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A Rapid and Efficient Method for Expansion of Human
Mesenchymal Stem Cells
SANNE K. BOTH, B.Sc.,
1
ADRIE J.C. VAN DER MUIJSENBERG, B.Sc.,
1
CLEMENS A. VAN BLITTERSWIJK, Ph.D.,
1
JAN DE BOER, Ph.D.,
1
and JOOST D. DE BRUIJN, Ph.D.
2
ABSTRACT
During the past decade, there has been much interest in the use of human mesenchymal stem cells (hMSCs)
in bone tissue engineering. HMSCs can be obtained relatively easily and expanded rapidly in culture, but
for clinical purposes large numbers are often needed and the cost should be kept to a minimum. A rapid
and efficient culturing protocol would therefore be beneficial. In this study, we examined the effect of
different medium compositions on the expansion and osteogenic differentiation of bone marrow–derived
hMSCs from 19 donors. We also investigated the effect of low seeding density and dexamethasone on both
hMSCs expansion and their in vitro and in vivo osteogenic differentiation capacity. HMSCs seeded at a
density of 100 cells/cm
2
had a significantly higher growth rate than at 5000 cell/cm
2
, which was further
improved by the addition of dexamethasone. Expanded hMSCs were characterized in vitro on the basis
of positive staining for CD29, CD44, CD105, and CD166. The in vitro osteogenic potential of expanded
hMSCs was assessed by flow cytometric staining for alkaline phosphatase. In vivo bone-forming potential
of the hMSCs was assessed by seeding the cells in ceramic scaffolds, followed by subcutaneous implan-
tation in nude mice and histopathologic assessment of de novo bone formation after 6-week implantation.
Expanded hMSCs from all donors displayed similar osteogenic potential independent of the culture con-
ditions. On the basis of these results we have developed an efficient method to culture hMSCs by seeding
the cells at 100 cells/cm
2
in an a-minimal essential medium–based medium containing dexamethasone.
INTRODUCTION
THE CURRENT AND MOST FREQUENTLY USED APPROACH for
the treatment of large bone defects is autologous or
allogeneic bone grafting. Both methods have distinct draw-
backs, including donor site pain and limited availability for
autograft, and the risk of immune reaction and disease trans-
mission for allograft. The creation of a tissue-engineered
implant, which consists of a suitable biomaterial scaffold
covered with the patient’s own osteogenic cells, could pro-
vide an alternative approach.
Human bone marrow contains a multipotent cell popu-
lation, which has the ability to differentiate into several
mesenchymal lineages,
1
including bone, cartilage, fat, and
muscle.
2
These cells were initially isolated by their ad-
herence to tissue culture surfaces
3,4
and are referred to as
colony-forming unit fibroblasts, marrow stromal cells, or
mesenchymal stem cells. Because of their multipotent na-
ture, these cells have attracted much interest for use in
cell and gene therapy, as well as for treating large bone
defects.
5,6
When grown under osteogenic differentiation
conditions, it has been shown that human mesenchymal
stem cells (hMSCs) not only possess in vitro osteogenic
potential
7,8
but also can form bone tissue in vivo, as dem-
onstrated by subcutaneous implantation in immunodeficient
mice.
9–11
1
Institute for Biomedical Technology, University of Twente, Bilthoven, the Netherlands.
2
Department of Materials, Queen Mary University of London, London, United Kingdom.
TISSUE ENGINEERING
Volume 13, Number 1, 2007
#Mary Ann Liebert, Inc.
DOI: 10.1089/ten.2005.0513
3
Several markers have been suggested for the identifica-
tion and osteogenic differentiation of hMSCs.
10–14
The
hMSCs are positive for the surface expressed antigen en-
doglin receptors CD105 and CD44, the activated leukocyte
cell adhesion molecule CD166, and b-integrin CD29.
12
Developmental markers such as alkaline phosphatase (ALP)
and STRO-1 can be used to characterize osteogenic and un-
differentiated hMSCs, respectively.
13–15
Several culture medium supplements that affect the ex-
pansion and differentiation of hMSCs have been reported.
For instance, basic fibroblastic growth factor and lithium
can enhance the proliferation of hMSCs.
15,16
The addition
of the synthetic glucocorticoid dexamethasone to the me-
dium will induce hMSCs into the osteogenic lineage, thereby
changing the shape of the cells
17
and increasing the ex-
pression of osteoblast markers such as ALP.
7,18,19
Although in vitro osteogenic differentiation of hMSCs and
proof of principle for the creation of an autologous tissue-
engineered implant has been shown,
9,20,21
we still lack a
well-defined protocol for the isolation and expansion of these
cells to make this technique available for clinical purposes.
Surgical skills, volume of the aspirated bone marrow cells,
22
and the donor age and variation in growth rate between dif-
ferent donors significantly influence the creation of a suc-
cessful living implant.
9,20,21
Besides commitment of hMSCs
to the osteogenic lineage, large numbers of cells are needed
for creating a tissue-engineered implant that can fill large
bone defects. Since expansion of hMSCs is labor intensive
and therefore expensive, the use of an optimal culture system
is desirable. For example, on the basis of using a scaffold
containing 8 million cells/mL,
23
20 mL of tissue-engineered
implant used for spinal fusion surgery would require about
160 million cells. By using previously established proto-
cols,
9,19,24
it would take 15–29 days and approximately 24
person-hours to reach enough cells. Although this protocol
was previously used to create tissue-engineered living bone
equivalents, which were also used in a clinical trial (data
submitted separately to PLoS Medicine, December 2006), it
was time consuming and labor intensive because of the
amount of subculturing needed. There is therefore a need
for an optimal protocol for expansion and osteogenic differ-
entiation of hMSCs.
20
In this report, we describe a method for
the expansion of osteogenic hMSCs under optimized con-
ditions to allow treatment of bone defects.
MATERIALS AND METHODS
Harvest and culture of hMSCs
Bone marrow aspirates were obtained from 19 donors who
were undergoing total hip replacement surgery and had given
informed consent (donor information is provided in Table 1).
Nucleated cells were counted in the aspirate and plated at a
density of 500,000 cells/cm
2
in a-minimal essential medium
(aMEM) proliferation medium (see below; donors 1–15) or
aMEM osteogenic medium (see below; donors 11–15). The
hMSCs from donors 16–19 were harvested and proliferated
as described above and then cryopreserved. After defrosting,
TABLE 1. INFORMATION ON DONORS ,EXPERIMENTS, AND NUMBER OF DAYS NEEDED FOR HUMAN MESENCHYMAL STEM CELLS
TO REACH 20010
6
CELLS WHEN SEEDED AT 100 AND 5000 CELLS/CM
2
MEM
versus
100 cells/cm
2
versus 5000 þ/
Days needed to reach
20010
6
cells
Donor Aspiration site Sex Age DMEM cells/cm
2
Dexamethasone In vivo 100 cells 5000 cells
1 Crista F 72 HH15 18
2 Crista F 87 HH19 24
3 Crista F 61 HH21 29
4 Crista F 63 HH16 17
5 Crista F 69 HH19 23
6 Acetabulum M 29 HH12 15
7 Acetabulum F 63 HH15 19
8 Acetabulum M 56 HH12 19
9 Iliac crest F 28 HH14 20
10 Acetabulum F 25 HH13 16
11 Crista F 77 HHH14 15
12 Acetabulum F 44 HHH16 18
13 Iliac crest F 26 HHH14 16
14 Acetabulum M 48 HHH16 18
15 Iliac crest M 56 HHH16 19
16 Acetabulum F 28 H
17 Acetabulum M 49 H
18 Iliac Crest M 42 H
19 Iliac crest F 36 H
Abbreviations: aMEM, a-minimal essential medium; DMEM, Delbecco’s modified Eagle medium; F, female; M, male.
4BOTH ET AL.
they were plated at 5000 cells/cm
2
and grown in the different
culture media as described below (n¼5 for each donor).
The aMEM proliferation medium contained minimal es-
sential medium (GIBCO, Carlsbad, CA), 10% fetal bovine
serum of a selected batch (FBS; BioWhittaker, Australia,
lot: 0S025F); 0.2 mM L-ascorbic acid-2-phosphate (Sigma,
St. Louis, MO), penicillin G (100 U/mL, Invitrogen, Carls-
bad, CA); streptomycin (100 mg/mL, Invitrogen); 2 mM L-
glutamine (Sigma) and 1ng/mL basic fibroblast growth
factor (Instruchemie, Delfzijl, the Netherlands). The aMEM
osteogenic medium was composed of the proliferation me-
dium supplemented with 10 nM dexamethasone (Sigma).
Delbecco’s modified Eagle medium (DMEM) proliferation
medium comprised 10% FBS (BioWhittaker), 1% nones-
sential amino acids 100 (Sigma), penicillin G (100 U/mL;
Invitrogen), streptomycin (100 mg/mL; Invitrogen), 2 mM L-
glutamine (Sigma), 1 mM sodium pyruvate (GIBCO), 1 mM
Hepes (GIBCO) and 1 ng/mL basic fibroblast growth factor
(Instruchemie). DMEM osteogenic medium was composed
of DMEM proliferation medium supplemented with 10 nM
dexamethasone (Sigma). The batch of FBS selected for the
hMSCs in this study was tested in vitro for cell growth,
expression of ALP, by FACS and the enzymatic expression
of ALP and in vivo for bone formation of the hMSCs. The
cells were grown at 37
8
C in a humidified atmosphere of 5%
carbon dioxide. Cultures were refreshed twice a week. When
the cells reached near confluence (80–90%), they were wa-
shed with phosphate-buffered saline (PBS; Sigma), detached
with 0.05% trypsin (GIBCO), containing 1 mM ethylene-
diaminetetraacetic acid and subcultured.
Flow cytometry
For flow cytometry, hMSCs from donors 1–15 were
detached and centrifuged at 100 gfor 10 min at 48C. The
cell pellets were resuspended in PBS containing 5% bovine
serum albumin (Sigma) and 0.05% sodium azide (NaN
3
;
Sigma) and stored on ice for 15 min. Next, the cells were
incubated in block buffer containing control mouse anti-
human IgG2a (Dako, Glostrup, Denmark); anti-ALP anti-
body (hybridoma B4-78), anti-CD29 monoclonal antibody
(Dako), anti-CD44 (Pharming, Leiden, the Netherlands)
antibody, anti-CD105 (Dako) antibody, or anti-CD166
(RDI, Concord, MA) monoclonal antibody. The cells were
incubated on ice for 45 min and washed three times with
PBS containing 1% BSA and 0.05% NaN
3
(wash buffer).
Incubation with the secondary antibody, goat anti-mouse-
IgG–fluorescein isothiocyanate (Dako), was performed
for 45 min on ice. After the hMSCs were washed 3 times
with wash buffer, the cells were incubated for 10 min with
10 mL Viaprobe (Becton Dickinson, Franklin Lakes, NJ) for
live-dead staining. The cell suspension was analyzed by us-
ing a flow cytometer (Becton Dickinson). From each sam-
ple, 10,000 events were collected in duplicate and the data
were analyzed via Cell Quest software (Becton Dickinson).
For statistical analysis of the percentages of positive cells
of the donors, a Student t-test was performed; significance
was determined at a pvalue less than .05.
ALP activity
The enzyme activity of ALP was measured in triplicate
for donors 16–19 by washing the cells with PBS and in-
cubating them for 10–15 minutes in a 20 mM paranitro-
phenol solution (Sigma) in 1 M diethanolamine (Sigma),
1 mM magnesium chloride (Sigma). The activity of the ALP
enzyme was measured with a microplate reader (BIOTEC
Instruments, Switzerland) at 310 nm and normalized for cell
number.
In vivo implantation
To assess the in vivo bone-forming capacity of various
passages of hMSCs from donors 1–15, they were seeded
onto porous biphasic calcium phosphate scaffolds (2–4 mm)
and implanted in nude mice. The hMSCs were seeded by
adding a 100-mL drop cell suspension, containing 200,000
cells, on each biphasic calcium phosphate scaffold (n¼6 per
experiment). After 2 h of adherence, the medium was added
and scaffolds were cultured for 7 days in aMEM osteogenic
medium. Before implantation, the particles were placed in
serum-free medium for approximately 1–2 h and washed 3
times with PBS. Immunodeficient mice (HsdCpb:NMRI-nu,
Harlan) were anesthetized by an intramuscular injection of
0.05 mL ketamine, xylazine, and atropine mixture. Before
surgery, the skin on both lateral sites of the spine was
cleaned with 70% alcohol, and 6 subcutaneous pockets were
created in each mouse. In each pocket, 3 tissue-engineered
samples were inserted and each culture condition was di-
vided over three mice. Six weeks postoperatively, the ani-
mals were euthanized by carbon dioxide. The explanted
samples were fixated in 1.5% glutaraldehyde (Sigma) in 0.14
M cacodylic acid buffer, dehydrated by increasing ethanol
steps, and embedded in methylmethacrylate (Sigma). Sec-
tions were made by using a diamond saw (Leica SP1600,
Wetzlar, Germany) and stained with 1% methylene blue
(Sigma) and 0.3% basic fuchsin solution (Sigma).
25
RESULTS
Effect of culture medium on hMSC growth
The cells of donors 16–19 were cultured for 2 passages
in the 2 different basal media. When hMSCs were proli-
ferated in aMEM for 4 days, the number of cells obtained
was significantly higher for all 4 donors compared with
hMSC proliferated in a DMEM proliferation medium,
regardless of the passage number (n¼4 for each donor,
P<.05) (Fig. 1A).
To investigate the effect of different media on the oste-
ogenic potential of hMSCs, the cells were cultured for 7
days in osteogenic aMEM-based and DMEM-based media.
EXPANSION OF MESENCHYMAL STEM CELLS 5
When grown in aMEM-based medium, all 4 donors (16–
19) displayed increased ALP enzyme activity when dexa-
methasone had been added (differentiation medium). In
DMEM-based medium, this difference was less apparent
(Fig. 1B). In addition, overall ALP enzyme activity was
higher when the cells were cultured in aMEM medium as
compared with DMEM.
Effect of seeding density and dexamethasone
on hMSC proliferation
With all 15 donors tested, the cultures seeded at a density
of 100 cells/cm
2
reached 200 million cells within 12–21
days of passage one (Fig. 1). In contrast, hMSCs seeded at
5000 cells/cm
2
, as described in our previous culturing
protocol,
9,19
had to be subcultured for at least 2 passages
(three passages for most donors) and cultured for 15–29
days before 200 million cells were reached (Fig. 2, Table 1).
By using the lower seeding density, the cultures there-
fore reached 200 million cells approximately 4.1 days
earlier compared with cells seeded at 5000 cells/cm
2
. Dexa-
methasone stimulates differentiation of hMSCs, and some
hMSCs culture protocols use it throughout the entire culture
period.
26
Therefore, we investigated the effect of dexa-
methasone on hMSC proliferation by culturing them in
proliferation medium or osteogenic medium throughout the
entire proliferation period. The number of cells obtained
from the primary culture was higher for all five donors when
grown in osteogenic medium compared with proliferation
medium and also at later passages independent of the
seeding density (Fig. 3). Furthermore, dexamethasone ap-
peared to stimulate hMSC proliferation at later passages as
well.
Osteogenic differentiation
Cells grown at 100 cells/cm
2
in the presence of dexa-
methasone resulted in the fastest and most labor-efficient
method to expand hMSCs. To confirm that hMSCs expanded
by using this protocol retained their osteogenic potential, we
first analyzed the effect of seeding density and dexameth-
asone on the hMSC phenotype with a panel of hMSC
markers. The hMSC markers CD29, CD44, CD105, and
CD166 were expressed in hMSCs of all donors (1–15) in all
culture conditions, and the expression varied between 95%
and 100% (Fig. 4). To investigate whether the culture con-
ditions influenced the osteogenic potential of the hMSCs,
we measured expression of the osteogenic marker ALP.
From previous experiments (data not shown), we deter-
mined that after approximately 7 days of culture in the
presence of dexamethasone, ALP expression of the cells
reached its optimum. To assess whether the continuous
presence of dexamethasone influenced the osteogenic po-
tential of the hMSCs, cells previously cultured without
dexamethasone were also cultured with dexamethasone for
Number of cells (log)
1E+13
1E+12
1E+11
1E+10
1E+09
1E+08
10000000
10000000 5 10 15
Days
20 25 30 35
5000 cells
5000 cells + dex
100 cells
100 cells + dex
FIG. 2. The effect of seeding density and culture medium on
human mesenchymal stem cell proliferation. Progressive cell
numbers are expressed against culture time. The cells were seeded
at 5000 or 100 cells/cm
2
in proliferation and osteogenic media
(n¼5 for each donor). Error bars for each passage were too small
to be visualized in the graph. Because of variants in proliferation
rate between donors, a representative graph of the growth curve of
donor 2 is given. dex: dexamethasone.
1e+6
8e+6
6e+6
Amount of cells
4e+6
2e+6
0Donor 16 Donor 17 Donor 18 Donor 19
DMEM
Alpha MEM A B
DMEM
Alpha MEM
Donor 16 Donor 17 Donor 18 Donor 19
6
5
4
3
2
1
0
Fold induction of ALP
FIG. 1. (A) Proliferation of human mesenchymal stem cells (hMSCs) in a-minimal essential medium (aMEM) versus Delbecco’s
modified Eagle medium (DMEM). When hMSCs were proliferated in aMEM for 4 days, the number of cells obtained was significantly
higher for all 4 donors compared with hMSCs proliferated in a DMEM proliferation medium, regardless of the passage number (n¼4;
P<.05). (B) Effect of aMEM or DMEM medium on alkaline phosphate (ALP) enzyme activity. The hMSCs were proliferated in
aMEM and DMEM proliferation and differentiation media, and the ALP enzyme activity was measured as activity per cell. The fold
increase in ALP enzyme activity of the proliferation versus osteogenic medium is displayed in the graph.
6BOTH ET AL.
7 days. At both seeding densities, ALP expression signifi-
cantly increased when the cells were cultured in the pres-
ence of dexamethasone compared with cells cultured in
proliferation medium (Figure 3). ALP induction was sim-
ilar between cells seeded at different densities and those
with comparable population doublings. The induction of
ALP by dexamethasone did not significantly, regardless of
whether hMSCs had been cultured in previous passages in
the presence of dexamethasone or only for 7 days.
In vivo bone-forming potential of hMSCs
After 6 weeks of implantation, the samples of all donors
exhibited direct deposition of bone tissue, irrespective of
seeding density or presence of dexamethasone in the cul-
ture medium. The newly formed bone comprised osteocytes
embedded within the bone matrix and layers of osteoblasts
(Fig. 5). Besides fibrous tissue and newly formed bone, no
other connective tissue formation was observed with any of
the donors. Microscopic observation was performed blinded
by 3 independent observers, who each examined 5–8 slides
of each implanted condition. Between the different donors
the amount of newly formed bone varied, but not within the
different culture conditions from a single donor.
DISCUSSION
In this study, we demonstrate that the use of an aMEM-
based medium is superior for both expansion and the
osteogenic differentiation of hMSCs, as compared with a
DMEM-based medium. The expansion of hMSCs at low
density results in a higher proliferation rate compared with
cells proliferated at a higher density, which is further stim-
ulated by the addition of dexamethasone. Neither seeding
densities nor the addition of dexamethasone interferes with
the osteogenic in vitro and in vivo potential of the hMSCs.
The use of aMEM or DMEM medium for the expansion
of hMSCs has been investigated before by Jaiswal et al.
7
Interestingly, they found that fold increase of ALP enzyme
activity was much higher with DMEM culture, a finding in
contrast with our results. Moreover, they also stated that
mineralization was sparsely detected in hMSCs cultured in
aMEM osteogenic medium, which also conflicts with pre-
vious results.
27
In this paper, we report that hMSCs pro-
liferate faster at lower seeding densities. To investigate the
optimal density, we tested the growth rate of the hMSCs
also at densities lower than 100 cells/cm
2
and concluded that
the growth rate at those lower densities was even higher
then when seeded at 100 cells/cm
2
(data not shown). Un-
fortunately, at these low densities the cells form a few iso-
lated cell colonies, which do not fill up the entire culture
flask. Therefore, we concluded that seeding 100 cells/cm
2
represents the most efficient expansion. To create an even
better expansion environment for the hMSCs, it might be
beneficial to refresh not the entire culture media but part of
it. With refreshing the entire culture, media components
secreted by the cells will be removed and the cells will have
to adept to the new environment. When cells are seeded at
low density, they display a lag phase of 3–5 days before
going into a rapid exceptional growth phase. At the end
of the lag phase it has been reported that the cells secrete
5000 cells 100 cells
100
80
60
%positive cells
40
20
0
control medium
7 days + dex
entire culture + dex
FIG. 3. The effect of seeding density and culture medium on the
expression of alkaline phosphate (ALP) of human mesenchymal
stem cells (hMSCs). The percentage of positive cells expressing
ALP of donors 6–11 were measured after proliferating the cells at
5000 and 100 cells/cm
2
. Cells proliferated without dexamethasone
(dex) were cultured without (A) and with (B) dexamethasone for
7 days, or they were continuously cultured in the presence of
dexamethasone (C). ALP activity increased significantly for all 6
donors when dexamethasone was present in the media, regardless
of the proliferation medium previously used (P<.05). Prolifer-
ating hMSCs with or without dexamethasone had no significant
influence on the ALP expression; the seeding densities also had no
significant effect.
120
100
80
60
%positive cells
40
20
0
CD29 CD44 CD109 CD166
100 cells/cm2
5000 cells/cm2
FIG. 4. The effect of seeding density on the expression of the
human mesenchymal stem cell (hMSC)–related markers CD29,
CD44, CD105, and CD166. Cells of donor 7 were cultured at a
seeding density of 100 cells/cm
2
and 5000 cells/cm
2
in prolifer-
ation medium until 200 million cells was reached.
EXPANSION OF MESENCHYMAL STEM CELLS 7
high levels of dickkopf-1 (DKK-1), inhibitor of the Wnt-
signaling pathway.
28
High levels of DKK-1 allow the hMSCs
to re-enter the cell cycle and therefore be responsible for
the rapid growth. However, these data contradict the results
of de Boer et al.
16
which demonstrate that the addition
of lithium, a stimulant of the Wnt-signaling pathway, in-
creases the expansion rate of hMSCs. No real conclusion
can be made as to what causes the cells to grow at a higher
rate when seeded at low densities; it is very likely that
several different factors contribute to this phenomenon.
The effect of increased expansion of hMSCs cultured at
lower densities has also been reported by Colter et al.
29
They reported that there might be a minor population of
small agranular cells, also referred to as recycling stem
cells, that give rise to a new population of small densely
granular cells and are thereby responsible for the rapid
expansion when hMSCs are seeded at lower densities. They
also reported that in replated low-density passages, their
hMSCs retained their ability to generate single cell–derived
colonies and thereby retained their multipotentiality for
differentiation. This is in contrast with an observation we
made. We observed that the osteogenic potential of the
cells both in vitro and in vivo seemed to be decreasing with
repeated passages of the hMSCs, indicating that the cells
might lose their multipotentiality for differentiation. This
change can be observed in vitro by flow cytometry. It has
been reported by Mendes et al.
18
that an increase in ALP
induction by dexamethasone is related to the occurrence of
bone formation in vivo. The addition of dexamethasone to
the medium is not a new concept. Rat MSC cultures are
always expanded in the presence of dexamethasone,
26,30
but for hMSCs dexamethasone is almost only used for dif-
ferentiation of the cells, not for expansion. We clearly
show herein that dexamethasone does not only influ-
ence the osteogenic potential of hMSCs but lso enhances
their proliferation rate, without interfering with the in vivo
bone-forming capacities of the hMSCs.
In conclusion, these results indicate that the optimal way
to rapidly expand hMSCs is by seeding the cells at a density
of 100 cells/cm
2
in an aMEM-based medium containing
dexamethasone.
ACKNOWLEDGMENT
The authors would like to acknowledge the orthopedic
departments from the University Hospital of Utrecht, Ac-
ademic Hospital of Maastricht, and the Sint Maartens clinic
in Nijmegen for providing bone marrow aspirates for this
study.
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Address reprint requests to:
Joost de Bruijn, Ph.D.
Department of Materials
Queen Mary University of London
Mile End Road
London E1 4NS
United Kingdom
E-mail: j.d.debruijn@gmul.ac.uk
EXPANSION OF MESENCHYMAL STEM CELLS 9
