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Cytotherapy
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Rapid expansion of human adipose-derived stromalcells preserving multipotency

H. Suga a; T. Shigeura b; D. Matsumoto a; K. Inoue a; H. Kato a; N. Aoi a; S.
Murase b; K. Sato b; K. Gonda a; I. Koshima a; K. Yoshimura aaDepartment of Plastic Surgery, University of Tokyo School of Medicine, Tokyo,
JapanbDivision of Research and Development, Biomaster Inc., Kanagawa, Japan
Online Publication Date: 01 January 2007
To cite this Article: Suga, H., Shigeura, T., Matsumoto, D., Inoue, K., Kato, H.,
Aoi, N., Murase, S., Sato, K., Gonda, K., Koshima, I. and Yoshimura, K. (2007)'Rapid expansion of human adipose-derived stromal cells preserving multipotency',
Cytotherapy, 9:8, 738 - 745
To link to this article: DOI: 10.1080/14653240701679873
URL: http://dx.doi.org/10.1080/14653240701679873

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Cytotherapy (2007) Vol. 9, No. 8, 738745

Rapid expansion of human adipose-derived stromalcells preserving multipotency
H Suga1, T Shigeura2, D Matsumoto1, K Inoue1, H Kato1, N Aoi1, S Murase2,
K Sato2, K Gonda1, I Koshima1 and K Yoshimura1
1

Department of Plastic Surgery, University of Tokyo School of Medicine, Tokyo, Japan and
2
Division of Research and Development, Biomaster Inc., Kanagawa, Japan

Background
Adipose-derived stromal (stem) cells (ASC) have been shown to be ofgreat therapeutic use in pre-clinical studies in diverse fields, but astandard expansion method has not been established. We investigatedthe effects of an endothelial growth medium (EGM-2) on ASC,focusing on proliferation and differentiation potentials.
Methods
ASC were cultured in EGM-2 and DMEM. Doubling time and total
cell number were compared between the two media. The proliferative
effect of each growth factor supplemented in EGM-2 was also
examined. Cultured cells in each medium were examined for surface
marker expression using flow cytometry. Differentiation into theadipogenic, chondrogenic and osteogenic lineages was analyzed after
culture in each medium.
Results
ASC cultured with EGM-2 proliferated much more rapidly (105 times
in 2 weeks) and reached the stationary phase earlier than thosecultured with DMEM. Among the supplements contained in EGM-2,

Introduction
Human adherent stromal cells isolated from adipose tissue
have been shown to have multipotency [1,2], able todifferentiate not only into mesenchymal lineages, including endothelial cells [36] and cardiomyocytes [79] but
also into neural cells [2] and hepatocytes [10]. These cells
have been referred to by various names, including preadipocytes, vascular stromal cells, adipose-derived mesenchymal progenitor cells and adipose stromal cells. In
this study, we refer to the cells as adipose-derived stromal

only fibroblast growth factor-2 (FGF-2) significantly promoted
proliferation of ASC, although the proliferative effect of FGF-2 wasmuch less than that of EGM-2, suggesting a synergism among othersupplement factors. Flow cytometry and differentiation assays suggested
that ASC cultured in EGM-2 preserved immunophenotype anddifferentiation capacity for at least three mesenchymal lineages(adipogenic, chondrogenic and osteogenic), similar to those cultured
with DMEM.
Discussion
The present expansion method markedly accelerates proliferation ofASC, preserving their multipotent differentiation capacities, and lays
the groundwork for establishing a practical route to mega-expansion of
ASC for clinical applications.
Keywords
adipose-derived stem cells, differentiation, endothelial growth medium,
fibroblast growth factor-2, mesenchymal stromal cells, multipotency,
pre-adipocytes, proliferation, vascular endothelial growth factor.

(stem) cells (ASC). The characteristics of ASC have beenstudied extensively [1115], as well as the potential
clinical applications of ASC [37,16]. In addition, clinical
trials have already begun involving enhancement of bone
and adipose regeneration and angiogenesis [1720].
Adipose tissue is thought to be a promising source ofmultipotent stromal cells because it can be harvested inrelatively large quantities (100 mL1 L) using liposuction, with minimal morbidity. Although ASC may be usedclinically without cell expansion, because of the large

Correspondence to: Kotaro Yoshimura, MD, Department of Plastic Surgery, University of Tokyo School of Medicine, 7-3-1, Hongo, Bunkyo-Ku,
Tokyo 113-8655, Japan. E-mail: yoshimura-pla@h.u-tokyo.ac.jp.
– 2007 ISCT

DOI: 10.1080/14653240701679873

Downloaded By: [University of Tokyo/TOKYO DAIGAKU] At: 08:43 5 December 2007

Rapid expansion of human adipose-derived stromal cells

quantities, it is of great value to culture and expand ASC
safely and effectively, without losing their multipotency,
for manipulation and further development of cell-based
therapies. There have been some reports indicating
enhanced proliferation of human ASC using specificculture media with supplements. It has been shown thatfibroblast growth factor-2 (FGF-2) is released by ASC,enhances proliferation [2124] and maintains the adipogenic potential of ASC [21]. It has been suggested that
FGF-1 and epidermal growth factor (EGF) act as
stimulators of both ASC proliferation and differentiation[2527]. Platelet-derived growth factor (PDGF) [25,28],
tumor necrosis factor-a (TNF-a) [29] and insulin-likegrowth factor-1 (IGF-1) [29] have also been shown topromote ASC proliferation, and the former two factors
were suggested to have inhibiting effects on ASC differentiation [25,29]. However, it has not been shown whether
ASC expanded by these methods preserves their multipotency or not.
In this study, we investigated the effects of an endothelial growth medium (EGM-2† ; Cambrex, Walkersville,
MD, USA) on culturing human ASC, focusing on
proliferation and differentiation potentials, and foundthat it markedly expands ASC, preserving multipotency.
EGM-2 is usually used to support the growth ofendothelial cells. In recent studies, EGM-2 has beenused for culture of non-endothelial cells [16,30,31]. ASC
have usually been cultured in DMEM or DMEM/F12
medium, and the effects of EGM-2 on ASC have not beenreported.

Methods
Cell isolation and culture
We obtained liposuction aspirates from 12 healthy female
donors undergoing liposuction of the abdomen or thighs,
after informed consent using an IRB-approved protocol.
The stromal vascular fraction containing ASC was isolated
from the fatty portion of liposuction aspirates, as described
previously [15]. Briefly, the aspirated fat was washed with
PBS and digested on a shaker at 378C in PBS containing
0.075% collagenase for 30 min. Mature adipocytes andconnective tissues were separated from pellets by centrifugation (800 g, 10 min). The cell pellets were resuspended,filtered with a 100-mm mesh (Millipore, Billerica, MA,
USA), plated at a density of 5 105 nucleated cells/100-mm
dish, and cultured at 378C in an atmosphere of 5% CO2 inhumid air. The culture medium was (1) DMEM (Nissui739

Pharmaceutical, Tokyo, Japan) containing 10% FBS or (2)EGM-2 containing 2% FBS. Endothelial basal medium(EBM; Cambrex) is the basal medium for EGM-2. EGM-2
does not contain any animal-derived factors but does contain
FGF-2, vascular endothelial growth factor (VEGF), IGF-1,
EGF, ascorbic acid, hydrocortisone, GA-1000 (gentamicin
and amphotericin-B) and heparin, although the concentration of each agent is not disclosed. Freshly isolated cells were
cultured for 7 days and the first culture was defined as
passage 0. The medium was replaced every 3 days. Cells
were passaged every week by trypsinization.

Measurement of doubling time and total cellnumberDuring cell culture in each medium, the doubling time wasmeasured at passages 0, 1, 2 and 3 by seeding ASC at adensity of 1 105 cells/10-cm dish. After cells reached thelogarithmic growth phase, they were sequentially trypsinized every 48 h and counted with a cell counter(NucleoCounter; Chemometec, Allerod, Denmark). The
doubling time was calculated according to the following
formula: doubling time 48 h/log2(N2/N1), where N1 is
the first cell count and N2 is the cell count 48 h later. The
total cell number after the initiation of culture in eachmedium was also measured by seeding ASC (passage 0) at
a density of 1 104 cells/3.5-cm dish and culturing thecells until they reached the stationary phase.

Measurement of the proliferative effect of
supplemented growth factors
To examine the proliferative effect of each growth factor
supplemented in EGM-2, ASC were cultured in medium
supplemented with a single growth factor (VEGF, EGF,
IGF-1 or FGF-2). EBM containing 2% FBS was used as
the control medium. ASC (passage 0) were seeded at adensity of 1 104 cells/well in a 6-well plate. Cells were
cultured in the control medium (2% FBS), supplemented
medium (0.1, 1 or 10 ng/mL of each growth factor) (2%FBS), DMEM (10% FBS) or EGM-2 (2% FBS). The
number of cells after 7 days of culture was counted using acell counter.

Flow cytometry of cultured cells
Cultured cells in each medium were examined for surface
marker expression using flow cytometry. The following
MAb conjugated to fluorochromes were used: anti-CD29
PE, CD31PE, CD34PE, CD45PE, CD90PE, CD146

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740

H Suga et al.

PE (BD Biosciences, San Diego, CA, USA), CD105PE(Serotec, Oxford, UK) and Flk-1-PE (Techne, Minneapolis,
MN, USA). Control MAb were included for all fluorochromes. Cells were incubated with directly conjugated
MAb for 30 min, then washed and fixed in 1% paraformaldehyde. Cells were analyzed using an LSR II (Becton
Dickinson, San Jose, CA, USA) flow cytometry system. Dataacquisition and analysis were then performed (Cell Quest
software; Becton Dickinson). Gates were set based on
staining with combinations of relevant and irrelevant MAb
so that no more than 0.1% of cells were positive using
irrelevant Ab.

Statistical analyzes
Results are expressed as mean9SEM. Welch’s t-test wasused to compare each parameter. A value of P B0.05 wasconsidered significant.

Results
Doubling time and total cell number

Induced differentiation of cultured cells

The doubling time of ASC cultured with EGM-2 wassignificantly shorter than that of cells cultured with DMEM
at each passage (19.392.1 h vs. 39.896.8 h at passage 0;
15.691.1 h vs. 55.193.5 h at passage 1; 20.390.7 h vs.
52.092.4 h at passage 2; 26.591.1 h vs. 54.396.4 h atpassage 3; Figure 1A). The total cell number showed that
ASC cultured with EGM-2 proliferated more rapidly andreached the stationary phase earlier than those cultured

After culture in each medium for 2 weeks, differentiation
into the adipogenic, chondrogenic and osteogenic lineages
was examined. For adipogenic differentiation, cells wereincubated for 4 weeks in DMEM containing 10% FBS
supplemented with 0.5 mm isobutyl-methylxanthine(Sigma, St Louis, MO, USA), 1 mm dexamethasone,
10 mm insulin (Sigma) and 200 mm indomethacin. Adipogenic differentiation was visualized with Oil Red O
staining. For quantitative analysis of lipid droplets, wemeasured Nile Red fluorescence using AdipoRedTM (Cambrex), with excitation at 485 nm and emission at 535 nm.
For chondrogenic differentiation, cells were incubatedfor 4 weeks in DMEM containing 1% FBS supplemented
with 6.25 mg/mL insulin, 10 ng/mL TGF-b1 and 50 nm
ascorbate-2-phosphate. Chondrogenic differentiation was
visualized with Alcian Blue staining. For quantitative
analysis, a micromass culture system was used, as reported
previously [32]. Cells were plated in a 15-mL tube andcultured in the chondrogenic medium for 3 weeks. Thenthe diameter of a micromass was measured.
For osteogenic differentiation, cells were incubated for 4
weeks in DMEM containing 10% FBS supplemented with
0.1 mm dexamethasone, 50 mm ascorbate-2-phosphate and10 mm b-glycerophosphate (Nacalai Tesque, Kyoto, Japan).
Osteogenic differentiation was visualized with von Kossa
staining. For quantitative analysis of total calcium, calcium
deposition was evaluated based on the ortho-cresolphthalein complexone (OCPC) method with the Calcium C-Test
Wako Kit (Wako Chemicals, Osaka, Japan) according to the
manufacturer’s instructions.

Figure 1. (A) Doubling time at passages 03. The doubling time of
ASC cultured with EGM-2 was significantly shorter than for thosecultured with DMEM at each passage. (B) Total cell number andpopulation doubling level after the initiation of culture with DMEM
or EGM-2. ASC cultured in EGM-2 proliferated more rapidly andreached the stationary phase earlier than those cultured in DMEM.
After 40 days of culture with EGM-2, the result was obtained from one
sample.

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Rapid expansion of human adipose-derived stromal cellswith DMEM (40 days vs. 200 days), although the maximum
population doubling level of ASC was similar when
cultured with EGM-2 or DMEM (3540 with EGM-2 vs.
4045 with DMEM; Figure 1B). Differentiation assays were
performed using ASC cultured with each medium for 2
weeks and, at that stage, ASC cultured with EGM-2
supposed to be 105 times (1010 vs. 105) compared withthose cultured with DMEM (Figure 1B).

Proliferative effect of each growth factor
At the concentrations tested, VEGF, EGF and IGF-1
showed no significant proliferative effect on ASC cultured
in EBM containing 2% FBS. FGF-2, at a density of 0.1, 1
or 10 ng/mL, significantly promoted proliferation of ASC
compared with the control. However, the proliferative
effect of FGF-2 was much less than that of EGM-2
containing all of the growth factors, indicating a synergistic
effect of supplemented growth factors (Figure 2).

Flow cytometry
Flow cytometry of ASC cultured in DMEM and EGM-2
showed no significant differences, except for CD105 at
passages 1, 2 and 3 (Table 1). Both cell populations
uniformly expressed mesenchymal markers (CD29 andCD90) and were devoid of hematopoietic cell marker
CD45. Expressions of CD34 (stem cell marker), CD31
(endothelial cell marker), CD146 (endothelial cell andvascular mural cell marker) and Flk-1 (VEGFR-2) were

Figure 2. Cell number after 7 days of culture in EBM (2% FBS)
supplemented with one of the following growth factors: VEGF, EGF,
IGF-1 or FGF-2 (n 3). FGF-2, at a density of 0.1, 1 or 10 ng/mL,
significantly promoted proliferation of ASC compared with control
medium. The numbers of cells cultured in DMEM (10% FBS) or
EGM-2 (2% FBS) are also indicated. *PB0.05.

741

similar in both cell populations, and CD34 expression of
ASC markedly decreased at passage 1 in both media.

Differentiation capacity
Both cell populations cultured in DMEM and EGM-2 for
2 weeks had similar capacities for differentiating into
adipogenic, chondrogenic and osteogenic lineages. No
morphologic differences between the two cell populations
were observed during and after differentiation (Figure 3A).
Quantitative analyzes (lipid droplets in adipogenic differentiation, micromass diameter in chondrogenic differentiation and total calcium content in osteogenic
differentiation) also showed no significant differences
between the two cell populations (Figure 3B).

Discussion
In this study, EGM-2 expanded ASC very rapidly while
preserving their multipotency for at least 2 weeks; theproliferative efficiency of EGM-2 was 105 times that of
DMEM in the first 2 weeks. A doubling time of ASC
shorter than that shown in this study (1520 h) has notbeen reported previously in the literature. EGM-2 contains 2% FBS and various growth factors, including FGF2, VEGF, IGF-1 and EGF. The highly boosting effects of
EGM-2 on ASC proliferation are suggested to result fromthe supplemented growth factors and other unknown
synergistic effects, as discussed below. ASC cultured with
EGM-2 proliferated much more rapidly and reached thestationary phase earlier than those cultured with DMEM
(40 days vs. 200 days), although the maximum population
doubling levels were similar between the two culture
media (Figure 1B). The results suggest that the majority of
ASC may have a limited capacity for self-renewal.
Serum concentrations can affect the proliferation activity of ASC. We have reported previously that the doublingtime of ASC cultured with 15% FBS was significantly
shorter than that of cells cultured with 10% FBS, although
the culture media was M199 supplemented with FGF-1 inthat study [15]. FBS is made by coagulations of fetal bovine
whole blood; thus it is supposed to contain not only IGF-1,
which is regularly present in serum, including plateletpoor plasma-derived serum, but also platelet-derived
cytokines, such as PDGF and EGF [33]. In the present
study, however, the doubling time of ASC cultured in
DMEM was significantly longer than that for ASC
cultured in EGM-2, in spite of the higher FBS concentration (10%) in DMEM compared with EGM-2 (2%). This

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742

H Suga et al.

Table 1. Flow cytometry analyzes of cell surface marker Ag. Expression of mesenchymal markers (CD29, CD90, CD105),
endothelial markers (CD31, CD146, Flk-1), a stem cell marker (CD34) and a hematopoietic marker (CD45) of ASC cultured
with DMEM or EGM-2 was quantitatively examined at passages 03

Passage 0

Percentage of positive cells for each surface marker
Passage 1
Passage 2

Passage 3

CD29
DMEM
EGM-2

98.490.1
99.490.0

98.890.4
99.390.1

99.190.2
99.590.1

99.490.2
94.591.5

CD31
DMEM
EGM-2

0.890.3
0.390.2

0.490.1
0.590.2

0.390.20.290.0

0.190.1
0.290.1

CD34
DMEM
EGM-2

60.0916.5
57.9916.6

4.191.50.290.1

0.490.1
0.090.0

1.290.3
0.290.1

CD45
DMEM
EGM-2

2.390.3
0.990.1

0.790.4
0.790.6

0.390.1
0.190.0

0.690.4
0.490.2

CD90
DMEM
EGM-2

98.890.5
99.290.3

99.790.1
99.290.3

99.790.1
97.090.1

99.790.0
81.894.3

CD105
DMEM
EGM-2

75.198.5
53.7918.9

92.394.2
37.299.3

92.791.4
24.2916.3

93.291.8
22.996.2

CD146
DMEM
EGM-2

7.593.8
3.292.2

25.4913.3
7.491.8

9.693.5
6.392.4

15.996.8
2.291.6

Flk-1
DMEM
EGM-2

5.190.9
9.194.6

3.991.3
7.890.1

3.291.4
4.893.2

2.090.6
1.190.3

result suggests that the growth factors added to EGM-2
have greater effects on the proliferation activity of ASC
than serum concentrations. In fact, a recent report hassuggested that platelet-derived growth factors (not designated, but assumed to be PDGF and EGF) may reduce theproliferation activity and adipogenic differentiation capacity of ASC [23].
Among the growth factors contained in EGM-2, supplementation with IGF-1, EGF or VEGF (0.1, 1 or 10 ng/mLof each growth factor) did not significantly promote theproliferative activity of ASC in EBM cultures containing
2% FBS. The growth factors had some proliferative effects
on ASC when added to serum-free media or SPPP, asreported previously [5,23,29]; however, any effects in thisstudy using a low concentration of FBS with IGF-1 and
EGF were subtle or masked. In this study, only FGF-2
showed a statistically significant promoting effect on ASC
proliferation, which has been suggested in previous studies[2124]. The results strongly suggest that FGF-2 is a
critical growth factor for supplementation of serumcontaining culture media. A previous study suggested that
FGF-2 plays a critical role in self-renewal of ASC [21]. It
has also been shown that FGF-2 added to SPPP increases
the proliferation activity and adipogenic differentiation
capacity [23]. Another study reported efficient proliferation of ASC transfected with the FGF-2 gene [34].

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Rapid expansion of human adipose-derived stromal cells

743

Figure 3. (A) Microscopic results of cell differentiation. Both cell populations cultured in DMEM and EGM-2 for 2 weeks had similar capacities
to differentiate into adipogenic, chondrogenic and osteogenic lineages. Adipogenic, chondrogenic and osteogenic differentiations were visualized with
Oil Red O, Alcian Blue and von Kossa staining, respectively. Scale bar 100 mm. (B) Quantitative analyzes of cell differentiation. Differentiation
potentials were evaluated by lipid droplet contents (adipogenic), micromass diameter (chondrogenic) and total calcium contents (osteogenic). No
statistical significances were observed (adipogenic P0.31, chondrogenic P0.68, osteogenic P0.55). NS, no significant difference.

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744

H Suga et al.

However, in 7 days, EGM-2 expanded ASC significantly
and several-fold more compared with FGF-2-supplemented EBM (Figure 2) and thus the effect of EGM-2 on ASC
proliferation cannot be explained solely based on theinfluence of FGF-2 alone. It is likely that synergistic effectsof the various growth factors and other factors contributed
to the exceptional efficiency.
We have previously reported surface marker expressionof freshly isolated and cultured ASC [15], although M-199
was used in the study. In the present study, the character ofASC expanded with EGM-2 did not appear to change
significantly. ASC cultured with EGM-2 preserved differentiation capacities similar to those with DMEM, into atleast three mesenchymal lineages: adipogenic, chondrogenic and osteogenic. In addition, flow cytometry of both
populations showed no significant differences, except for
CD105, and presented no increase of differentiation
markers, such as CD31, suggesting that ASC remain in
an undifferentiated and proliferating state. These results
suggest that EGM-2 accelerates expansion of ASC mainly
by facilitating proliferation of undifferentiated cells.
Although expression of CD105 has been used as one ofthe definitive markers of mesenchymal stromal cells, it
may not strongly correlate with the multipotency.
Recent reports have shown that ASC can differentiate
into endothelial cells in vitro under certain culture
conditions using endothelial growth media and also invivo [36]. ASC may be essentially common progenitors of
adipocytes and vascular cells [5]. In most of the in vitro
studies, a semi-solid medium, such as methylcellulose andMatrigel, was used, which may be key to endothelial
differentiation of ASC [35]. Although EGM-2 containing
VEGF was originally a medium for expanding endothelial
cells, and ASC express Flk-1, a VEGF receptor, endothelial cell marker expression of ASC was not enhanced by
EGM-2 in our study using cell culture on a plastic dish. In
addition, hypoxic conditions have various influences on
ASC [3639], one of which is enhancing ASC secretion ofangiogenic factors such as VEGF and HGF [36]. In thestudies showing endothelial differentiation of ASC in vivo,
ASC were transplanted to the ischemic hind limb or underother ischemic conditions [46], so a hypoxic condition
may be an important factor in endothelial differentiationin vivo.
A number of pre-clinical studies with human ASC havebeen reported; in most, undifferentiated ASC were used,rather than ASC differentiated into a specific lineage,

although the functional mechanism of transplanted ASC
varied among studies. Transplanted ASC survive as
undifferentiated cells and act as tissue-specific progenitors
or provider cells of soluble factors in some studies [3,7,16].
In others, transplanted ASC differentiate into a specificlineage, such as bone and vessels, according to thecircumstances of recipient sites [3,7,17]. For the therapeutic use of ASC, expansion of undifferentiated cells, rather
than their differentiation into a specific lineage, is likely to
be of great importance in the processing of the cells before
transplantation. In clinical practice, a safer and more rapid
expansion method is required in view of time and costrequirements. Although neither EBM nor EGM-2 isapproved for clinical use at present, EGM-2 does notcontain animal-derived factors and the FBS used in thisstudy can easily be replaced with autologous serum or
human allogenic serum. The present expansion methodwith EGM-2 has an exceptional efficiency and lays thegroundwork for establishing a practical route to megaexpansion of ASC for clinical applications.

Acknowledgements
We are very grateful to Ayako Kurata, Akiko Matsuura andSatomi Kawarasaki for their technical assistance.
The authors declare that they have no competing financial
interests.

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