use in the
previous report (22). Therefore, UC can be considered as
an alternative source of MSCs for clinical applications.
Gong et al. reported on banking UCMSCs for clini-
cal use (12). MSCs for human clinical use should not
only be strictly qualified for cell quality but must also be
infection free. Therefore, all procedures of clinical-grade
MSCs should be managed in a qualified manner, such as
under GTP conditions. However, not all physicians have
the facilities to guarantee the quality of cells for clinical
applications. In this study, all UCMSCs provided from
the UCMSC bank under GTP conditions were free of
contamination. Therefore, MSC banks that cryopreserve
manufactured unrelated MSCs may help solve time,
donor variability, and contamination problems. Like CB
banks (28), MSC banks may have a ready supply of “off-
the-shelf” cells available for clinical use.
Chemotherapy and/or radiotherapy prior to HSCT dam-
ages the marrow stroma (10). MSCs play an important role
in providing the specialized BM microenvironment and
secret cytokines for hematopoiesis (33). Additionally, MSCs
(8,15,25,31,35), including BMMSCs and UCMSCs, which
we previously reported (34), possess immunosuppressive
effects, so MSC infusion may suppress immune function
in recipients and, therefore, enhance engraftment of donor
HSCs. Cotransplantation of BMMSCs and HSCs was shown
to promote engraftment after HSCT (4,16,18,21,22,27). In
the present study, we found that patients receiving cotrans-
plantation of CB and UCMSCs resulted in faster engraft-
ment of ANC and platelets than those receiving CBT alone.
Further studies are needed to confirm the cotransplantation
of UCMSCs in the fast engraftment, the effect on GVHD,
and long-term survival after CBT.
In conclusion, UCMSCs are easier to obtain without
harm to the donor and proliferate faster than BMMSCs,
which indicates that they may be ideal candidates for cell-
based therapy. No severe adverse effects were noted in this
human clinical application of UCMSCs. “Off-the-shelf”
UCMSCs may make UCMSCs more available for clinical
applications. Accordingly, application of ex vivo expanded
Table 3. Comparison Between Patients Cotransplanted With CB and UCMSCs and Those Transplanted With CB Alone
Age at transplant (years), median (range)
Male [No. (%)]
Original disease [No. (%)]
CB cell dose
TNCs dose (×107/kg), median (range)
CD34+ cell dose (×105/kg), median (range)
Days to ANC > 0.5 × 109/L, median (range)
Days to platelet count > 20 × 109/L, median (range)
GVHD [No. (%)]
Acute GVHD, grades I–II
Acute GVHD, grades III–IV
Chronic GVHD, limited
Chronic GVHD, extensive
Outcome [No. (%)]
Transplant related mortality
Overall survival rate
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANC, absolute neutrophil count; CB, cord blood; CBT, cord blood transplantation;
GVHD, graft-versus-host disease; TNCs, total nucleated cells; UCMSCs, umbilical cord-derived mesenchymal stem cells. *p < 0.05.
2050 WU ET AL.
banked UCMSCs in humans appears to be feasible and
safe. Cotransplantation of UCMSCs was found to enhance
hematopoiesis after CBT. Further studies are warranted.
ACKNOWLEDGMENTS: This work was supported by the
Ministry of Economic Affairs in Taiwan and grants from the
China Medial University Hospital (DMR-102-035) and Chung
Shan Medical University Hospital (CSH-2013-A-006). The authors
declare no conflicts of interest.
N.; Hirshberg, A.; Scheithauer, B. W.; Cohen,
Y.; Loewenthal, R.; Trakhtenbrot, L.; Paz, N.; Koren-
Michowitz, M.; Waldman, D.; Leider-Trejo, L.; Toren, A.;
Constantini, S.; Rechavi, G. Donor-derived brain tumor fol-
lowing neural stem cell transplantation in an ataxia telangi-
ectasia patient. PLoS Med. 6:221–231; 2009.
Antonucci, I.; Stuppia, L.; Kaneko, Y.; Yu, S.; Tajiri, N.;
Bae, E. C.; Chheda, S. H.; Weinbren, N. L.; Borlongan, C. V.
Amniotic fluid as a rich source of mesenchymal stromal cells
for transplantation therapy. Cell Transplant. 20:789–795;
Baksh, D.; Yao, R.; Tuan, R. S. Comparison of proliferative
and multilineage differentiation potential of human mesen-
chymal stem cells derived umbilical cord and bone marrow.
Stem Cells 25:1384–1392; 2007.
Ball, L. M.; Bernardo, M. E.; Roelofs, H.; Lankester, A.;
Cometa, A.; Egeler, R. M.; Locatelli, F.; Fibbe, W. E.
Cotransplantation of ex vivo expanded mesenchymal stem
cells accelerates lymphocyte recovery and may reduce the
risk of graft failure in haploidentical hematopoietic stem
cell transplantation. Blood 110:2764–2767; 2007.
Chang, J. W.; Hung, S. P.; Wu, H. H.; Wu, W. M.; Yang, A. H.;
Tsai, H. L.; Yang, L. Y.; Lee, O. K. Therapeutic effects of
umbilical cord blood-derived mesenchymal stem cell trans-
plantation in experimental lupus nephritis. Cell Transplant.
Chao, Y. H.; Peng, C. T.; Harn, H. J.; Chan, C. K.; Wu, K. H.
Poor potential of proliferation and differentiation in bone
marrow mesenchymal stem cells derived from children with
severe aplastic anemia. Ann. Hematol. 89:715–723; 2010.
Chao, Y. H.; Tsai, C.; Peng, C. T.; Wu, H. P.; Chan, C. K.;
Weng, T.; Wu, K. H. Cotransplantation of umbilical cord
MSCs to enhance engraftment of hematopoietic stem cells
in patients with severe aplastic anemia. Bone Marrow Trans-
plant. 46:1391–1392; 2011.
Deuse, T.; Stubbendorff, M.; Tang-Quan, K.; Phillips, N.; Kay,
M. A.; Eiermann, T.; Phan, T. T.; Volk, H. D.; Reichenspurner,
H.; Robbins, R. C.; Schrepfer, S. Immunogenicity and immuno-
modulatory properties of umbilical cord lining mesenchymal
stem cells. Cell Transplant. 20:655–667; 2011.
Ding, D. C.; Shyu, W. C.; Lin, S. Z. Mesenchymal stem
cells. Cell Transplant. 20:5–14; 2011.
Galotto, M.; Berisso, G.; Delfino, L.; Podesta, M.; Ottaggio,
L.; Dallorso, S.; Dufour, C.; Ferrara, G. B.; Abbondandolo,
A.; Dini, G.; Bacigalupo, A.; Cancedda, R.; Quarto, R.
Stromal damage as consequence of high-dose chemo/radio-
therapy in bone marrow transplant recipients. Exp. Hematol.
Gluckman, E. History of cord blood transplantation. Bone
Marrow Transplant. 44:621–626; 2009.
Gong, W.; Han, Z.; Zhao, H.; Wang, Y.; Wang, J.; Zhong,
J.; Wang, B.; Wang, S.; Wang, Y.; Sun, L.; Han, Z. Banking
human umbilical cord derived mesenchymal stromal cells
for clinical use. Cell Transplant. 21:207–216; 2012.
Hunt, D. P.; Irvine, K. A.; Webber, D. J.; Compston, D. A.;
Blakemore, W. F.; Chandran, S. Effects of direct transplanta-
tion of multipotent mesenchymal stromal/stem cells into the
demyelinated spinal cord. Cell Transplant. 17:865–873; 2008.
Iop, L.; Chiavegato, A.; Callegari, A.; Bollini, S.; Piccoli,
M.; Pozzobon, M.; Rossi, C. A.; Calamelli, S.; Chiavegato,
D.; Gerosa, G.; De Coppi, P.; Sartore, S. Different cardio-
vascular potential of adult- and fetal-type mesenchymal
stem cells in a rat model of heart cryoinjury. Cell Transplant.
Keyser, K. A.; Beagles, K. E.; Kiem, H. P. Comparison of
mesenchymal stem cells from different tissues to suppress
T-cell activation. Cell Transplant. 16:555–562; 2007.
Koç, O. N.; Gerson, S. L.; Cooper, B. W.; Dyhouse, S. M.;
Haynesworth, S. E.; Caplan, A. I.; Lazarus, H. M. Rapid
hematopoietic recovery after coinfusion of autologous-blood
stem cells and culture-expanded marrow mesenchymal stem
cells in advanced breast cancer patients receiving high-dose
chemotherapy. J. Clin. Oncol. 18:307–316; 2000.
Koldehoff, M.; Steckel, N. K.; Hlinka, M.; Beelen, D. W.;
Elmaagacli, A. H. Quantitative analysis of chimerism after
allogeneic stem cell transplantation by real-time polymerase
chain reaction with single nucleotide polymorphisms, stan-
dard tandem repeats, and Y-chromosome-specific sequences.
Am. J. Hematol. 81:735–746; 2006.
Lazarus, H. M.; Koc, O. N.; Devine, S. M.; Curtin, P.;
Maziarz, R. T.; Holland, H. K.; Shpall, E. J.; McCarthy,
P.; Atkinson, K.; Cooper, B. W.; Gerson, S. L.; Laughlin,
M. J.; Loberiza, F. R.; Moseley, Jr., A. B.; Bacigalupo, A.
Cotransplantation of HLA-identical sibling culture- expanded
mesenchymal stem cells and hematopoietic stem cells in hema-
tologic malignancy patients. Biol. Blood Marrow Transplant.
Le Blanc, K.; Frassoni, F.; Ball, L.; Locatelli, F.; Roelofs,
H.; Lewis, I.; Lanino, E.; Sundberg, B.; Bernardo, M. E.;
Remberger, M.; Dini, G.; Egeler, R. M.; Bacigalupo, A.;
Fibbe, W.; Ringdén, O. Mesenchymal stem cells for treat-
ment of steroid-resistant, severe, acute graft-versus-host
disease: A phase II study. Lancet 371:1579–1586; 2008.
Le Blanc, K.; Rasmusson, I.; Sundberg, B.; Götherström, C.;
Hassan, M.; Uzunel, M.; Ringdén, O. Treatment of severe
acute graft-versus-host disease with third-party haploidenti-
cal mesenchymal stem cells. Lancet 363:1439–1441; 2004.
Le Blanc, K.; Samuelsson, H.; Gustafsson, B.; Remberger,
M.; Sundberg, B.; Arvidson, J.; Ljungman, P.; Lönnies, H.;
Nava, S.; Ringdén, O. Transplantation of mesenchymal stem
cells to enhance engraftment of hematopoietic stem cells.
Leukemia 21:1733–1738; 2007.
Macmillan, M. L.; Blazar, B. R.; DeFor, T. E.; Wagner, J. E.
Transplantation of ex-vivo culture-expanded parental hap-
loidentical mesenchymal stem cells to promote engraftment
in pediatric recipients of unrelated donor umbilical cord
blood: Results of a phase I–II clinical trial. Bone Marrow
Transplant. 43:447–454; 2009.
Magatti, M.; De Munari, S.; Vertua, E.; Nassauto, C.;
Albertini, A.; Wengler, G. S.; Parolini, O. Amniotic mes-
enchymal tissue cells inhibit dendritic cell differentiation
of peripheral blood and amnion resident monocytes. Cell
Transplant. 18:899–914; 2009.
Marchetti, S.; Santangelo, R.; Manzara, S.; D’Onghia, S.;
Fadda, G.; Cattani, P. Comparison of real-time PCR and
pp 65 antigen assays for monitoring the development of
UCMSCs ENHANCE CORD BLOOD TRANSPLANTATION 2051 Download full-text
Cytomegalovirus disease in recipients of solid organ and bone
marrow transplants. New Microbiol. 34:157–164; 2011.
Nauta, A. J.; Fibbe, W. E. Immunomodulatory properties of
mesenchymal stromal cells. Blood 110:3499–3506; 2007.
Pittenger, M. F.; Mackay, A. M.; Beck, S. C.; Jaiswal, R. K.;
Douglas, R.; Mosca, J. D.; Moorman, M. A.; Simonetti, D. W.;
Craig, S.; Marshak, D. R. Multilineage potential of adult
human mesenchymal stem cells. Science 284:143–147; 1999.
Poloni, A.; Leoni, P.; Buscemi, L.; Balducci, F.; Pasquini,
R.; Masia, M. C.; Viola, N.; Costantino, E.; Discepoli, G.;
Corradini, P.; Tagliabracci, A.; Olivieri, A. Engraftment
capacity of mesenchymal cells following hematopoietic stem
cell transplantation in patients receiving reduced-intensity
conditioning regimen. Leukemia 20:329–335; 2006.
Querol, S.; Rubinstein, P.; Marsh, S. G.;
Madrigal, J. A. Cord blood banking: “Providing cord blood
banking for a nation.” Br. J. Haematol. 147:227–235; 2009.
Ringdén, O.; Uzunel, M.; Rasmusson, I.; Remberger, M.;
Sundberg, B.; Lönnies, H.; Marschall, H. U.; Dlugosz, A.;
Szakos, A.; Hassan, Z.; Omazic, B.; Aschan, J.; Barkholt,
L.; Le Blanc, K. Mesenchymal stem cells for treatment of
therapy-resistant graft-versus-host disease. Transplantation
Rubinstein, P.; Carrier, C.; Scaradavou, A.; Kurtzberg, J.;
Adamson, J.; Migliaccio, A. R.; Berkowitz, R. L.; Cabbad,
M.; Dobrila, N. L.; Taylor, P. E.; Rosenfield, R. E.; Stevens,
28. Goldman, J.;
C. E. Outcomes among 562 recipients of placental-blood
transplants from unrelated donors. N. Engl. J. Med. 339:1565–
Sattler, C.; Steinsdoerfer, M.; Offers, M.; Fischer, E.;
Schierl, R.; Heseler, K.; Däubener, W.; Seissler, J. Inhibition
of T cell proliferation by murine multipotent mesenchymal
stromal cells is mediated by CD39 expression and adenos-
ine generation. Cell Transplant. 20:1221–1230; 2011.
Secco, M.; Zucconi, E.; Vieira, N. M.; Fogaça, L. L.;
Cerqueira, A.; Carvalho, M. D.; Jazedje, T.; Okamoto,
O. K.; Muotri, A. R.; Zatz, M. Multipotent stem cells from
umbilical cord: Cord is richer than blood! Stem Cells 26:
Verfaillie, C. M. Direct contact between human primitive
hematopoietic progenitors and bone marrow stroma is
not required for long-term in vitro hematopoiesis. Blood
Wu, K. H.; Chan, C. K.; Tsai, C.; Chang, Y. H.;
Chiu, T. H.; Ho, M.; Peng, C. T.; Wu, H. P.; Huang, J. L.
Effective treatment of severe steroid-resistant acute graft-
versus-host disease with umbilical cord-derived mesenchy-
mal stem cells. Transplantation 91:1412–1416; 2011.
Yagi, H.; Soto-Gutierrez, A.; Parekkadan, B.; Kitagawa,
Y.; Tompkins, R. G.; Kobayashi, N.; Yarmush, M. L.
Mesenchymal stem cells: Mechanisms of immunomodula-
tion and homing. Cell Transplant. 19:667–679; 2010.
34. Sieber, M.;