Beruflich Dokumente
Kultur Dokumente
Laboratory of Biology,
School of Medicine,
University of Athens, Athens,
Greece; bCell and Gene
Therapy Laboratory, Center
of Basic Research II, and
c
Biotechnology Laboratory,
Center of Basic Research,
Biomedical Research
Foundation of the Academy
of Athens, Athens, Greece;
d
First Department of
Obstetrics and Gynecology,
School of Medicine,
University of Athens, Athens,
Greece
Correspondence: Maria G.
Roubelakis, Ph.D., Laboratory of
Biology, Medical School,
University of Athens, Mikras
Asias, 75 Athens 115 27, Greece.
Telephone: 30-210-7462145 or
30-210-6597013; E-Mail:
roubel@med.uoa.gr
Received April 18, 2013; accepted
for publication August 30, 2013;
first published online in SCTM
EXPRESS December 4, 2013.
AlphaMed Press
1066-5099/2013/$20.00/0
http://dx.doi.org/
10.5966/sctm.2013-0081
ABSTRACT
MicroRNAs (miRNAs) have recently been shown to act as regulatory signals for maintaining stemness
and for determining the fate of adult and fetal stem cells, such as human mesenchymal stem cells
(hMSCs). hMSCs constitute a population of multipotent stem cells that can be expanded easily in culture and are able to differentiate into many lineages. We have isolated two subpopulations of fetal
mesenchymal stem cells (MSCs) from amniotic fluid (AF) known as spindle-shaped (SS) and roundshaped (RS) cells and characterized them on the basis of their phenotypes, pluripotency, proliferation
rates, and differentiation potentials. In this study, we analyzed the miRNA profile of MSCs derived
from AF, bone marrow (BM), and umbilical cord blood (UCB). We initially identified 67 different
miRNAs that were expressed in all three types of MSCs but at different levels, depending on the source.
A more detailed analysis revealed that miR-21 was expressed at higher levels in RS-AF-MSCs and BMMSCs compared with SS-AF-MSCs. We further demonstrated for the first time a direct interaction
between miR-21 and the pluripotency marker Sox2. The induction of miR-21 strongly inhibited Sox2
expression in SS-AF-MSCs, resulting in reduced clonogenic and proliferative potential and cell cycle
arrest. Strikingly, the opposite effect was observed upon miR-21 inhibition in RS-AF-MSCs and BMMSCs, which led to an enhanced proliferation rate. Finally, miR-21 induction accelerated osteogenesis
and impaired adipogenesis and chondrogenesis in SS-AF-MSCs. Therefore, these findings suggest
that miR-21 might specifically function by regulating Sox2 expression in human MSCs and might also
act as a key molecule determining MSC proliferation and differentiation. STEM CELLS TRANSLATIONAL
MEDICINE 2014;3:5468
INTRODUCTION
Human mesenchymal stem cells (MSCs) represent a multipotent stem cell population that is
able to self-renew and differentiate into multiple
cell lineages [1]. Adult MSCs derived from bone
marrow (BM) have been widely studied; however,
recent attention has focused on MSCs derived
from fetal sources, such as amniotic fluid (AF)
[27] or umbilical cord blood (UCB) [8, 9]. Our
group has recently identified MSCs from human
second trimester AF obtained during routine amniocenteses for prenatal diagnosis [4, 5]. Two
morphologically distinct adherent cell types were
isolated, which were termed spindle-shaped (SS)
and round-shaped (RS) cells. The two cell types
exhibited differences in phenotype, pluripotency,
proliferation rate, differentiation potential, and
proteomic profile [4]. Both subpopulations expressed mesenchymal stem cell markers such as
CD73, CD105, CD166, and integrins CD29 and
CD49e at similar levels. However, SS-AF-MSCs
expressed higher levels of CD90 and CD44 antigens compared with RS-AF-MSCs [4, 10]. SS-AFMSCs express pluripotency markers such as
such as cell proliferation [16, 17], differentiation [18, 19], cell cycle
[20, 21], and apoptosis [22].
Because stem cell populations derived from different sources
manifest specific miRNA signatures, in this study we attempted to
identify and compare the miRNA profiles of AF-MSCs, BM-MSCs,
and UCB-MSCs to systematically clarify the post-transcriptional
regulation of MSCs from various sources. In particular, we wanted
to obtain further insight into the miRNA patterns that are characteristic of AF-MSC subpopulations and to identify specific miRNAs
that may play an important role in their molecular identity. More
importantly, we provide functional evidence that miR-21 has
a key role in both subpopulations of AF-MSCs and is involved in
the suppression of the transcription factor Sox2. In addition, our
findings suggest that the induction of miR-21 expression inhibits
the expression of other pluripotency genes and alters the proliferation rate, the cell cycle, and the differentiation properties of
AF-MSCs.
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the intensity-dependent differences between the dyes. The expression matrix contains normalized Hy3/Hy5 ratios (log2 transformed) from all hybridizations. The subset of differentially
expressed miRNAs was used to construct an unsupervised hierarchical clustering of the different samples (Exiqon, A/S). For in silico
data analysis, we used the GOmir program (http://www.
bioacademy.gr/bioinformatics/projects/GOmir/), which integrates miRNA target prediction and functional analysis by combining the predicted target genes from the TargetScan (http://
www.targetscan.org/), miRanda (http://www.microrna.org/
microrna/home.do), RNAhybrid (http://bibiserv.techfak.unibielefeld.de/rnahybrid/), and PicTar (http://pictar.mdc-berlin.
de/) computational tools and provides a full gene description
and functional analysis for each target gene [25, 26].
Western Blots
A detailed protocol is provided in the supplemental online data.
56
(Erasmus University Medical Center, Rotterdam, The Netherlands) using Lipofectamine 2000 reagent according to the manufacturers protocol (Invitrogen). An equal concentration of
scrambled miR-21 antagonist (Exiqon A/S) or pLKO.1-sh-control
(Erasmus MC) was used as a control. Thirty hours after transfection, total RNA and protein were extracted. For functional assays,
pLKO.1-sh-Sox2 (sh-Sox2) or pLKO.1-sh-control (sh-control) viruses were produced by transient transfection of HEK293T cells,
using a four-plasmid lentiviral expression system, as previously described. Selection was performed using puromycin (Sigma-Aldrich)
at 0.5 mg/ml for 5 days.
Apoptosis Assay
One million transduced SS-AF-MSCs expressing miR-21, EF1, shSox2, or sh-control were assessed by annexin V-fluorescein isothiocyanate staining (BD Biosciences), according to the manufacturers instructions. 7-Aminoactinomycin D (Sigma-Aldrich) was
used for live-dead cell discrimination. Flow cytometry was performed using the Beckman Coulter Cytomics FC 500 flow cytometer (Beckman Coulter). Three independent experiments were
performed, and the mean 6 SEM of each experiment was
calculated.
SS-AF-MSCs expressing miR-21 or EF1 were stained with propidium iodide. The cells were fixed in ice-cold 70% ethanol for at
least 16 hours at 4C. After fixation, the cells were incubated with
2 mg/ml RNase A (Sigma-Aldrich) at 37C for 40 minutes. Then,
50 mg/ml propidium iodide (Sigma-Aldrich) was added, and the
cells were analyzed by flow cytometry. The nuclear debris, and
overlapping nuclei were gated out. The data are presented as
the mean 6 SEM of at least three independent experiments.
Senescence-associated b-galactosidase (SA b-gal) staining
was performed using a cellular senescence assay kit (Merck
KGaA). Briefly, 5 3 104 transduced SS-AF-MSCs expressing miR21 or EF1 were fixed in 13 fixing solution and stained for SA
b-gal activity overnight. The positive (blue) cells were counted
and were expressed relative to the number of positive cells in
SS-AF-MSCs expressing EF1. The data are presented as the
mean 6 SEM of at least three independent experiments.
The clonogenic potential of SS-AF-MSCs expressing miR-21 or shSox2 was evaluated by performing a fibroblast colony-forming
unit (CFU-F) assay. Specifically, transduced SS-AF-MSCs expressing miR-21, EF1, sh-Sox2, or sh-control were plated at three clonal
densities (70, 140, and 280 cells) in 60-mm plates for 14 days. The
CFU-Fs were quantified by Giemsa staining. The number of CFU-Fs
was estimated per 100 MSCs initially plated based on a linear regression analysis of the three different initial cell concentrations.
The data are presented as the mean 6 SEM of at least three independent experiments.
Statistics
The unpaired Students t test method was used to determine the
statistical significance, and p values are indicated in the figures,
where * represents p , .05, ** represents p , .01, and *** represents p , .001.
RESULTS
MTS Proliferation Assay
To determine the effect of miR-21 on proliferation, the transduced SS-AF-MSCs expressing miR-21, EF1, sh-Sox2, or shcontrol were plated at a density of 103 cells per well in 96-well
plates and cultured for 3, 7, or 10 days. RS-AF-MSCs or BMMSCs transfected with miR-21 antagonist or the scrambled sequence were plated at a density of 103 cells per well in 96-well
plates and cultured for 24 or 48 hours. At each time point, the appropriate amount of MTS reagent (Promega) was added to each
well and the cells were incubated for 3 hours, according to the
manufacturers instructions. The absorbance was recorded at
490 nm with an enzyme-linked immunosorbent assay plate
reader (ELX 800; Biotek Instruments Inc., Winooski, VT, http://
www.biotek.com/). The percentage increase in proliferation
was calculated using the following formula: [(OD dayx 2 OD
day0)/(OD day0) 3 100]. Three independent experiments were
performed, each including five replicates, and the mean 6 SEM
of each experiment was calculated.
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Figure 1. miRNA profiles of AF-MSCs, BM-MSCs, and UCB-MSCs. (A): Hierarchical clustered heat map illustrating the differential miRNA expression profiles of AF-MSCs, BM-MSCs, and UCB-MSCs compared with the pool of all samples (statistically significant differences, p , .05). (B):
Validation of the microarray through real-time PCR expression analysis of random miRNAs. The data are presented as mean 6 SEM. (C): Representative photos of the cultured UCB-MSC (Ci), BM-MSC (Cii), SS-AF-MSC (Ciii), and RS-AF-MSC (Civ) morphology. Magnification, 310. Abbreviations: AF, amniotic fluid; BM, bone marrow; miRNA, microRNA; MSC, mesenchymal stem cell; PCR, polymerase chain reaction; RS, roundshaped; SS, spindle-shaped; UCB, umbilical cord blood.
expression analysis, the calculated p values were based on Students t test. In addition, the Benjamini and Hochberg multiple
testing adjustment method was applied to the p values. A detailed
analysis of the expression levels of the detected miRNA in AFMSCs, BM-MSCs, and UCB-MSCs is presented in supplemental
online Fig. 1. Specifically, 16 miRNAs in AF-MSCs, 9 in BMMSCs, and 44 in UCB-MSCs were expressed at higher levels, respectively. Similarly, 51 miRNAs in AF-MSCs, 58 in BM-MSCs,
and 23 in UCB-MSCs were expressed at lower levels, respectively.
Furthermore, some miRNAs, such as miR-143, miR-487, miR-326,
and miR-199*, were downregulated, whereas no miRNA was
upregulated in all three MSC categories. The fold expression difference was calculated for each group versus the pool of samples.
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Figure 2. miR-21 expression in SS-AF-MSCs and RS-AF-MSCs. (A): Differentially expressed miRNAs in SS-AF-MSCs versus RS-AF-MSCs,
according to miRNA microarray data. (B): miR-21 expression levels in SS-AF-MSCs and RS-AF-MSCs were estimated by real-time PCR.
The values shown are the means 6 SEM of three samples. (Ci): Analysis of Sox2 expression in SS-AF-MSCs and RS-AF-MSCs by real-time
PCR. The results were normalized to the human glyceraldehyde 3-phosphate dehydrogenase-positive control and then to the SS-AF-MSCs.
(Cii): Western blot analysis of Sox2 in cell extracts from SS-AF-MSCs and RS-AF-MSCs. A protein band of 37 kDa corresponding to Sox2
was detected. b-Actin was used as a positive control for equal loading. Quantification was performed using Quantity One software, and
the results were normalized to the b-actin-positive control and then to the SS-AF-MSCs. The values shown are the mean 6 SEM of three
independent experiments (*, p , .05; **, p , .01; Students t test). (D): Working hypothesis for the role of miR-21 in SS-AF-MSCs and
RS-AF-MSCs. Abbreviations: AF, amniotic fluid; miRNA, microRNA; MSCs, mesenchymal stem cells; PCR, polymerase chain reaction;
RS, round-shaped; SS, spindle-shaped.
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Figure 3. miR-21 induction in SS-AF-MSCs decreases the expression levels of Sox2, Oct4, and Nanog. (Ai): NGFR expression in SS-AF-MSCs
transduced with miR-21 or EF1 lentivirus at MOI of 4 before and after cell sorting. (Aii): Real-time PCR for miR-21 detection in SS-AF-MSCs
transduced with miR-21 lentivirus at MOI of 4 after sorting for NGFR. The results were normalized to the RNU44 internal control and then
to the EF1-transduced cells. (B): Real-time PCR expression analysis of Sox2 (Bi) and Western blot analysis of Sox2 (Bii) in SS-AF-MSCs expressing
miR-21 compared with EF1-transduced cells. (C): Real-time PCR expression analysis of Oct4 (Ci) and Western blot analysis of Oct4 (Cii) in SS-AFMSCs expressing miR-21 compared with EF1-transduced cells. (D): Real-time PCR expression analysis of Nanog (Di) and Western blot analysis of
Nanog (Dii) in SS-AF-MSCs expressing miR-21 compared with EF1-transduced cells. The real-time PCR results were normalized to the human
glyceraldehyde 3-phosphate dehydrogenase-positive control and then to the EF1-transduced SS-AF-MSCs. Western blot quantification was
performed using Quantity One software, and the results were normalized to the b-actin-positive control and then to EF1-transduced SSAF-MSCs. The values presented are the mean 6 SEM of three independent experiments (*, p , .05; **, p , .01; Students t test). Abbreviations:
AF, amniotic fluid; EF1, elongation factor 1; MSCs, mesenchymal stem cells; NGFR, nerve growth factor receptor; PCR, polymerase chain reaction; PE, phycoerythrin; SS, spindle-shaped.
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Figure 4. miR-21 induction or Sox2 inhibition alters the growth of SS-AF-MSCs. (Ai): The clonogenic potential of SS-AF-MSCs expressing miR-21
was determined by CFU-F assay. The mean numbers 6 SEM of CFU-F per 100 cells in a 14-day clonogenic assay are presented. The values presented are the mean 6 SEM of three independent experiments (*, p , .05; Students t test). (Aii): Representative images of the colonies formed.
(Aiii): Comparative analysis of the growth rates of miR-21 versus EF1-transduced SS-AF-MSCs during 10 days in culture. The values presented are
the mean 6 SEM of three independent experiments (*, p , .05; **, p , .01; Students t test). (Aiv): Apoptosis was examined by FACS analysis of
annexin V staining in miR-21 and EF1-transduced SS-AF-MSCs. (Av): 7AAD was used for live-dead cell discrimination. (Bi): Western blot analysis
of Sox2 in SS-AF-MSCs transduced with sh-Sox2 or sh-control viruses. Quantification was performed using Quantity One software, and the results
were normalized to the b-actin positive control and then to the SS-AF-MSCs. (Bii): The clonogenic potential of SS-AF-MSCs transduced with shSox2 or sh-control virus was determined by CFU-F assay. The mean numbers 6 SEM of CFU-F per 100 cells in a 14-day clonogenic assay are
presented. The values presented are the mean 6 SEM of three independent experiments (**, p , .01; ***, p , .001; Students t test). (Biii):
Representative images of the colonies formed. (Biv): Comparative analysis of the growth rates of sh-Sox2 versus sh-control transduced SS-AFMSCs during 10 days in culture. The values presented are the mean 6 SEM of three independent experiments (*, p , .05; **, p , .01; ***, p ,
.001; Students t test). (Bv): Apoptosis was examined by FACS analysis of annexin V staining in sh-Sox2 and sh-control transduced SS-AF-MSCs.
(Bvi): 7AAD was used for live-dead cell discrimination. Abbreviations: 7AAD, 7-aminoactinomycin D; AF, amniotic fluid; CFU-F, fibroblast colonyforming unit; EF1, elongation factor 1; FACS, fluorescence-activated cell sorting; MSCs, mesenchymal stem cells; NGFR, nerve growth factor
receptor; PCR, polymerase chain reaction; PE, phycoerythrin; SS, spindle-shaped.
Figure continues on next page.
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sh-Sox2 virus (Fig. 4Bi). In particular, Sox2 downregulation in SSAF-MSCs reduced their clonogenic potential (3.1 CFU-F 6 0.1)
compared with SS-AF-MSCs transduced with sh-control virus
(14.3 CFU-F 6 1.1) (Fig. 4Bii, 4Biii) and also decreased their proliferation rate during ten days in culture (Fig. 4Biv). Finally, only
3.8% of the sh-Sox2-transduced SS-AF-MSCs were observed to
be positive for annexin V, as determined by FACS (Fig. 4Bv, 4Bvi).
To gain further insight into the mechanisms underlying the altered proliferation of SS-AF-MSCs on miR-21 induction, cell cycle
analysis was performed by propidium iodide staining (Fig. 5Ai).
After miR-21 induction in SS-AF-MSCs, the proportion of cells in
G0/G1 phase increased (69.45% 6 0.85%), whereas the portion
in S phase decreased (6.25% 6 1.25%), compared with EF1transduced cells (G0/G1: 53.5% 6 5 and S: 8.3% 6 2.7%) (Fig.
5Aii). Moreover, G1 cell cycle arrest was demonstrated mainly
through a decrease in G1-specific cyclin D1 at both the RNA
(Fig. 5Bi) and protein levels (Fig. 5Ci). Consistently, miR-21 induction was sufficient to reduce levels of cyclin E1 and cyclin A at the
protein level (Fig. 5Cii, 5Ciii). Accordingly, an increase in the expression of cyclin-dependent kinase inhibitors (CDKIs) such as
p27 (CDKN1B) and p18 (CDKN2C) was also evident after miR-21
expression in SS-AF-MSCs (Fig. 5Bii). In addition, the expression
level of Cdc25A (cell division cycle 25A) was decreased in SSAF-MSCs expressing miR-21 compared with the expression level
of EF1-transduced cells (Fig. 5Biii). Finally, SS-AF-MSCs expressing
miR-21 induced SA b-gal activity (Fig. 5D). These observations
suggest that miR-21 may regulate the proliferation of SS-AFMSCs through a combined effect on different components of
the cell cycle machinery.
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Figure 5. Cell cycle analysis in SS-AF-MSCs expressing miR-21. (Ai, Aii): Propidium iodide staining of SS-AF-MSCs expressing miR-21 and EF1transduced cells, as determined by FACS. The values shown are the means 6 SEM of three independent experiments. (Bi): Real-time PCR
expression analysis of cyclin D1, cyclin E1, cyclin A2, and cyclin B2 in SS-AF-MSCs expressing miR-21 compared with EF1-transduced cells.
(Bii): Real-time PCR expression analysis of p18 and p27 in SS-AF-MSCs expressing miR-21 compared with EF1-transduced cells. (Biii): Real-time
PCR expression analysis of Cdc25A in SS-AF-MSCs expressing miR-21 compared with EF1-transduced cells. The real-time PCR results were normalized to the human b-actin positive control and then to the EF1-transduced SS-AF-MSCs. The values presented are the mean 6 SEM of three
independent experiments. (C): Western blot analysis of cyclin D1, cyclin E1, and cyclin A in SS-AF-MSCs expressing miR-21 or EF1. Quantification
was performed using Quantity One software, and the results were normalized to the b-actin positive control and then to the EF1-transduced SSAF-MSCs. The values presented are the mean 6 SEM of three independent experiments (*, p , .05; Students t test). (D): Representative images
of SS-AF-MSCs expressing EF1 (Di) or miR-21 (Dii) assayed for SA b-gal activity. Magnification, 320. Graph shows relative levels of SA b-galpositive cells of three independent experiments ( *, p , .05; **, p , .01; Students t test). Abbreviations: AF, amniotic fluid; CFU-F,
fibroblast colony-forming unit; EF1, elongation factor 1; FACS, fluorescence-activated cell sorting; MSCs, mesenchymal stem cells; NGFR,
nerve growth factor receptor; PCR, polymerase chain reaction; PE, phycoerythrin; SA b-gal, senescence-associated b-galactosidase; SS,
spindle-shaped.
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Figure 6. Inhibition of miR-21 in RS-AF-MSCs and BM-MSCs. (Ai): Real-time PCR analysis of miR-21 in RS-AF-MSCs transfected with miR-21
antagonist or the scrambled sequence of the miR-21 antagonist. The results were normalized to the RNU44 internal control and then to
the RS-AF-MSC expression levels. (Aii): Comparative analysis of the growth rates of RS-AF-MSCs transfected with miR-21 antagonist or the
scrambled sequence 24 and 48 hours after transfection. (Aiii): Western blot analysis of Sox2, Oct4, and Nanog in RS-AF-MSCs transfected with
miR-21 antagonist or the scrambled sequence. (Aiv): Western blot analysis of Sox2 in RS-AF-MSCs that were transfected with scramble or miR-21
antagonist and subsequently treated with pLKO.1-sh-Sox2 or pLKO.1-sh-control for 32 hours. (Bi): Real-time PCR analysis of miR-21 in BM-MSCs
transfected with the miR-21 antagonist or the scrambled sequence. The results were normalized to the RNU44 internal control and then to the
BM-MSC expression level. (Bii): Comparative analysis of the growth rates of BM-MSCs transfected with the miR-21 antagonist or the scrambled
sequence 24 and 48 hours after transfection. (Biii): Western blot analysis of Sox2, Oct4, and Nanog in BM-MSCs transfected with the miR-21
antagonist or the scrambled sequence. (Biv): Western blot analysis of Sox2 in BM-MSCs that were transfected with scramble or miR-21 antagonist and subsequently treated with pLKO.1-sh-Sox2 or pLKO.1-sh-control for 32 hours. Western blot quantification was performed using Quantity One software, and the results were normalized to the b-actin positive control and then to the RS-AF-MSCs or BM-MSCs, as appropriate. The
values presented are the mean 6 SEM of three independent experiments (*, p , .05; **, p , .01; ***, p , .001; Students t test). Abbreviations:
AF, amniotic fluid; BM, bone marrow; MSCs, mesenchymal stem cells; PCR, polymerase chain reaction; RS, round-shaped.
only reduces the cell proliferation rate but also accelerates osteogenesis and decelerates adipogenesis and chondrogenesis.
To analyze the role of Sox2 inhibition in SS-AF-MSC differentiation properties, adipogenic, osteogenic, and chondrogenic
induction studies were also performed. More oil droplets were
S TEM C ELLS T RANSLATIONAL M EDICINE
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Figure 7. miR-21 induction alters the differentiation potential of SS-AF-MSCs. (Ai): Oil Red O staining for adipogenic differentiation in transduced SS-AF-MSCs expressing miR-21 or EF1. Magnification, 320. (Aii): Quantitative analysis of Oil Red O staining in SS-AF-MSCs expressing miR21 and EF1-transduced cells at different time points. (Aiii): Real-time PCR expression analysis of PPARg in SS-AF-MSCs expressing miR-21 compared with EF1-transduced cells. (Bi): Alizarin red S staining for osteogenic differentiation of SS-AF-MSCs expressing miR-21 and of EF1-transduced cells. Magnification, 320. (Bii): Quantitative analysis of Alizarin red S staining in SS-AF-MSCs expressing miR-21 and EF1-transduced cells.
(Biii): Real-time PCR expression analysis of Runx2 and osteocalcin in SS-AF-MSCs expressing miR-21 compared with EF1-transduced cells. (Ci):
Pellets formed by SS-AF-MSCs expressing miR-21 and EF1-transduced cells. (Cii): Alcian Blue and collagen IV staining for chondrogenic differentiation in SS-AF-MSCs expressing miR-21 and EF1-transduced cells. Magnification, 310. (Ciii): Real-time PCR expression analysis of aggrecan
and Sox9 in SS-AF-MSCs expressing miR-21 compared with EF1-transduced cells. The results of the real-time PCR analysis were normalized to the
human b-actin-positive control and then to the EF1-transduced SS-AF-MSCs. The values presented are the mean 6 SEM of three independent
experiments (*, p , .05; **, p , .01; Students t test). Abbreviations: AF, amniotic fluid; EF1, elongation factor 1; MSCs, mesenchymal stem cells;
PCR, polymerase chain reaction; PPARg, peroxisome proliferator-activated receptor-g; SS, spindle-shaped.
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staining (supplemental online Fig. 4Ai, 4Aii). Consistently, the relative expression of adipogenic differentiation marker PPARg was
increased in SS-AF-MSCs transduced with sh-Sox2 virus compared
66
with SS-AF-MSCs transduced with sh-control virus during differentiation, as determined by real-time PCR analysis (supplemental
online Fig. 4Aiii). Osteogenic differentiation was decreased when
Sox2 was inhibited in SS-AF-MSCs (supplemental online Fig. 4Bi,
4Bii). In agreement, the expression of osteogenic molecular markers
such as osteocalcin was decreased in SS-AF-MSCs transduced with
sh-Sox2 virus compared with SS-AF-MSCs transduced with shcontrol virus (supplemental online Fig. 4Biii). Additionally, after
chondrogenic induction, the pellets formed after Sox2 inhibition
in SS-AF-MSCs were larger than those formed by SS-AF-MSCs
transduced with sh-control virus (supplemental online Fig.
4Ci, 4Cii). Expression levels of the chondrogenic markers Sox9
and aggrecan were also increased after Sox2 inhibition in SS-AFMSCs (supplemental online Fig. 4Ciii). Thus, it is evident that
Sox2 and miR-21 act through independent mechanisms and alter
the differentiation properties of SS-AF-MSCs.
DISCUSSION
MicroRNAs and their role in the determination of stem cell fate
have recently attracted intense interest. Many studies have already been conducted in MSCs in an attempt to define the miRNAs that regulate the unique properties of MSCs [3335]. Recent
interest has been focused on different MSC populations from fetal and adult sources [10, 36, 37]. The best characterized cells to
date are adult BM-MSCs, but recently, fetal MSCs such as AFMSCs and UCB-MSCs have also attracted increased attention
[4, 5, 3840]. Previously, we successfully isolated and characterized karyotypically normal subpopulations of AF-MSCs and performed a systematic phenotypic, molecular, and proteomic
analysis [5, 6]. Interestingly, our studies have shown that SSAF-MSCs may represent a valuable tool in cell therapy because
they are able to induce liver repair [6] or serve as delivery vehicles
for anticancer agents in vivo [2].
In this study, we defined the miRNA profiles of MSCs derived
from fetal sources such as amniotic fluid and umbilical cord blood
and directly compared them with the profile of BM-MSCs. Specifically, 67 miRNAs were differentially expressed in the three MSC
sources. On the basis of the detailed analysis, no common miRNAs
were detected as upregulated in all the populations, but miR-143,
miR-487, miR-326, and miR-199* were downregulated in all the
sample categories tested. These miRNAs have been shown to target genes that regulate various cellular processes that are critical
for stem cells, such as proliferation, differentiation, and cell survival. For example, miR-143 has been found to target genes such
as ERK5 (extracellular-signal-regulated kinase 5) [41], DNMT3A
(DNA methyltransferase 3A) [42], FNDC3b (fibronectin type III domain containing 3B) [43], and Bcl-2 (B-cell lymphoma 2) [44], as
indicated by the miRecords database [26]. To further study the
source of AF-MSCs, a more detailed analysis was performed
for the two subpopulations of AF-MSCs, which resulted in the
identification of 32 differentially expressed miRNAs between
SS-AF-MSCs and RS-AF-MSCs. These two populations exhibited
differences in morphology, proliferation rate and differentiation properties, suggesting that the differential expression of
miRNAs may regulate these functions. One of the most interesting upregulated miRNAs in RS-AF-MSCs compared with SS-AFMSCs was miR-21. The role of miR-21 has been studied in various
fields, including development, oncology, stem cell biology, and
aging [45]. Interestingly, miR-21 has an oncogenic role in several
cancers and is thought to be a potential disease biomarker [46,
67
CONCLUSION
Although many studies have indicated that miR-21 is an oncomir
that regulates proliferation and apoptosis, we observed a contrasting role in AF-MSCs. Our data suggest that miR-21 may act
as a regulator of the clonogenic potential, proliferation rate,
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ACKNOWLEDGMENTS
This research has been cofinanced by the European Union (European Social Fund) and Greek national funds through the Operational Program Education and Lifelong Learning of the
National Strategic Reference Framework Research Funding Program: Heracleitus II, Investing in Knowledge Society through
the European Social Fund. We are grateful to Dr. Helen A. Papadaki (Medical School, University of Crete) and Dr. A. Stravoropoulos
(Cord Blood Bank, BRFAA) for providing BM-MSCs and umbilical
cord blood samples, respectively. We also thank Prof. L. Naldini,
Dr. Panagiotis Politis, and Dr. Elena Siapati for providing plasmids
and reagents, and for discussions, and we thank Dr. Ariana Gavriil
for technical assistance with FACS.
AUTHOR CONTRIBUTIONS
O.T.: experimental design, experimental procedures, data analysis, manuscript writing; D.Z.: experimental procedures, data analysis; V.B.: experimental procedures, data analysis, review of
manuscript; K.I.P. and A.A.: provision of amniotic fluid samples;
N.P.A.: conception, financial support, review of manuscript;
M.G.R.: conception and design, financial support, experimental
procedures, data analysis, manuscript writing.
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