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

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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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