Post on 09-May-2023
ARTICLE IN PRESS
www.elsevier.com/locate/yexcr
YEXCR-06733; No. of pages: 8; 4C: 4
DTD 5
Experimental Cell Researc
Functional studies of mesenchymal stem cells derived from
adult human adipose tissue
Andrea Dickera,1, Katarina Le Blancb,c,1, Gaby Astroma, Vanessa van Harmelena,
Cecilia Gotherstromb, Lennart Blomqvista, Peter Arnera, Mikael Rydena,*
aDepartment of Medicine, M63, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, SwedenbDepartment of Laboratory Medicine, Division of Immunology, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden
cCenter for allogeneic stem cell transplantation, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden
Received 4 February 2005, revised version received 20 April 2005
Abstract
Recent evidence suggests that cells with the properties of human mesenchymal stem cells (hMSCs) can be derived from adult peripheral
tissues, including adipose tissue, muscle and dermis. We isolated hMSCs from the stromal–vascular portion of subcutaneous adipose tissue
from seven adult subjects. These cells could be readily differentiated into cells of the chondrocyte, osteocyte and adipocyte lineage
demonstrating their multipotency. We studied the functional properties of hMSCs-derived adipocytes and compared them with adipocytes
differentiated from hMSCs obtained from bone marrow (BM-hMSC). The two cell types displayed similar lipolytic capacity upon stimulation
with catecholamines, including a pronounced antilipolytic effect mediated through a2A-adrenoceptors, a typical trait in human but not rodent
fat cells. Furthermore, both cell types secreted the fat cell-specific factors leptin and adiponectin in comparable amounts per time unit. The fat
tissue-derived hMSCs retained their differentiation capacity up to at least fifteen passages. We conclude that hMSCs derived from adult
human adipose tissue can be differentiated into fully functional adipocytes with a similar, if not identical, phenotype as that observed in cells
derived from BM-hMSCs. Human adipose-tissue-derived MSCs could therefore constitute an efficient and easily obtainable renewable
cellular source for studies of adipocyte biology.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Mesenchymal stem cell; Adipocyte; Lipolysis; Leptin; Adiponectin
Introduction
Human mesenchymal stem cells (hMSCs) display multi-
potency with the ability under the correct conditions to
differentiate into lineages of mesenchymal tissues including
muscle, bone, cartilage and fat [1]. This property renders
them interesting as a model system for studies of differ-
entiation pathways and potentially useful for cell and gene
therapy. MSCs were first characterized in bone marrow, but
0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2005.04.029
Abbreviations: hMSC, human mesenchymal stem cell; TG, triglyceride;
AR, Adrenoceptor; Adr, adrenaline; Y, Yohimbine; BM, bone marrow.
* Corresponding author. Fax: +46 8 585 838 50.
E-mail address: mikael.ryden@medhs.ki.se (M. Ryden).1 These two authors contributed equally to the work presented herein.
a wealth of studies have demonstrated the presence of
uncommitted MSCs progenitor cells in the connective tissue
of several organs including muscle, adipose tissue and
trabecular bone (for a review see [2]). The role of these cells
is not entirely clear, but they are generally believed to
constitute a reserve cellular fraction for tissue maintenance
and repair. Although several authors have shown that
adipose tissue hMSC can differentiate into morphologically
clearly discernable cell types [3], few studies have
demonstrated that the functional properties of hMSCs
differentiated in vitro are equivalent to those of primary
cell cultures from the same organ. This is partly due to the
fact that there are no well-established methods to assess the
functional properties of chondrocyte/osteocyte/myocyte
function in vitro. In this respect, differentiation into the
h xx (2005) xxx – xxx
ARTICLE IN PRESSA. Dicker et al. / Experimental Cell Research xx (2005) xxx–xxx2
adipocyte lineage has a clear advantage since the functional
properties of these cells can be easily investigated. Mammal
adipocytes display at least two unique traits compared to
non-fat cells. They store energy in the form of triglycerides
(TGs) which are released via lipolysis in the form of
glycerol and free fatty acids and they secrete fat-specific
proteins, in particular leptin and adiponectin into the
circulation [4].
In experimental fat cell research, immortalized cell lines
are of paramount importance. Although several murine fat
cell lines are available, to date, no human white fat cell line
has been described. Since there are important species-
specific differences in adipocyte function, there is a strong
need for a human white fat cell line. It is quite possible that
hMSCs may be of value in this respect, providing they can
retain their capacity to differentiate into fat cells that
maintain human-specific adipocyte characteristics. In a
recent paper, we could in fact demonstrate that hMSCs
derived from fetal liver and adult human bone marrow
could be differentiated into functional adipocytes with
characteristics similar to those observed in primary cultures
of human preadipocytes (which are lineage-committed
progenitor cells) [5]. Due to ethical considerations related
to the use of fetal tissues and the somewhat laborious and
painful procedure associated with bone marrow aspiration,
it would be ideal to have an easily accessible source of
hMSCs in adult subjects. Investigators have recently shown
that hMSC with retained pluripotentiality can be derived
from subcutaneous adipose tissue [6–12]. It is not known
whether hMSCs present in adult fat are also able to
differentiate into functional cells. Since subcutaneous lip-
oaspirates are very easy to perform and associated with
minimal risks, adipose tissue could constitute an important
source for hMSCs. However, this warrants a detailed
analysis and comparison with hMSCs derived from bone
marrow. We determined whether subcutaneous adipose
tissue of adult humans contains hMSCs and whether these
cells display functional characteristics of adipocytes that
can be maintained for numerous passages. Furthermore, we
compared the phenotype of these cells with adipocytes
derived from BM-hMSCs.
Table 1
Mesenchymal stem cells in adult human adipose tissue
Subject number
(fat tissue donor)
Age
(years)
BMI
(kg/m2)
Gender Subject number
(BM donor)
Age
(years)
1 24 29.8 F 1 25
2 27 21.9 F 2 25
3 50 61.5 M 3 57
4 28 24.2 M 4 61
5 28 23.5 F 5 69
6 38 21.6 F
7 32 28.4 F
To the left is a summary of age and BMI of the different subjects from
which fat stroma was isolated for subsequent preparation of adipose-tissue-
derived hMSC. To the right are the ages of the bone-marrow donors (no
anthropometric data available).
Materials and methods
Human MSC culture
Subcutaneous fat was obtained from seven subjects (two
men, five women) undergoing cosmetic lipoaspiration. They
were aged between 24 and 50 years, with a body mass index
(BMI) ranging from 21.6 to 61.5 kg/m2. Although
adipocytes derived from the subject with the highest BMI
displayed somewhat lower levels of GPDH, in this limited
material, there was no apparent effect of BMI on either cell
differentiation or adipocyte function (data not shown). Bone
marrow aspirates were obtained from five normal-weight
subjects, aged between 25 and 69 years. Cells from these
subjects have been examined in detail in other studies from
our groups. Unfortunately, no anthropometric data were
obtained from these donors. The individual patient data are
detailed in Table 1. The hospital’s committee of ethics
approved this study, and informed consent was obtained
from all subjects. Mature adipocytes were separated out
according to Rodbell et al. [13]. The remaining stromal–
vascular portion was used for subsequent isolation of MSC
as described previously [5]. Cells were plated at a density of
160,000/cm2 and allowed to proliferate in Dulbecco’s
Modified Eagle’s Medium–Low Glucose (DMEM-LG)
(Life Technologies, Gaithersburg, MD), supplemented with
10% fetal bovine serum (FBS) (Sigma, St. Louis, MO,
USA) and 1% antibiotic–antimycotic solution (Life Tech-
nologies, Gaithersburg, MD). The serum lot used was
selected on the basis of optimal MSC proliferation. In
continued passages, cells were plated at 4000/cm2. Cultures
were maintained at 37-C in a humidified atmosphere
containing 5% CO2 and passaged at 60–70% confluency
for at least fifteen passages. Isolated MSCs were analyzed
by flow cytometry. Detached cells were washed and re-
suspended in PBS. Approximately 105 cells were incubated
on ice for 30 min with conjugated, monoclonal antibodies
against CD34, CD14 and CD45 (Becton Dickinson, San
Jose, CA, USA), CD44 (Immunotech, Marseilles, France),
CD29 (Coulter, Miami, FL, USA), CD 166 and CD105
(Ansell, Bayport, MN, USA) and CD73 (PharMingen,
Uppsala, Sweden). Nonspecific fluorescence was deter-
mined, using equal aliquots of cell preparation incubated
with anti-mouse monoclonal antibodies (Becton Dickinson).
Finally, the cells were assayed in a flow cytometer
(FACSort, Beckton Dickinson), and the data analyzed with
Cellquest software (Becton Dickinson).
Cell lineage differentiation
Both types of hMSCs were differentiated into the
adipogenic, osteogenic and chondrogenic lineage as
described previously [5]. To promote chondrogenic differ-
ARTICLE IN PRESSA. Dicker et al. / Experimental Cell Research xx (2005) xxx–xxx 3
entiation, 7.5 � 105 mesenchymal cells were pelleted in a
15-mL polypropylene tube and cultured in Dulbecco’s
Modified Eagles Medium–High Glucose (DMEM-HG)
(Life Technologies) with insulin (6.25 Ag/mL), transferrin
(6.25 Ag/mL), linolenic acid (5,33 Ag/ml), BSA (1.25 mg/
ml), pyruvate (1 mmol/L), ascorbate-2-phosphate (0.17
mmol/L), dexamethasone (0,1 Amol/L), proline (0.35
mmol/L) and selenous acid (6.25 ng/mL). Differentiation
was induced by the addition of TGF-h3 (0.01 Ag/mL) (R&D
Systems, Minneapolis, MN). The medium was replaced
every 3–4 days for 21 days. Pellets were formalin fixed,
embedded in paraffin, examined morphologically and
stained for the presence of glucosaminoglycans with
Alacian green. For osteogenic differentiation, 3.1 � 103
cells per cm2 were grown in MSC medium supplemented
with dexamethasone (0.1 Amol/L), ascorbic acid (0.05
mmol/L) and glycerophosphate (10 mmol/L). The medium
was replaced every 3–4 days for 21 days. Osteocytes were
stained with Alizarin Red. Differentiation of preadipocytes
from both bone marrow and adult adipose tissue was
performed as described previously [5] and assessed by
determining glycerol-3-phosphate-dehydrogenase (GPDH)
activity and staining with the triglyceride-specific dye Oil
Red O [5].
Lipolysis
Lipolysis experiments were performed on differentiated
adipocytes as described previously [14]. In brief, cells were
washed with DMEM/NUT.MIX.F-12 medium and then
incubated in duplicates for 3 h at 37-C with DMEM/
NUT.MIX.F-12 medium containing 20 g/L BSA. The
following concentrations were used for each agent: 0.1
mM adrenaline and 0.1 mM of the a2-adrenoceptor blocker
yohimbine. Incubation without drugs was made to deter-
mine basal lipolysis. Following incubation, medium was
removed and kept at �20-C for subsequent measurement of
glycerol concentration (an index of lipolysis) using a
bioluminescence method [15]. For protein measurements,
cells were lysed in a pre-chilled protein lysis buffer
containing 50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton
X-100, 0.1 M PMSF and protease inhibitors as described
previously [16]. Protein content was assayed using BCA
Protein Assay Reagent Kit (PIERCE, Rockford, IL,USA) on
96-well microtiter plates with BSA as a standard. Measure-
ments were performed on a spectrophotometer in accord-
ance to the guidelines given in the kit. GPDH and protein
concentrations were almost identical in the different wells,
therefore glycerol release could be expressed in absolute
terms.
Adipokine release from differentiated adipocytes
Adipokine release was measured in conditioned media
from adipocytes of several passages. Medium was collected
after a 48-h incubation, and release was expressed per 24 h.
Leptin was measured according to the manufacturer’s
protocol using the Quantikine Human Leptin Immunoassay
(R&D Systems, Minneapolis, MM, USA). Adiponectin
release was determined using a human adiponectin RIA-
kit (Linco Research, Inc, St Charles, MO).
Statistical analysis
Values are given as mean T standard error (SE). They werecompared with the non-parametric test Kruskal–Wallis.
Drugs and chemicals
Bovine serum albumin (BSA) fraction V (A-9418),
glucose, glycerol kinase, adrenaline, Oil Red O and
yohimbine were obtained from Sigma Chemical (Sigma,
St. Louis, MO, USA). All chemicals used were of the
highest grade of purity that was commercially available.
Results
Phenotype of MSC derived from adipose tissue and bone
marrow
Cultured MSCs from both sources were strongly positive
for the endoglin receptor CD105, CD73, CD44, CD90 and
h-1-integrin 29 (Fig. 1). Flow cytometry was negative for
the endothelial marker CD31, CD80, the lipopolysacharide
receptor CD14 and the leukocyte common antigen CD45.
While bone-marrow-derived MSCs were negative for
hematopoietic stem cell marker CD34, a slight positivity
(15–20%) was observed in adipocyte-derived MSC which
is in agreement with findings in a recent report [17].
Adipose-tissue-derived hMSCs display multipotent
differentiation
The multipotency of the MSCs isolated from adult
subcutaneous adipose tissue was demonstrated by incubat-
ing the cells in media promoting differentiation into the
osteogenic, chondrogenic and adipogenic lineage, respec-
tively. Incubation in the presence of dexamethasone, h-glycerophosphate and ascorbate induced cell aggregation
and matrix production, which stained positive with the
calcium-specific marker Alizarin Red (Fig. 2A). Pelleted
cells cultured with TGF-h developed typical morphological
features of chondrocytes and a multilayered proteoglycan-
rich matrix which stained positive with Alacian green (Fig.
2B). Finally, MSCs incubated with insulin and the PPAR-g
agonist Roziglitazone differentiated into adipocyte-like
cells, which stained positive with the triglyceride specific
dye Oil Red O (Fig. 2C). The MSC-derived adipocytes
were also analyzed for the activity of the fat cell specific
enzyme glycerol-3-phosphate dehydrogenase (GPDH). The
activity levels were similar to that observed in human
ARTICLE IN PRESS
Fig. 2. Differentiation of hMSC derived from adult s.c. fat tissue. Cells were
incubated in the presence of specific differentiation agents. (A) Alizarin
staining shows the presence of bone tissue and osteocytes. (B) Staining with
Alacian green demonstrates the deposition of proteoglycans and the
morphological characteristics of chondrocytes. (C) Differentiation into the
adipocyte lineage was demonstrated by staining with Oil Red O.
Fig. 1. Epitope analysis of hMSC. Representative flow cytometric analysis
of cultured MSCs derived from adipose tissue (left panel) and bone marrow
(right panel) with monoclonal antibodies against CD45 (A), CD34 (B),
CD73 (C), CD90 (D) and CD105 (E). Dashed lines indicate ISO type-
matched mouse IgG antibody controls staining. All MSCs were negative for
CD45 and positive for CD73, CD90 and CD105. Adipose-tissue-derived
MSCs were positive for CD34, whereas bone-marrow-derived MSCs were
negative.
A. Dicker et al. / Experimental Cell Research xx (2005) xxx–xxx4
preadipocytes and adipocytes derived from bone-marrow-
derived MSCs (data not shown).
Adipose-tissue-derived hMSC adipocytes display lipolysis
similar to that of bone-marrow-derived hMSCs
A hallmark of adipocyte differentiation is the ability to
release stored TG through lipolysis. The lipolytic capacity
of adipose-tissue-derived hMSC was determined by assess-
ing glycerol release in the medium after stimulation with
lipolytic agents and expressing it relative to that observed in
medium alone (basal lipolysis), i.e. responsiveness. A 3-h
ARTICLE IN PRESS
Fig. 4. Secretion of adipocyte specific proteins. Leptin (A) and adiponectin
(B) release was measured and corrected for the incubation time in
conditioned media from adipose-tissue-derived hMSC adipocytes (r) and
BM-derived hMSC-adipocytes (o).
A. Dicker et al. / Experimental Cell Research xx (2005) xxx–xxx 5
incubation with 0.1 mM adrenalin (Adr) resulted in a small
but non-significant increase in lipolysis compared with
control cells (167 T 33% of basal lipolysis, Fig. 3A). The
weak effect of the endogenous catecholamine appeared to
be due to a potent stimulation of the antilipolytic a2A-
adrenoceptor (AR) since co-incubation with the a2-AR
antagonist yohimbine (Y) (0.1 mM) increased lipolysis
substantially (1561 T 1039%, P = 0.0148, Fig. 3A). The
lipolytic capacity of these cells was compared with that of
adipocytes differentiated from hMSCs of adult bone marrow
(BM). In agreement with previous studies [5], the a2-AR
effect was even more pronounced in these cells since
adrenalin reduced lipolysis (51.4 T 7.6%, Fig. 3B). Co-
incubation with yohimbine resulted in a significant increase
in lipolysis (449 T 64%, P = 0.0073, Fig. 3B). To correct for
differences in cell number and differentiation, the lipolytic
rate was also corrected for the activity of the adipogenic
enzyme glycerol-3-phosphate dehydrogenase. However, this
did not significantly alter the results.
Adipose-tissue-derived hMSC adipocytes secrete leptin and
adiponectin
The secretion of the fat cell-specific proteins leptin and
adiponectin was assessed in various passages from differ-
entiated adipocytes of adipose-tissue-derived hMSCs and
compared with that observed in adipocytes from BM-
Fig. 3. Lipolysis in differentiated adipocytes from hMSCs. Glycerol release
in the medium was measured in differentiated adipocytes from adipose-
tissue-derived hMSCs (A) or BM-derived hMSCs (B). Basal lipolysis was
set to 100% for each experiment, which was repeated at least four times
with cells from different donors. Note that adrenaline-induced lipolysis is
significantly augmented in the presence of yohimbine due to the strong
antilipolytic effect of a2A-AR. (*statistically significant over basal lipolysis
P < 0.05).
derived hMSCs. Both proteins were released into the
medium in a time-dependent manner (Fig. 4). Secretion
was expressed as release per 24 h. Adipose-tissue-derived
hMSCs released leptin at levels 2–4 times higher than that
of bone-marrow-derived hMSCs. In contrast, adiponectin
release was similar in the two cell types.
Fig. 5. Adipocyte differentiation of hMSC derived from adipose tissue is
constant through multiple passages. (A) GPDH activity was measured from
cells at different passages. Experiments were performed on cells from the
same subject, and a representative example from one of the hMSC lines is
depicted. (B) Glycerol release was assessed after stimulation with adrena-
line (N) or adrenaline + yohimbine (n) and expressed relative to that of
unstimulated cells (basal) incubated in parallel. Experiments were
performed on cells from the same subject at the indicated passages.
ARTICLE IN PRESSA. Dicker et al. / Experimental Cell Research xx (2005) xxx–xxx6
Adipose-tissue-derived hMSCs retain adipocyte
differentiation capacity through multiple passages
The functional differentiation capacity of hMSCs ob-
tained from adipose tissue was tested by analyzing GPDH
activity and lipolytic capacity in cells from several passages.
A representative example is depicted in Fig. 5, demonstrat-
ing that GPDH activity (Fig. 5A) and stimulated lipolysis
(Fig. 5B, expressed as glycerol release in adrenaline- or
adrenaline+yohimbine-treated cells, respectively over con-
trol cells) were similar for at least fifteen passages. Thus,
adrenaline, a natural catecholamine stimulated spontaneous
(basal) lipolysis approximately 1- to 3-fold in cells from
each passage. The addition of the a2-adrenoceptor inhibitor
yohimbine (which counteracts the antilipolytic effect of
catecholamines) augmented adrenaline-stimulated lipolysis
2- to 4-fold in each experiment. It is well-established that a
pronounced a2-adrenoceptor activity is characteristic of
human but not murine adipocytes [5].
Discussion
In this work, we have assessed the differentiation
capacity of hMSCs derived from the stromal compartment
of adult adipose tissue. In accordance with recent studies
from other groups, we show that these cells display
multipotency and can be differentiated into several different
lineages. However, in contrast to other reports, we have also
determined the function of these cells induced into the
adipogenic lineage. We chose to study the function of
adipocytes since, in contrast to osteocytes and chondrocytes,
there are well-established assays to study these cells and
determine their functional capacity in vitro. We demonstrate
that fat cells developed from these immature precursors are
fully functional and virtually indistinguishable from adipo-
cytes obtained from bone-marrow-derived hMSCs. More-
over, their functional differentiation capacity is constant
through multiple passages. This would suggest that hMSCs
in adult adipose tissue of humans are functionally identical
to those obtained from bone marrow aspirates. This implies
that adipose tissue could constitute an easily accessible
source to harvest hMSC and that these cells constitute an
efficient and virtually endless source of in vitro differ-
entiated adipocytes from a single donor.
The presence of immature precursors in adult human
tissue is interesting from several points of view and could
benefit several areas of research. In particular, human
adipocyte research is hampered by the lack of human fat
cell lines, and most studies are therefore performed on
immortalized rodent cell lines such as 3T3-L1 or primary
cultures of human preadipocytes. The former are murine
cells that display several distinct metabolic and signal
transduction differences compared with human cells [16].
Preadipocytes are fibroblast-like cells isolated from the
stromal portion of human fat tissue which are differentiated
in vitro into cells displaying functional traits identical to
those of mature fat cells [14, 18]. However, these cultures
are set up by plating the entire stromal compartment and are
therefore often hampered by contamination by other cell
types (endothelial cells, fibroblasts, smooth muscle cells,
etc.). Furthermore, since these cells are immediately differ-
entiated (in order to minimize the proliferation of contam-
inating cells), they cannot be expanded and display only a
limited life span. Adipocyte research is now focusing on
molecular and cellular mechanisms underlying obesity and
its consequences. A better understanding of these processes
requires large amounts of cells with a metabolism displaying
high homology to mature human adipocytes. Human MSCs
provide us with an optimal renewable system to study
mature adipocyte phenotypes as well as cell differentiation
steps including the possibility to assess points of differential
cell lineage determination. We show that fat-tissue-derived
hMSCs retain their capacity to differentiate into fat cells
(GPDH activity) for numerous passages and that the newly
developed adipocytes retain their human-specific lipolytic
properties (characterized by a pronounced a2-adrenoceptor
effect that can be counteracted by the receptor-specific
inhibitor yohimbine).
Fat cell lipolysis is a hallmark of developed adipocytes,
and lipolytic capacity in vitro is predictive of in vivo
lipolysis [19]. Lipolysis is dysregulated in several common
disorders of insulin resistance in man, including obesity,
dyslipidemia, type 2 diabetes, the metabolic syndrome and
polycystic ovarian syndrome [20]. Studies in the last
decades have shown that the close relationship between
lipolysis and insulin resistance is due to the elevated release
of free fatty acids (FFA) to the circulation. FFA have
multiple effects, visceral lipolysis leads to FFA release into
the draining portal vein with direct effects on the liver
leading to increased output of glucose and VLDL trigly-
cerides. Increased FFA release from subcutaneous adipose
tissue affects muscle (decreased glucose uptake and insulin
sensitivity) and probably also negatively affects insulin
release from the pancreas (for a review see [20]). The
pathogenic processes that influence fat cell lipolysis in these
conditions are not understood, and it has been difficult to
determine whether the observed lipolytic dysregulation is of
primary or secondary origin. The fibrous stroma which
surrounds fat cells has recently attracted much attention. In
obesity, the stromal compartment is lined with inflammatory
cells that interact with and influence adipocyte function
[21]. Moreover, obesity (as well as other insulin resistant
conditions) develops slowly in humans, and prospective
studies of fat cell function are therefore not feasible. In order
to circumvent these issues, we have previously set up
primary preadipocyte cultures from obese and non-obese
subjects. These cells have not yet been adipocytes in vivo,
and they are differentiated in vitro under standardized
conditions. By comparing these in vitro generated adipo-
cytes with freshly isolated mature fat cells from the same
subject, we have been able to differentiate between primary
ARTICLE IN PRESSA. Dicker et al. / Experimental Cell Research xx (2005) xxx–xxx 7
and secondary defects in obese subjects [14]. However, in
order to obtain reproducible results, extensive cell culture
work has to be performed. This is mainly due to the
interindividual differences between cells from different
subjects and the varying cellular contamination discussed
previously. Other disadvantages of this primary cell culture
system are that cell numbers cannot be increased effectively
by proliferation, thereby limiting the expandability of the
relatively small tissue samples and that differentiated cells
cannot be frozen for subsequent follow-up studies. In this
respect, fat-derived hMSCs have important advantages since
they constitute a homogenous proliferating cell population
that can be propagated and cryopreserved. This enables
researchers to study hMSC-derived adipocytes and build up
fat cell banks from subjects with different phenotypic
alterations and then compare these processes in vitro.
Hopefully, this will enable scientists in the metabolic field
to determine whether defects in lipolysis (and possibly other
traits such as lipogenesis and glucose uptake) are of primary
or secondary origin in several common conditions.
The presence of a small proportion of multipotent
progenitor cells in the stroma of peripheral organs suggests
that a cellular reserve exists which can be activated upon
tissue damage. However, although animal models have
shown that MSCs can be recruited in different damage
models, to date, there are few reports in humans demonstrat-
ing that these progenitor cells are of clinical value, although
in a recent case report, adipose-tissue-derived MSCs were
successfully used in the treatment of a rectovaginal fistula in
a patient with Crohn’s disease [22]. The role of these cells is
therefore not entirely understood, are they dormant remains
from earlier developmental stages or do they continue to
proliferate at a slow pace, replacing a proportion of
malfunctioning or degenerating cells? Further studies to
elucidate the importance of these cells are warranted.
In summary, we have demonstrated that the MSCs
present in adult adipose tissue can be differentiated into
fat cells which appear to be functionally indistinguishable
from adipocytes derived from bone marrow harvested
hMSCs. Since these adipose-tissue-derived hMSCs retain
their ability to differentiate into fat cells with intact cell-
specific characteristics for numerous passages, they con-
stitute an almost endless source of adipocytes for exper-
imental research on cells from a single donor. Given the
increasing research on white adipose tissue and its role in
development of insulin resistance and other common
disorders, this cellular system is a valuable addition to
researchers in the experimental metabolic field.
Acknowledgments
This work was supported by the foundations of Ake
Wiberg, Tore Nilsson, Signe and Olof Wallenius, Magnus
Bergvall and Novo Nordisk, the Swedish Research Council,
the Swedish Diabetes Association, the Tobias Foundation,
the Swedish Cancer Society, the Children’s Cancer Foun-
dation, the Stockholm Cancer Society, the Swedish Society
of Medicine, and Karolinska Institutet.
References
[1] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas,
J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak,
Multilineage potential of adult human mesenchymal stem cells,
Science 284 (1999) 143–147.
[2] F.P. Barry, J.M. Murphy, Mesenchymal stem cells: clinical applica-
tions and biological characterization, Int. J. Biochem. Cell Biol. 36
(2004) 568–584.
[3] P.A. Zuk, M. Zhu, H. Mizuno, J. Huang, J.W. Futrell, A.J. Katz, P.
Benhaim, H.P. Lorenz, M.H. Hedrick, Multilineage cells from human
adipose tissue: implications for cell-based therapies, Tissue Eng. 7
(2001) 211–228.
[4] P. Arner, The adipocyte in insulin resistance: key molecules and the
impact of the thiazolidinediones, Trends Endocrinol. Metab. 14 (2003)
137–145.
[5] M. Ryden, A. Dicker, C. Gotherstrom, G. Astrom, C. Tammik, P.
Arner, K. Le Blanc, Functional characterization of human mesenchy-
mal stem cell-derived adipocytes, Biochem. Biophys. Res. Commun.
311 (2003) 391–397.
[6] D.A. De Ugarte, K. Morizono, A. Elbarbary, Z. Alfonso, P.A. Zuk, M.
Zhu, J.L. Dragoo, P. Ashjian, B. Thomas, P. Benhaim, I. Chen, J.
Fraser, M.H. Hedrick, Comparison of multi-lineage cells from human
adipose tissue and bone marrow, Cells Tissues Organs 174 (2003)
101–109.
[7] F. Guilak, H.A. Awad, B. Fermor, H.A. Leddy, J.M. Gimble, Adipose-
derived adult stem cells for cartilage tissue engineering, Biorheology
41 (2004) 389–399.
[8] Y.D. Halvorsen, A. Bond, A. Sen, D.M. Franklin, Y.R. Lea-Currie, D.
Sujkowski, P.N. Ellis, W.O. Wilkison, J.M. Gimble, Thiazolidine-
diones and glucocorticoids synergistically induce differentiation of
human adipose tissue stromal cells: biochemical, cellular, and
molecular analysis, Metabolism 50 (2001) 407–413.
[9] H. Hattori, M. Sato, K. Masuoka, M. Ishihara, T. Kikuchi, T.
Matsui, B. Takase, T. Ishizuka, M. Kikuchi, K. Fujikawa,
Osteogenic potential of human adipose tissue-derived stromal cells
as an alternative stem cell source, Cells Tissues Organs 178 (2004)
2–12.
[10] R.H. Lee, B. Kim, I. Choi, H. Kim, H.S. Choi, K. Suh, Y.C. Bae, J.S.
Jung, Characterization and expression analysis of mesenchymal stem
cells from human bone marrow and adipose tissue, Cell. Physiol.
Biochem. 14 (2004) 311–324.
[11] A.-M. Rodriguez, C. Elabd, F. Delteil, J. Astier, C. Vernochet, P.
Saint-Marc, J. Guesnet, A. Guezennec, E.-Z. Amri, C. Dani, G.
Ailhaud, Adipocyte differentiation of multipotent cells established
from human adipose tissue, Biochem. Biophys. Res. Commun. 315
(2004) 255–263.
[12] A. Sen, Y.R. Lea-Currie, D. Sujkowska, D.M. Franklin, W.O.
Wilkison, Y.D. Halvorsen, J.M. Gimble, Adipogenic potential of
human adipose derived stromal cells from multiple donors is
heterogeneous, J. Cell Biochem. 81 (2001) 312–319.
[13] M. Rodbell, Metabolism of isolated fat cells: I. Effects of hormones
on glucose metabolism and lipolysis, J. Biol. Chem. 239 (1964)
375–380.
[14] V. van Harmelen, A. Dicker, M. Ryden, H. Hauner, F. Lonnqvist, E.
Naslund, P. Arner, Increased lipolysis and decreased leptin production
by human omental as compared with subcutaneous preadipocytes,
Diabetes 51 (2002) 2029–2036.
[15] J. Hellmer, C. Marcus, T. Sonnenfeld, P. Arner, Mechanisms for
differences in lipolysis between human subcutaneous and omental fat
cells, J. Clin. Endocrinol. Metab. 75 (1992) 15–20.
ARTICLE IN PRESSA. Dicker et al. / Experimental Cell Research xx (2005) xxx–xxx8
[16] M. Ryden, A. Dicker, V. van Harmelen, H. Hauner, M. Brunnberg, L.
Perbeck, F. Lonnqvist, P. Arner, Mapping of early signaling events in
tumor necrosis factor-alpha-mediated lipolysis in human fat cells,
J. Biol. Chem. 277 (2002) 1085–1091.
[17] A.C. Boquest, A. Shahdadfar, K. Fronsdal, O. Sigurjonsson, S.H.
Tunheim, P. Collas, J.E. Brinchmann, Isolation and transcription
profiling of purified uncultured human stromal stem cells: alteration of
gene expression after in vitro cell culture, Mol. Biol. Cell 16 (2005)
1131–1141.
[18] H. Hauner, G. Entenmann, M. Wabitsch, D. Gaillard, G. Ailhaud, R.
Negrel, E.F. Pfeiffer, Promoting effect of glucocorticoids on the
differentiation of human adipocyte precursor cells cultured in a
chemically defined medium, J. Clin. Invest. 84 (1989) 1663–1670.
[19] M. Kolehmainen, J.J. Ohisalo, J.M. Kaartinen, V. Tuononen, M.
Paakkonen, E. Poikolainen, E. Alhava, M.I. Uusitupa, Concordance of
in vivo microdialysis and in vitro techniques in the studies of adipose
tissue metabolism, Int. J. Obes. Relat. Metab. Disord. 24 (2000)
1426–1432.
[20] P. Arner, Insulin resistance in type 2 diabetes: role of fatty acids,
Diabetes/Metab. Res. Rev. 18 (Suppl. 2) (2002) S5–S9.
[21] H. Xu, G.T. Barnes, Q. Yang, G. Tan, D. Yang, C.J. Chou, J. Sole, A.
Nichols, J.S. Ross, L.A. Tartaglia, H. Chen, Chronic inflammation in
fat plays a crucial role in the development of obesity-related insulin
resistance, J. Clin. Invest. 112 (2003) 1821–1830.
[22] D. Garcia-Olmo, M. Garcia-Arranz, L.G. Garcia, E.S. Cuellar,
I.F. Blanco, L.A. Prianes, J.A. Montes, F.L. Pinto, D.H.
Marcos, L. Garcia-Sancho, Autologous stem cell transplantation
for treatment of rectovaginal fistula in perianal Crohn’s disease:
a new cell-based therapy, Int. J. Colorectal Dis. 18 (2003)
451–454.