Post on 25-Apr-2023
ORIGINAL PAPER
Decrease of apoptosis markers during adipogenic differentiationof mesenchymal stem cells from human adipose tissue
Debora Lo Furno • Adriana C. E. Graziano • Silvia Caggia • Rosario E. Perrotta •
Maria Stella Tarico • Rosario Giuffrida • Venera Cardile
Published online: 12 March 2013
� Springer Science+Business Media New York 2013
Abstract Although the proliferation and differentiation
of mesenchymal stem cells (MSCs) from adipose tissue
(AT) have been widely studied, relatively little information
is available on the underlying mechanism of apoptosis
during the adipogenic differentiation. Thus, the aim of this
study was to analyze how the expression of some apoptotic
markers is affected by in vitro expansion during adipogenic
differentiation of AT-MSCs. The cultures incubated or not
with adipogenic medium were investigated by Western blot
at 7, 14, 21, and 28 days for the production of p53, AKT,
pAKT, Bax, PDCD4 and PTEN. MSCs were recognized
for their immunoreactivity to MSC-specific cell types
markers by immunocytochemical procedure. The effec-
tiveness of adipogenic differentiation was assessed by
staining with Sudan III and examination of adipogenic
markers expression, such as PPAR-c and FABP, at dif-
ferent time points by Western blot. The adipogenic dif-
ferentiation medium led to the appearance, after 7 days, of
larger rounded cells presenting numerous vacuoles con-
taining lipids in which it was evident a red–orange staining,
that increased in size in a time-dependent manner, parallel
to an increase of the levels of expression of PPAR-c and
FABP. More than 50 % of human MSCs were fully dif-
ferentiated into adipocytes within the four-week induction
period. The results showed that during adipogenic differ-
entiation of AT-MSCs the PI3K/AKT signaling pathway is
activated and that p53, PTEN, PDCD4, and Bax proteins
are down-regulated in time-dependent manner. Our data
provide new information on the behavior of some apoptotic
markers during adipogenic differentiation of AT-MSCs to
apply for tissues repair and regeneration.
Keywords Adipogenesis � Apoptosis � Cell death �Differentiation � Human cultures � MSC
Introduction
Until the 1990s, adipose tissue (AT) received little atten-
tion, because it was considered to lack any specific meta-
bolic activity and to be an inert storage depot with access to
stored triacylglycerols being regulated mainly by adrener-
gic stimulation. This view remained prevalent until
research focused attention on the relevant role of adipose
tissue as a source of metabolic fuel. In recent years, interest
in the biology of adipose tissue has emerged in relation to
the discovery of a host of adipocyte-derived factors that
contribute to energy homeostasis. It is now well established
that several growth factors and cytokines secreted by adi-
pose tissue play a key role in cell differentiation, energy
metabolism, and insulin resistance. Today, adipose tissue
represents a valid reservoir of mesenchymal progenitors
[1]. Adult stem cells can be found in all tissues and organs,
where they maintain homeostasis and respond to injury. In
the tissues and organs, adult stem cells are located in stem
cell niches, which provide supporting micro-environment
in the form of other cells and regulatory signals that
interact with and regulate the stem cells and stem cell
derived progenitors [2]. The sources of stem cells are
various, such as bone marrow, umbilical cord blood, adi-
pose tissue [3], peripheral blood, muscle, dermis, synovial
D. Lo Furno � A. C. E. Graziano � S. Caggia � R. Giuffrida �V. Cardile (&)
Department of Bio-medical Sciences, Section of Physiology,
University of Catania, V.le A. Doria 6, 95125 Catania, Italy
e-mail: cardile@unict.it
R. E. Perrotta � M. S. Tarico
Dipartimento di Specialita Medico-Chirurgiche,
Cannizzaro Hospital, 95100 Catania, Italy
123
Apoptosis (2013) 18:578–588
DOI 10.1007/s10495-013-0830-x
membrane, periosteum, and trabecular bone [4]. Compared
to bone marrow, the first recognized source of mesenchy-
mal stem cells (MSCs) [5], adipose tissue can be obtained
in larger volumes, at lower risks, less painful, and easier to
get as it is the waste product of liposuction [6]. The dis-
covery that adipose tissue is rich in adult stem cells capable
of differentiating into many lines has led to consider the
potential clinical applications for the repair of damaged
tissues and angiogenic therapy. MSCs have great potential
as therapeutic agents because they are easy to isolate and
can be expanded from patients without serious ethical or
technical problems. AT-MSCs have multi germ-line
potential and can be differentiated into cells of the meso-
dermal lineage such as osteogenic, chondrogenic, adipo-
genic, and myogenic lineages, and even into cells with
neuron-like morphology that express neuronal proteins [7].
In addition, AT-MSCs can differentiate into endothelium
and have angiogenic capacity [8]. In vitro differentiation of
MSCs into adipocytes has been demonstrated by increased
expression of adipokines, such as leptin and adiponectin,
and GLUT4 transporter, detected by immunofluorescent
techniques [9, 10].
Although the proliferation and differentiation of MSCs
have been widely studied [11, 12], little information is
available on the underlying mechanism of apoptosis in
MSCs. Programmed cell death or apoptosis is an intrinsic
death program that occurs in various physiological and
pathological situations which is highly conserved throughout
evolution. Apoptosis or self destruction is necessary for
normal development and homeostasis of multicellular
organisms. This process is regulated by many intracellular
and extracellular signals. Cellular disruption results from the
activation of a family of cysteine proteases known as casp-
ases. Caspases are synthesized as inactive proenzymes
which, upon activation, cleave various substrates in the
cytoplasm or nucleus leading to morphological changes and
cell death. Understanding the process of apoptosis is vital for
therapy development. Also adipose tissue reorganization is
an ongoing physiological process that involves both adipo-
genesis and apoptosis [13]. Thus, it is of considerable
importance to understand the mechanism by which adipo-
cyte differentiation is regulated. Little is, in fact, known
about either the physiologic regulators of cell death or the
apoptotic sensitivity of preadipocytes versus adipocytes.
Using the well-established immortalized murine 3T3-L1 cell
line model of adipogenesis, it was reported that as adipo-
genesis ensues, these cells acquire resistance to apoptosis
induced by growth factor deprivation [14]. Increased
expression of 2 cell survival genes, neuronal apoptosis
inhibitor protein (NAIP) and Bcl-2 is consistent with the
differentiation-dependent effect on survival [14]. Others
have confirmed the apoptotic sensitivity of 3T3-L1 preadi-
pocytes [15, 16]. In any case, the majority of research
examining changes in lipogenic gene expression has been
conducted in rodents and most information describing adi-
pogenesis utilizes the 3T3-L1 cell line [17–19].
We hypothesize that apoptosis profiles change also dur-
ing adipogenesis of AT-MSCs. Thus, the aim of this study
was to analyze how the expression of some apoptotic
markers is affected by in vitro expansion during adipogenic
differentiation of AT-MSCs. We intended to gain insight
into the molecular effects of growth of AT-MSCs even at
early passages that would have impact for the quality con-
trol of MSCs preparations used for therapeutic application.
Materials and methods
Patients
Adipose tissue was gathered from ten donors, five men and
five women (from 22 to 30 years of age and mean body
mass index of 27 ± 3.8) undergoing abdominal liposuction
procedures. Lipoaspirates were obtained under an approved
Institutional Review Board protocol and after informed
consent had been obtained from the patients at the Can-
nizzaro Hospital, Catania (Italy). The patients were not
smokers and occasionally taking non-steroidal anti-
inflammatory drugs (NSAIDs). The women did not take
estrogen replacement therapy.
Isolation and culture of human MSCs from adipose
tissue
The raw lipoaspirate (50–100 ml) was washed with sterile
phosphate-buffered saline (PBS; Invitrogen, Milan, Italy) to
remove red blood cells and debris, and incubated for 3 h at
37� C with an equal volume of serum-free Dulbecco’s mod-
ified Eagle’s medium (DMEM)-low glucose (DMEM-lg;
PAA Laboratories, Pasching, Austria) containing 0.075 % of
type I collagenase (Invitrogen, Milan, Italy). Collagenase
activity was then inactivated by an equal volume of DMEM-
lg containing 10 % of heat-inactivated fetal bovine serum
(FBS; Invitrogen, Milan, Italy). Successively, the digested
lipoaspirate was centrifuged at 350 g for 10 min. The pellets
were re-suspended in PBS (plus penicillin/streptomycin 1 %)
and filtered through a 100 lm nylon cell strainer (Falcon BD
Biosciences, Milan, Italy). The filtered cells were again
centrifuged at 350 g for 10 min, plated in 75 cm2 culture
flasks (Falcon BD Biosciences) with DMEM-lg (10 % FBS,
penicillin/streptomycin 1 %) containing 1 % of MSC growth
medium (MSCGS; ScienCell Research Laboratories, Milan,
Italy) and incubated at 37 �C with 5 % CO2 for expansion.
Twenty-four hours after the initial plating, non-adherent cells
were removed by intensely washing the plates. After 3 days
culture medium was changed and the cells maintained for 7,
Apoptosis (2013) 18:578–588 579
123
14, 21 and 28 day with 2 weekly medium changes. When the
cells reached confluence they were trypsinized, washed in
PBS, resuspended in medium, and replated onto 75 cm2
flasks. After the first passage some cultures were used to
identify specific surface markers by immunocytochemistry.
The effectiveness of differentiation was assessed by histo-
chemical staining with Sudan III and by Western blot for
peroxisome proliferator-activated receptor gamma (PPAR-c)
and fatty acid binding protein 4 (FABP4). Control cultures
without the differentiation stimuli were carried out in parallel
to the experiments and stained and PPAR-c and FABP4
analyzed in the same manner.
Determination of MSC markers
In order to identify MSCs derived from lipoaspirate,
immunocytochemical procedures were carried out using
several cell surface markers. In particular, they were rec-
ognized for their immunoreactivity to MSC-specific cell
type markers, rather than hematopoietic stem cell markers.
After reaching confluence (80 % of total flask surface), all
subpopulations were trypsinized (trypsin-EDTA; Sigma-
Aldrich, Milan, Italy) and subcultured in a 12-well culture
dish for 2 days. Immunocytochemistry was then carried out
on MSCs. Briefly, cells were first washed with PBS, then
fixed with 4 % paraformaldheyde in PBS for 30 min and
incubated for 30 min with a 5 % solution of normal goat
serum (Sigma-Aldrich, Milan, Italy). They were subse-
quently incubated overnight at 4 �C with primary antibodies
(Millipore, Milan, Italy): CD44, 1:200 dilution; CD90,
1:100; CD105, 1:100; CD14, 1:200; CD34, 1:200; CD45,
1:200. The following day, cells were washed with PBS and
incubated for one hour at room temperature with Cy3-con-
jugated goat anti-rabbit secondary antibody or Cy3-conju-
gated goat anti-mouse secondary antibody or fluorescein
isothiocyanate-conjugated goat anti-mouse secondary anti-
body (Millipore, Milan, Italy). Cells were then washed with
PBS, and incubated with DAPI (1:10,000; Invitrogen). As a
control, the specificity of immunostaining was verified by
omitting incubation with the primary or secondary antibody.
Digital images were acquired using a Leica DMRB
fluorescence microscope (Leica Microsystems Srl, Milan,
Italy) equipped with a computer-assisted Nikon digital
camera (Nital SpA, Turin, Italy). Immunoreactivity was
evaluated taking into account the signal-to-noise ratio of
immunofluorescence.
Induced differentiation of human MSCs in adipocytes
The ability of MSCs to differentiate towards the adipogenic
line was examined in experiments where these were put in
culture for 7, 14, 21 and 28 days Some cells, as a control,
were incubated in DMEM-lg (10 % FBS, 1 % penicillin/
streptomycin, 1 % of MSC growth supplement). Some
cells were maintained with adipogenic medium (human
MesenCult MSC Basal Medium plus Adipogenic Stimu-
latory Supplement; cat. #05401 and #05403, respectively;
StemCell Technologies). Some experiments were also
performed to assess the nature of the vacuoles and, in
particular, their content in lipids. To this purpose, cells
were treated with Sudan III stain (Sigma-Aldrich, Milan,
Italy). To further characterize differentiated adipocytes,
cell lysates at 7, 14, 21, and 28 days were probed by
Western blot for PPAR-c and FABP4.
Staining of cultures
Sudan III is a dye having high affinity to fats, therefore it is
used to demonstrate triglycerides, lipids, and lipoproteins in
liquids, frozen sections, paraffin sections, and cells. To
identify differentiated adipocytes, the medium was removed
from the 6-wells microplate, the cultures were treated with
10 % formalin and incubated for 1 h at room temperature.
Hence the formalin was removed, the wells washed with
60 % ethanol, completely dried, hydro-alcoholic solution of
Sudan III added for 10 min, and washed with distilled water.
An adipocyte colony was defined as a group of five or more
Sudan III-stained adipocytes.
Determination of adipogenic and apoptotic markers
The expression of PPAR-c, FABP4, p53, AKT, pAKT,
Bax, PDCD4 and PTEN was evaluated by Western blot
analysis at 7, 14, 21, and 28 days. Adipogenic medium-
treated and untreated cells were trypsinized, centrifuged
and washed twice with PBS. Then, the pellets were
resuspended with lysis buffer (M-PER� Mammalian Pro-
tein Extraction Reagent, Thermo scientific, PIERCE Bio-
technology) supplemented with a cocktail of protease
inhibitor (complete, Mini, Protease Inhibitor Cocktail
Tablets, Roche) according to manufacturer’s instructions.
Forty micrograms of protein were loaded in the gel
(4–12 % Novex Bis–Tris gel, Invitrogen) and, after elec-
trophoresis, transferred to nitrocellulose membranes, using
a wet system. After transfer, the membranes were stained
with Ponceau Red to assess whether the proteins were
transferred correctly. Membranes were blocked in PBS
containing 0.01 % Tween-20 (PBST) and 5 % non-fat dry
milk at room temperature for 1 h. Then, they were incu-
bated at 4 �C overnight with mouse anti-PPAR-c (E-8;
sc-7273; Santa Cruz Biotechnology, Santa Cruz, CA,)
(dilution 1:300), rabbit anti-FABP4 (D25B3; #3544; Cell
Signalling Technology) (dilution 1:1,000), -p53 (FL-393;
sc-6243; Santa Cruz Biotechnology, Santa Cruz, CA)
(1:300 dilution), -AKT (#9272; Cell Signaling) (1:1,000
dilution), -pAKT (Ser473, 193H12; #4058; Cell Signaling)
580 Apoptosis (2013) 18:578–588
123
(1:1,000 dilution), -Bax (B3428, Sigma–Aldrich) (1:2,000
dilution), -PDCD4 (A00957; Sigma–Aldrich) (1:1,000
dilution), mouse monoclonal anti-PTEN (10264-2G;
Sigma–Aldrich) (1:1,000 dilution), and rabbit polyclonal
anti-b-actin (A2066; Sigma–Aldrich) (1:5,000 dilution)
antibodies, diluted in PBST with 5 % non-fat dry milk. The
presence of antibodies was detected by horseradish per-
oxidase-conjugated secondary antibody using the enhanced
chemiluminescence detection Supersignal West Pico
Chemiluminescent Substrate (Pierce Chemical Co., Rock-
ford, IL). The signal intensity of primary antibody binding
was quantitatively analyzed with ImageJ software and was
normalized to a loading control b-actin (actin bands
showed in the Figs. 3, 5, and 7 are from the same blot).
Values were expressed as arbitrary densitometric units
(A.D.U.) corresponding (proportional) to signal intensity.
Short interfering RNA (siRNA) preparation
For the p53 silencing assay, cells were transfected with
control siRNA (sc-37007; Santa Cruz Biotechnology),
consisting of a scrambled sequence that not lead to the
specific degradation of any known cellular mRNA, and p53
siRNA (sc-29435; Santa Cruz Biotechnology) according to
the manufacturer’s protocol. In a six well tissue culture
plate, AT-MSCs (2 9 105 cells/well) were seeded onto
medium without antibiotics 1 day before transfection.
siRNA was mixed with siTransfection medium (sc-36868;
Santa Cruz Biotechnology) in one tube and siTransfection
reagent (sc-29528; Santa Cruz Biotechnology) to siTrans-
fection medium in another tube, and the contents of the
tubes were incubated at room temperature for 10 min.
After incubating, the mixtures are mixed and incubated at
room temperature for 30 min. After 30 min, the medium
covering the cells is replaced with new medium without
antibiotics and cells are treated with the mixtures. The cells
were incubated for 24 h. Protein levels were monitored by
Western-blot to check silencing efficiency. The expression
of p53 protein was strongly reduced by specific siRNA 48
hours after transfection and thus AT-MSCs were treated
with adipogenic medium.
Wild-type p53 gene transfection
Wild-type p53 gene (pC53-SN3) was transfected using
SuperFect Reagent (QIAGEN, Milan, Italy) according to
the instruction manual. In brief, on the day before trans-
fection, AT-MSCs were seeded in a 90-mm dish to 60 %
confluency and cultured at 37 �C and 5 % CO2 in an
incubator. To the time of transfection, pC53-SN3 and
pC53-SN3-null, 20 lg each were dissolved in TE, pH 7.4,
with medium without serum to a total volume of 150 ll.
SuperFect Transfection Reagent, 40 ll was added to the
DNA solution. The samples were incubated for 10 min at
room temperature. AT-MSCs cells were washed once with
phosphate-buffered saline (PBS) and 3 ml of medium with
15 % FCS and 400 lg/ml G418 sulfate (G418, Calbio-
chem, San Diego, CA) containing the transfection com-
plexes was added. Then, the cells were incubated with the
complexes for 2 h at 37 �C and washed once with PBS and
cultured with fresh medium (containing 15 % FCS and
G418). After 24 hours, media were removed, and the cells
were washed 3 times with cold PBS, scraped from dishes
with a scraper, and centrifuged to obtain a cell pellet. The
cells were plated for adipogenic differentiation and cul-
tured with adipogenic medium.
Statistical analysis
Each experiment was repeated at least three times in trip-
licate and the mean ± SEM for each value was calculated.
Statistical analysis of results [Student’s t test for paired and
unpaired data; variance analysis (ANOVA)] was performed
using the statistical software package SYSTAT, version 11
(Systat Inc., Evanston IL, USA). A difference was con-
sidered significant at p \ 0.05.
Results
After 3 days of culture, two types of adherent cells were
observed: a more numerous cell population consisting of
small flattened cells and a population consisting of a few
spindle-shape fibroblastoid cells identified after as MSCs.
After 1 week of culture, these MSCs became the predom-
inant cell type. After the second cell passage the MSCs
cultures appeared to be homogeneous and with a high
replicative potential. When they reached high confluence,
the cells lost their replicative potential and presented
morphological changes.
In the immunocytochemistry analysis performed after
the first passage, MSCs did not present labeling for the
hematopoietic line for CD45, CD14 and CD34 and were
positive for the following adhesion molecules: by CD44
(H-CAM), CD90 (Thy 1), and CD105 (Endoglin) (Fig. 1).
MSCs in adipogenic differentiation medium led to the
appearance, after 7 days, of larger rounded cells presenting
numerous fat vacuoles in the cytoplasm, that increased in
size in a time-dependent manner (the vacuoles were larger
after 28 days of culture). The number of these cells
increased continuously up to the 28th day of culture
(Fig. 2). Adipocytes were verified by the presence of
intracellular vesicles containing saturated neutral lipid by
means of staining with Sudan III. In the control plates there
was no specific staining while in the plates treated with
Apoptosis (2013) 18:578–588 581
123
adipogenic medium it was possible to appreciate the for-
mation of large vacuoles containing lipids in which it was
evident a red–orange staining (Fig. 2). More than 50 % of
human MSCs were fully differentiated into adipocytes
within the four-week induction period. Adipogenic differ-
entiation medium-treated cell lysates were also probed by
Western blot for PPAR-c and FABP4. Already at 7 days,
differentiated cell expressed a 3-fold higher level of PPAR-
c than controls (Fig. 3a), as well as an increase of FABP4
was observed (Fig. 3b). The levels of PPAR-c and FABP4
increased further over time (Fig. 3a, b). To verify the role
of p53 in adipogenic differentiation of AT-MSCs, over-
and down-expression of p53 has been performed, and the
effects on MSC differentiation have been examined. The
results have been showed in Table 1 as percentage of
Sudan III labeled cells and in Fig. 4 as protein expression
levels based on Western blot evaluation of p53 and adi-
pogenic markers PPAR-c and FABP4, respectively, at 7,
14, 21, 28 days.
After establishing differentiation procedures, Western
blot analysis was performed on samples at 7, 14, 21, and
28 days to detect expression differences of p53, AKT,
pAKT, Bax, PTEN, and PCD4.
Our results demonstrated that, compared to the controls
(not significantly different at 7, 14, 21, and 28 days), in the
cells incubated with adipogenic medium, p53 was down-
regulated during adipogenic differentiation of AT-MSCs,
suggesting a negative role in regulating adipogenesis
(Fig. 5). The comparison among the cells treated with
adipogenic medium showed that the p53 levels remain
constant at 7 and 14 days, but they were down-regulated in
later stages of differentiation, particularly at 28 days
(Fig. 5).
Since the intracellular signaling cascade involving
phosphoinositide 3-kinase (PI3K) and AKT is involved in
the regulation of many cellular processes, we investigated
whether these signaling pathways play a role in the adipo-
genesis of human AT-MSCs. As shown in Fig. 6, adipo-
genic medium induced a rapid and marked phosphorylation
and activation of AKT at 7 days, which increased in time-
dependent manner until 28 days. In order to verify that the
activation of AKT in human MSCs adipogenesis is PI3K
dependent, we investigated the effect of LY294002 (Life
Technologies), a selective inhibitor of PI3K, on induced
AKT activation. The treatment of adipogenic medium-
cultured AT-MSCs with 20 lM LY 294002 for 24 hours
blocked AKT phosphorylation and kinase activity (data not
shown). Consequently the adipogenic differentiation
appeared to be very slow and the results of Sudan III
staining and PPAR-c and FABP4 expression were sub-
stantially similar to those of cells with p53 inactivated
(Fig. 4).
The p53 gene represents a central integrator of signals
resulting in cell cycle arrest, DNA repair, and in certain
Fig. 1 CD44 (a), CD90 (b), CD105 (c) (positive markers), CD45 (d),
CD14 (e), and CD34 (f) (negative markers) mesenchymal stem cells
(MSCs) markers expression by immunocytochemistry and using a
Leica DMRB fluorescence microscope equipped with a computer
assisted Nikon digital camera. a–f Magnification 940; Scale bars50 lm. DAPI stained MSCs were superimposed to show cell nuclei
582 Apoptosis (2013) 18:578–588
123
instances, cell death. p53 accumulates and transactivates
downstream target genes responsible for feedback degra-
dation circuitry of p53, cell cycle, and apoptosis, such as
the pro-apoptotic Bcl-2 family member Bax, and others.
Among these others, in addition to Bax, we analyzed PTEN
and PDCD4. In Fig. 7 the results of expression of PTEN
were reported demonstrating that in cultures without adi-
pogenic medium (controls) PTEN was increased in time-
dependent manner; compared to the controls at the same
time point, PTEN was decreased in the cells treated with
adipogenic medium with values lower at 28 days.
Since PDCD4 plays a role in maintaining a low level of
p53 in unstressed cells, we verified whether PDCD4 is
directly involved in the adipogenic differentiation of
AT-MSCs. Adipogenic medium differentiating AT-MSCs
possessed significantly down-regulated levels of PDCD4
protein expression. The levels were elevated in control cells
at 7 days and were fairly constant over time. In contrast, in
cells treated with adipogenic medium the production of
PDCD4 decreased over time and was no longer detectable at
28 days (Fig. 8).
Then, we set out to investigate pro-apoptotic protein
Bax and results were reported in Fig. 9. Adipogenic med-
ium induced a significantly decrease of Bax expression in
MSCs already at 7 days while its expression significantly
increased in control MSCs.
To understand the cross-talk between the examined
signaling pathways, the effects of overexpression/suppres-
sion of p53 on PTEN, Bax, and PDCD4 levels were stud-
ied. The results are showed in Fig. 10 as protein expression
levels based on Western blot evaluation of PTEN (A), Bax
(B), and PDCD4 (C) at 7, 14, 21, 28 days.
Fig. 2 Sudan III staining of AT-MSCs at 7, 14, 21, and 28 days. In
the control plates there is no specific staining while in the plates
treated with adipogenic medium it is possible to appreciate the
formation of large vacuoles containing lipids in which it was evident a
red–orange staining (Color figure online)
Fig. 3 Effects of adipogenic medium on a PPAR-c and b FABP4
expression in AT-MSCs determined by Western blot at different time
points. Data show the relative expression (mean ± SEM) of calcu-
lated as arbitrary densitometric units (A.D.U.) collected from three
independent experiments (three individual donors). *p \ 0.05 com-
pared to untreated with adipogenic medium cells (C)
Apoptosis (2013) 18:578–588 583
123
Discussion
Mesenchymal stem cells (MSCs) have been the subject of
intensive investigation over the last decades because of
their multipotent ability to differentiate towards many cell
types. Investigation of MSCs in vitro offers a way to
address critical questions on differentiation or cell fate
determination by inducing them to a certain mature cell
type. Adipocytes, deriving from MSCs in vivo, play critical
roles in both normal physiology and the progression of
various disease states [20]. Therefore, the knowledge of the
pathways directly implicated in the cellular transformation
and the control of the culture conditions of MSCs are
needed to improve the use of these cells for therapeutic
applications.
In this study, we showed that the PI3K/AKT signaling
pathway is activated during the induction of adipogenesis
of human AT-MSCs, confirming the importance of this
signaling pathway in promoting adipogenesis [21, 22].
Expression of constitutively active form of AKT, the main
downstream effector of PI3K, in 3T3-L1 has been shown to
cause spontaneous adipocyte differentiation [23, 24]. In
agreement with previous findings, we confirmed that
adipocytic differentiation is characterized by a strong
activity of PI3K, as documented by the high level of
pAKT.
Moreover, our results demonstrated that p53, PTEN,
PDCD4, and Bax proteins are down-regulated during adi-
pogenic differentiation of AT-MSCs.
The activation status of the Akt pathway may be cor-
related with the expression and function of p53. The
function of p53 in development and its importance in the
control of differentiation processes have been previously
established [25–27] with some divergences. Some studies
suggest that p53 facilitates cell differentiation, whereas in
others it seems to be suppressive [28, 29]. Adipocytes arise
Table 1 Percentage of Sudan III labeled cells after over- and knockdown expression of p53 in AT-MSCs
AT-MSCs p53 status Sudan III % labeled cells at 7, 14, 21, 28 days, respectively
Adipogenic medium-treated Wild type 16 ± 1; 35 ± 3; 40 ± 2; 52 ± 4
Adipogenic medium-treated Inactivated 22 ± 2; 45 ± 2; 52 ± 4; 60 ± 2
Adipogenic medium-treated Overexpressed 4 ± 1; 6 ± 3; 7 ± 2; 11 ± 3
Fig. 4 p53 values and effects of over- and knockdown expression of
p53 on adipogenic markers PPAR-c and FABP4 in adipogenic-treated
AT-MSCs determined by Western blot at 7, 14, 21, and 28 days. Data
show the relative expression (mean ± SEM) of calculated as arbitrary
densitometric units (A.D.U.) collected from three independent
experiments (three individual donors). Asterisks and degrees denote
significant differences of values of PPAR-c and FABP4 compared to
untreated control (wild type), p53 inactivated, and p53 overexpressed,
respectively
584 Apoptosis (2013) 18:578–588
123
from mesenchymal stem cells by a sequential pathway of
distinct differentiation stages. Early studies demonstrated
that p53 is down-regulated during adipogenic differentia-
tion of 3T3-L1 preadipocytes [30] and exhibits a reduction
in its DNA-binding [31], suggesting a negative role in
regulating adipogenesis. In contrast, it was more recently
shown that the protein levels of p53 remain constant during
adipogenic differentiation of 3T3-L1 cells. Moreover, in
late stages of this differentiation, p53 is phosphorylated on
two N-teminal residues, which may indicate its activation
[32]. Proliferation, self-renewal and genomic stability are
tightly controlled in embryonic and adult stem cells [33]
and p53 is a protein with many important functions,
including cell cycle arrest and suppression of tumor for-
mation. p53 has a role in triggering apoptosis under certain
conditions; it has been reported that p53-mediated apop-
tosis could be delayed by active PI3K/AKT [34]. There-
fore, we investigated whether p53 was involved in
inhibition of some markers of apoptosis through the PI3K/
Akt pathway. Our data demonstrated that the p53 protein
level was not significantly different when the differentia-
tion program starts, but in 21 and 28 days differentiated
adipocytes it is completely down-regulated.
Fig. 5 Effects of adipogenic medium on p53 expression in AT-MSCs
determined by Western blot at 7, 14, 21, and 28 days. Data show the
relative expression (mean ± SEM) of calculated as arbitrary densi-
tometric units (A.D.U.) collected from three independent experiments
(three individual donors). *p \ 0.05 compared to untreated with
adipogenic medium cells (C)
Fig. 6 Effects of adipogenic medium on Akt/pAkt expression in
AT-MSCs determined by Western blot. Data show the relative
expression (mean ± SEM) of calculated as arbitrary densitometric
units (A.D.U.) collected from three independent experiments (three
individual donors). Asterisks and degrees denote significant differ-
ences of values of AKT and pAKT compared to untreated control (C),
respectively
Fig. 7 Effects of adipogenic medium on PTEN expression in
AT-MSCs determined by Western blot at 7, 14, 21, and 28 days.
Data show the relative expression (mean ± SEM) of calculated as
arbitrary densitometric units (A.D.U.) collected from three indepen-
dent experiments (three individual donors). *p \ 0.05 compared to
without adipogenic medium cultures (C)
Fig. 8 Effects of adipogenic medium on PDCD4 production in
AT-MSCs determined by Western blot at 7, 14, 21,and 28 days. Data
show the relative expression (mean ± SEM) of calculated as arbitrary
densitometric units (A.D.U.) collected from three independent
experiments (three individual donors). *p \ 0.05 compared to
without adipogenic medium cultures (C)
Apoptosis (2013) 18:578–588 585
123
As above reported, the p53 tumor suppressor gene is
involved in cell-cycle regulation, apoptosis, senescence,
and differentiation in several biological systems [35]. The
Bcl-2 family of proteins includes molecules that can pro-
mote or repress programmed cell death [36]. Bax is a
proapoptotic member of this family. The transcriptional
activation of p53 results in the expression of proapoptotic
proteins, including Bax and cell death protein [37, 38]. The
proapoptotic Bax protein induces cell death by acting on
the mitochondria. The Bax protein binds to the perme-
ability transition pore complex, which is involved in the
regulation of mitochondrial membrane permeability [39].
Bax shows extensive amino acid homology with Bcl-2 and,
in addition to forming homodimers, can form heterodimers
with Bcl-2. When Bax predominates, programmed cell
death is accelerated, and the death repressor activity of Bcl-
2 is countered; therefore, the ratio of Bcl-2 to Bax deter-
mines survival or death following an apoptotic stimulus
[40]. We observed that p53 down-regulation is related to a
strong decrease of bax gene expression. Hence, a p53
decrease could inhibit apoptosis by reducing the translo-
cation of Bax from nucleus to mitochondria, the release of
cytochrome C and the activation of caspase 3.
The PTEN gene provides instructions for making a
protein that is found in almost all tissues in the body. This
protein acts as a tumor suppressor, which means that it
helps regulate the cycle of cell division by keeping cells
from growing and dividing too rapidly or in an uncon-
trolled way. The PTEN protein modifies other proteins and
fats (lipids) by removing phosphate groups, which consist
of three oxygen atoms and one phosphorus atom. One of
the primary targets of PTEN is lipid phosphatidylinositol
triphosphate (PIP3), a direct product of PI3K [41, 42]. Loss
of PTEN function, either in murine embryonic stem (ES)
cells or in human cancer cell lines, results in accumulation
of PIP3 and activation of its downstream effectors, such as
AKT/protein kinase B [43, 44] and Rac-1/Cdc42 [45].
Activation of AKT stimulates cell cycle progression by
Fig. 9 Effects of adipogenic medium on Bax expression in
AT-MSCs determined by Western blot at 7, 14, 21, and 28 days.
Data show the relative expression (mean ± SEM) of calculated as
arbitrary densitometric units (A.D.U.) collected from three indepen-
dent experiments (three individual donors). *p \ 0.05, compared to
AT-MSCs untreated with adipogenic medium (C)
Fig. 10 Effects of over- and knowckdown expression of p53 on
apoptotic markers PTEN (a), Bax (b), and PDCD4 (c) in adipogenic
medium-treated AT-MSCs determined by Western blot at 7, 14, 21,
and 28 days. Data show the relative expression (mean ± SEM) of
calculated as arbitrary densitometric units (A.D.U.) collected from
three independent experiments (three individual donors). Asterisks,
bullet, and degrees denote significant differences of values of PTEN
(a), Bax (b), and PDCD4 (c) compared to untreated control (wildtype), p53 inactivated, and p53 overexpressed, respectively
586 Apoptosis (2013) 18:578–588
123
downregulation of p27, a major inhibitor for G1 cyclin-
dependent kinases [44]. Activated AKT/protein kinase B is
also a well characterized survival factor in vitro and pre-
vents cells from undergoing apoptosis by inhibiting the
proapoptotic factors BAD [44] and caspase 9 [46] as well
as the nuclear translocation of Forkhead transcription fac-
tors [47].
Recently, an important role for PTEN in maintaining
stem cells has emerged, which could affect how we inter-
pret its tumor suppressor function and target it. Conditional
deletion of PTEN in hematopoietic cells [48, 49] causes a
myeloproliferative disorder and leukemia, which is con-
sistent with its role as a tumor suppressor in many tissues.
The inhibitory effect of PTEN loss on normal stem cell
function might be tissue-specific, because deletion of
PTEN in neural stem cells increases proliferation and the
cells maintain their self-renewing capacity [50].
Programmed cell death 4 (PDCD4) is a newly identified
tumor suppressor gene involved in the apoptotic machinery
and in cell transformation and invasion [51]. Different
mechanisms are involved in PDCD4 dysregulation: among
them activation of AKT leads to PDCD4 phosphorylation
at serine 67 and 457, causing a nuclear-to-cytoplasmic
switch of the PDCD4 gene product [52]. A previous study
identified S6K1 as the kinase that, upon mitogen stimula-
tion, phosphorylates PDCD4 and targets it for ubiquitina-
tion and degradation [53]. Our data demonstrated a
reduction of the expression of PDCD4 in adipogenic
medium-treated AT-MSCs. However, the overactivation of
PI3K/AKT is implicated in cancer [54], and PDCD4 is a
tumor suppressor [55]. A preferred strategy to increase
adipogenic differentiation could involve interventions that
selectively promote PDCD4 degradation in differentiating
adipocytes without increased tumor risk in the tissues.
Probably, AKT phosphorylates PDCD4 in a PI3K-depen-
dent manner, causes nuclear translocation of PDCD4, and
inactivates PDCD4 in its function as an inhibitor of AP-1-
mediated transcription. Additional studies are necessary to
define the exact mechanism of this inhibition.
In summary, mesenchymal stem cells can be isolated
and expanded in vitro. This has a great interest for tissue
engineering and therapeutic applications. Learning about
the mechanisms regulating proliferation, self-renewal and
transformation is critical in order to improve the safe use of
these cells and to understand their possible implications
also in tumorigenic processes. Our in vitro results indicate
that PI3K/AKT messenger pathway is crucial for adipo-
genic differentiation of AT-MSCs; it is related to reduction
of p53 and down-regulation of some markers of apoptosis,
such as Bax, PTEN, and PDCD4. Our data provide new
information on the performance of some apoptotic markers
during adipogenic differentiation of AT-MSCs to apply for
tissues repair and regeneration.
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