Fetal and Neonatal Stem Cells

FETAL AND NEONATAL STEM CELLS

Sox2 Suppression by miR-21 Governs Human
Mesenchymal Stem Cell Properties
OURANIA TROHATOU,a,b DIMITRA ZAGOURA,a,b VASILIKI BITSIKA,a,c KALLIOPI I. PAPPA,d
ARISTIDIS ANTSAKLIS,d NICHOLAS P. ANAGNOU,a,b MARIA G. ROUBELAKISa,b
Key Words. Amniotic fluid mesenchymal stem cells x Bone marrow mesenchymal stem cells x
Umbilical cord blood mesenchymal stem cells x miR-21 x Sox2 x Cell cycle
a

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

Sox2 (SRY sex determination SRY region Y-box2),

Oct4 (octamer-binding transcription factor 4)
and the homeobox transcription factor Nanog
[4, 5, 11]. SS-AF-MSCs also exhibit a high proliferation rate in culture and differentiate in vitro not
only into mesoderm-derived cell types but also into endoderm-derived cells, such as hepatocytes
[46]. Indeed, we have previously demonstrated
that SS-AF-MSCs can be expanded in culture at
higher levels than BM-MSCs, and this occurs without karyotypic changes and with the maintenance
of their capacity to differentiate into osteogenic,
adipogenic, and chondrogenic cells [2, 4, 5].
MicroRNAs (miRNAs) are single-stranded noncoding RNA sequences of 1923 nucleotides that
act as post-transcriptional regulators of gene expression by base pairing to partially complementary sequences in the 39 untranslated region
(UTR) of multiple target mRNAs, resulting in silencing of the mRNA [1214]. Approximately
40%90% of human protein-coding genes are
predicted to be regulated by miRNAs at the translational level [15]. Recent studies have suggested
that miRNAs are involved in embryonic and adult
stem cell fate by regulating biological processes

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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.

MATERIALS AND METHODS

Isolation and Culture of Human MSCs
All protocols involving human subjects were approved by the Ethical Committee of Alexandra Hospital, Athens, Greece; the Bioethics Committee of the School of Medicine of the University
of Athens; and the Bioethics Committee of the Biomedical Research Foundation of the Academy of Athens (BRFAA). All samples were collected after informed consent was obtained from
each individual. The AF-MSCs were isolated and cultured according to methods described in previous studies [2, 46]. SS and RS
colonies were selected and subcultured [4]. BM samples from
healthy volunteers were obtained from posterior iliac crest aspirates, as described previously [5, 23]. Bone marrow mononuclear
cells were plated at a density of 2 3 105 cells per cm2 in Dulbeccos
modified Eagles medium (DMEM; Sigma-Aldrich, Gillingham,
U.K., http://www.sigmaaldrich.com) supplemented with 20% fetal
bovine serum (Gibco-BRL, Paisley, U.K., http://www.invitrogen.
com) and incubated at 37C in a 5% (v/v) CO2 humidified chamber
for approximately 20 days, until the first BM-MSC colonies
appeared in culture. UCB samples were processed within 24
hours. UCB-MSCs were isolated by Ficoll-Paque density centrifugation (Sigma-Aldrich) and cultured in DMEM (Gibco-BRL) containing 20% fetal bovine serum (Gibco-BRL), according to the
method described previously [24].

miRNA Microarray Analysis

Total RNA, including miRNAs, from three AF-MSC, three BMMSC, and two UCB-MSC samples were extracted using Trizol reagent according to the manufacturers protocol (Invitrogen, Paisley, U.K., http://www.invitrogen.com). The samples were labeled
using a miRCURY Hy3/Hy5 labeling kit (Exiqon A/S, Vedbaek Denmark, http://www.exiqon.com/ls) and hybridized on an miRCURY
LNA Array (v.8.1) (Exiqon A/S), which consists of control probes,
mismatch probes, and 427 capture probes that perfectly match all
of the human miRNAs registered and annotated in miRBase release 7.1 at the Wellcome Trust Sanger Institute. Analysis of
the scanned slides indicated that labeling was successful because
all of the capture probes for the control-spiked oligonucleotides
produced signals within the expected range. The quantified signals were normalized using the global Lowess (Locally Weighted
Scatterplot Smoothing) regression algorithm, which minimizes

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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].

Real-Time Quantitative Polymerase Chain Reaction

This experimental approach used a well-established protocol, the
details of which are provided in the supplemental online data.

Western Blots
A detailed protocol is provided in the supplemental online data.

Lentiviral Vector Construction and Transduction of

AF-MSCs
A four-plasmid lentiviral expression system containing pCCLsin.
cPPT.hEF1a.DLNGFR.Wpre [27], a kind gift from Dr. L. Naldini
(University School of Medicine, Milan, Italy), which includes the
elongation factor 1a (EF1a), was used to induce miR-21 expression. To construct the lentiviral vector expressing human premiR-21, the pre-miR-21 sequence was amplified by polymerase
chain reaction (PCR) from human genomic DNA. We cloned the
pre-miR-21 gene into the pCCLsin.cPPT.hEF1a.DLNGFR.Wpre
plasmid using the following primers: forward 59-TCGACTCGAGGTTCGATCTTAACAGG-39 and reverse 59-TCGAACGCGTACCAGACAGAAGGACC-39. miR-21 or EF1 (empty vector) viruses
were produced by transient transfection of HEK293T cells, as previously described [2, 28], followed by collection with Amicon Ultra
Centrifugal Filters-100K Units (Merck, Whitehouse Station, NY,
http://www.merck.com). The lentiviral titers were determined
by infection of HT1080 cells with serial dilutions of the concentrated viral stock. The plasmid contained a complementary
DNA for the low-affinity nerve growth factor receptor (DLNGFR)
as a positive expression marker. Nerve growth factor receptor
(NGFR)-positive cells were identified by fluorescence-activated
cell sorting (FACS) analysis using a PE-tagged mouse antihuman CD271 antibody (BD Biosciences, San Diego, CA, http://
www.bdbiosciences.com) and a Beckman Coulter Cytomics FC
500 flow cytometer (Beckman Coulter, Palo Alto, CA, http://
www.beckmancoulter.com). The titers ranged from 106 to 5 3
106 infectious units/ml. For transduction, 2.5 3 104 SS-AFMSCs or HEK293T cells per well were seeded into 12-well plates
and lentivirus was added at a multiplicity of infection (MOI) of 4.
After 7 days, the NGFR-positive cells were identified by flow
cytometry and sorted using a FACSAria cell sorter (BD Biosciences). In all cases, cell sorting was performed, and the efficiency
was determined to be greater than 98%.

miR-21 Inhibition and sh-Sox2 Transfection

The RS-AF-MSCs and BM-MSCs were transfected with 0.3 mM
miR-21 antagonist (Exiqon A/S) and/or 1 mg pLKO.1-sh-Sox2

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(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.

miR-21 in the Lineage Determination of Human MSCs

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.

Cell Cycle Analysis

Enhanced Green Fluorescence Protein Reporter Assay
The Sox2 39UTR enhanced green fluorescence protein (EGFP) reporter construct was generated from genomic DNA by PCR amplification of the human Sox2 39UTR sequence using the primers
described in supplemental online Table 1. The mutated form
of the Sox2 39UTR sequence was generated by site-directed mutagenesis (Promega, Madison, WI, http://www.promega.com)
according to the manufacturers instructions. The fragments were
inserted into the pEGFP-C2 vector (Promega) downstream of the
EGFP cassette. HEK293T cells were transduced with the miR-21
lentivirus, then transiently transfected with the pEGFP-C2 constructs (Sox2 39UTR or mutant Sox2 39UTR). Forty-eight hours
later, EGFP-positive cells were identified in all tested samples
by FACS analysis. Three independent experiments were performed, each including two replicates, and the mean 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.

Fibroblast Colony-Forming Unit Assay

Adipogenic, Osteogenic, and Chondrogenic

Differentiation

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.

Detailed protocols are provided in the supplemental online data.

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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Analysis of miRNA Patterns in AF-MSCs, BM-MSCs,

and UCB-MSCs
We previously performed a detailed molecular and proteomic
analysis of SS-AF-MSCs and RS-AF-MSCs and BM-MSCs that
revealed fundamental differences between their characteristics
[4, 5]. As a next step, we have attempted to analyze the posttranscriptional differences between the MSCs derived from AF,
BM, and UCB by evaluating and comparing their miRNA profiles.
Initially, we performed an miRNA microarray (miRCURY LNA Array
v.8.1) analysis of three AF-MSC (SS-AF-MSCs and RS-AF-MSCs),
three BM-MSC, and two UCB-MSC samples, each hybridized to
the captured probes against the pool of all the samples. The
miRNA profile revealed 67 differentially expressed miRNAs
among the three MSC sources, as shown in the heat map plot
(Fig. 1A). The expression matrix contains the normalized Hy3/
Hy5 ratios (log2 transformed) from all of the hybridizations
(supplemental online Table 2). miRNAs with p value , .05 were
considered to be significantly differentially expressed. For this
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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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To validate the microarray results, we performed real-time PCR

analysis of some of the differentially expressed miRNAs, such
as hsa-miR-221, hsa-miR-222, hsa-miR-210, and hsa-let-7d, which
confirmed the same trends in the respective miRNA expression
levels (Fig. 1B).

Differentially Expressed miRNAs in AF-MSC

Subpopulations
We recently isolated two morphologically different subpopulations of AF-MSCs, termed RS and SS AF-MSCs [4]. These cells have
distinct morphologies, exhibit phenotypic and molecular differences, and differ in their ability to differentiate into multiple cell
types. Notably, the proliferation rate of SS-AF-MSCs is higher than

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miR-21 in the Lineage Determination of Human MSCs

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.

that of RS-AF-MSCs [4]. In this study, a detailed miRNA microarray

analysis of the two AF-MSC subpopulations indicated a differential miRNA expression pattern. More specifically, 32 miRNAs were
differentially expressed between the SS-AF-MSCs and RS-AFMSCs (Fig. 2A). Interestingly, RS-AF-MSCs exhibited increased
expression of miR-21 (6.6-fold expression) compared with SS-AFMSCs (0.52-fold expression), as determined by both the miRNA
microarray results and real-time PCR analysis (Fig. 2B). An extensive bioinformatics analysis of miR-21 using GOmir software [25]
indicated a list of 55 predicted target genes supplemented with

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gene descriptions and functional analyses (supplemental online

Table 3). The transcription factor Sox2 was identified as a target
gene containing predicted miR-21 binding sites. Our group recently showed that SS-AF-MSCs exhibited higher expression levels of Sox2 compared with RS-AF-MSCs [4]. Similarly, in this study,
we confirmed Sox2 expression at the RNA and protein level in
both AF-MSC populations (Fig. 2Ci, 2Cii). Based on these findings,
we focused on the role of miR-21 and its binding partner Sox2 in
the proliferation rate and differentiation properties of SS-AFMSCs and RS-AF-MSCs (Fig. 2D).
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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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miR-21 in the Lineage Determination of Human MSCs

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.

miR-21 Binds Directly to the Sox2 39UTR

Based on the hypothesis that Sox2 might be a target of miR-21,
a GFP reporter assay was performed to demonstrate the potential binding. The Sox2 39UTR was fused to an enhanced green
fluorescence protein (EGFP) reporter gene (supplemental
online Fig. 2A) and EGFP expression was determined by FACS
analysis in HEK293T cells transduced with miR-21 lentivirus.
As shown in supplemental online Fig. 2A, miR-21 significantly repressed EGFP expression (EGFP expression: 15.1% 6 2.04%).

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Consistently, neither the Sox2 39UTR in the absence of miR-21

(EGFP expression: 41.125% 6 4.06%) nor the mutant Sox2
39UTR (containing a TC mutation at the miR-21 binding site)
in the presence of miR-21 (EGFP expression: 36.7% 6 2.81%)
had any effect, as determined by FACS analysis and immunofluorescence microscopy (supplemental online Fig. 2B). Notably,
the HEK293T cells did not exhibit endogenous miR-21 expression (supplemental online Fig. 3A). These experiments confirmed the direct interaction of miR-21 with the Sox2 39UTR.
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Figure 4. Continued from previous page.

We then sought to evaluate the expression levels of Sox2 in

SS-AF-MSCs expressing miR-21. We transduced SS-AF-MSCs with
miR-21 lentivirus at an MOI of 4 and sorted the cells for NGFR
(98% efficiency) (Fig. 3Ai). The miR-21 expression levels in the
transduced SS-AF-MSCs were 2.5- to 9.8-fold higher than in the
control cells (Fig. 3Aii). Interestingly, a statistically significant decrease in Sox2 at the RNA and protein level was observed in SS-AFMSCs expressing miR-21 compared with EF1-transduced cells
(Fig. 3B). This finding clearly indicates the direct interaction between miR-21 and the Sox2 39UTR, which results in translational
repression and target degradation.

miR-21 Induction Decreases Oct4 and Nanog Expression

in SS-AF-MSCs
Accumulating evidence suggests that Sox2 promotes pluripotency and represses differentiation in concert with Oct4 and
Nanog as a core transcription factor network [29, 30]. To determine whether Sox2 inhibition by miR-21 also affects these two
transcription factors, we performed real-time PCR and Western
blot analysis of Oct4 and Nanog expression in transduced
SS-AF-MSCs expressing miR-21 or EF1. Statistically significant

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decreases in Oct4 and Nanog were observed at both the mRNA

and protein levels in SS-AF-MSCs expressing miR-21 compared
with EF1-transduced cells (Fig. 3C, 3D). Oct4 and Nanog were
not predicted as potential targets of miR-21 by in silico target
prediction analysis, suggesting that the effect observed was
the result of an indirect mechanism.

miR-21 Induction Alters the Cell Growth Rate of

SS-AF-MSCs
To analyze the role of miR-21 in SS-AF-MSCs, a CFU-F assay was
performed to determine the clonogenic potential of these cells
(Fig. 4Ai, 4Aii). SS-AF-MSCs expressing miR-21 showed reduced
clonogenic potential (17.1 CFU-F 6 1.59) compared with EF1transduced cells (20.9 CFU-F 6 0.53). To further explore the role
of miR-21 in the proliferation rate of SS-AF-MSCs, MTS assays
were performed at 3, 7, and 10 days. miR-21 induction in
SS-AF-MSCs caused a significant decrease in cell proliferation
compared with EF1-transduced cells (Fig. 4Aiii). In addition, no apoptotic effect was observed in SS-AF-MSCs expressing miR-21, as
determined by annexin V and 7ADD staining (Fig. 4Aiv, 4Av). Similar results were observed after transduction of SS-AF-MSCs with

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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.

Inhibition of miR-21 Increases the Proliferation Rate of

RS-AF-MSCs and BM-MSCs Through Upregulation
of Sox2
As described above, RS-AF-MSCs express miR-21 at high levels.
Thus, we transfected these cells with a miR-21 antagonist to explore a possible transition to the SS-AF-MSC phenotype. After
transfection with the antagonist, miR-21 expression was decreased in RS-AF-MSCs compared with cells transfected with
a scrambled sequence of the miR-21 antagonist (Fig. 6Ai). The
proliferation rate of the RS-AF-MSCs was increased 24 hours after
miR-21 inhibition, as determined by the MTS assay (Fig. 6Aii). In
addition, reduction of miR-21 expression led to a significant increase in the expression levels of the pluripotency genes Sox2,
Oct4, and Nanog (Fig. 6Aiii). However, no morphological alterations were observed in RS-AF-MSCs. To test whether Sox2 expression difference was mediated through miR-21 directly, we

63

performed rescue experiments. RS-AF-MSCs were treated with

miR-21 antagonist followed by transfection with Sox2 shRNA.
As expected, transfection with sh-Sox2 reduced the increase of
Sox2 resulted from miR-21 antagonist treatment (Fig. 6Aiv).
BM-MSCs exhibit reduced proliferation during multiple passages [31], similar to RS-AF-MSCs, and both cell types express
miR-21 at high levels. BM-MSCs (passage 6) expressing miR-21
at high levels (supplemental online Fig. 3B) were also transfected
with the miR-21 antagonist or the scrambled sequence. Downregulation of miR-21 expression was observed (Fig. 6Bi), and the
proliferation rate of the BM-MSCs was increased 24 hours after
miR-21 inhibition, as shown by MTS assay (Fig. 6Bii). Increased expression levels of the pluripotency genes Sox2, Oct4, and Nanog
were also detected (Fig. 6Biii). The rescue experiment was also
performed for BM-MSCs. Likewise, transfection with sh-Sox2 reduced the increase of Sox2 that resulted from miR-21 antagonist
treatment (Fig. 6Biv).

miR-21 Induction Results in Altered Differentiation

Properties of SS-AF-MSCs
To evaluate the role of miR-21 in the differentiation potential of
SS-AF-MSCs, adipogenic, osteogenic, and chondrogenic induction
studies were performed. Transduced SS-AF-MSCs expressing
miR-21 or EF1 were cultured under adipogenic conditions for 3,
5, 7, 9, 14, and 21 days. Fewer oil droplets were observed in
SS-AF-MSCs expressing miR-21 compared with EF1-transduced
cells (Fig. 7Ai). Consistently, the relative expression of adipogenic
differentiation markers such as peroxisome proliferator activated
receptor-g (PPARg) was decreased in SS-AF-MSCs expressing
miR-21 compared with EF1-transduced cells during differentiation, as determined by real-time PCR analysis (Fig. 7Aiii).
After osteogenic induction, transduced SS-AF-MSCs expressing miR-21 or EF1 were stained for calcium deposits using Alizarin
red S reagent (Fig. 7Bi). In contrast to adipogenesis, osteogenic
differentiation was increased by miR-21 expression compared
with EF1-transduced cells (Fig. 7Bii). The expression of osteogenic
molecular markers such as osteocalcin and Runx2 was increased
in SS-AF-MSCs expressing miR-21 compared with EF1-transduced
cells (Fig. 7Biii).
After chondrogenic induction, the SS-AF-MSCs aggregated
and formed pellets (Fig. 7Ci). For histological evaluation, the pellets were cut into serial 3-mm sections and stained with Alcian
Blue (Fig. 7C). The pellets formed by the SS-AF-MSCs expressing
miR-21 were smaller than those formed by EF1-transduced cells.
Chondrogenic differentiation was also detected by immunofluorescence staining for collagen IV to confirm the production of col-

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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miR-21 in the Lineage Determination of Human MSCs

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.

lagen, which plays a role in the formation of a dense mass through

strong interactions (Fig. 7Cii) [32]. In addition, the expression levels of the chondrogenic markers Sox9 and aggrecan were decreased after miR-21 induction in SS-AF-MSCs (Fig. 7Ciii). These
results demonstrate that miR-21 induction in SS-AF-MSCs not

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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
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65

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.

observed in SS-AF-MSCs transduced with sh-Sox2 virus compared

with SS-AF-MSCs transduced with sh-control virus, resulting in an
increase in adipogenic differentiation determined by Oil Red O

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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

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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,

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miR-21 in the Lineage Determination of Human MSCs

47]. Furthermore, miR-21 has been implicated in the promotion

of tumor growth by targeting genes such as the tumor suppressor
Pdcd4 (programmed cell death 4), PTEN (phosphatase and tensin
homolog), or TPM1 (tropomyosin 1) in several tumor types [46,
48, 49]. In addition to its oncogenic effect, miR-21 exhibits an antiangiogenic function by targeting the RhoB transcript in endothelial cells [50]. In mouse embryonic stem cells, RE1 silencing
transcription factor is responsible for the transcriptional inhibition of miR-21, resulting in the suppression of self-renewal and
pluripotency as a result of the loss of expression of the critical regulators Oct4, Sox2, Nanog, and c-Myc [16].
After extensive comparative bioinformatics analysis using
GOmir software, Sox2 was identified as a predicted target of
miR-21. In this study, we proved for the first time that Sox2 is negatively regulated by miR-21 at the post-transcriptional level by
means of a specific target site within the 39UTR. miR-21 induction
in SS-AF-MSCs led to a significant reduction in Sox2 expression at
the RNA and protein levels, further supporting a direct interaction. Ours is also the first study to demonstrate that miR-21
may reduce Oct4 and Nanog expression, likely through an indirect
mechanism, because miR-21 is not believed to target any of these
transcription factors, according to in silico analysis. These three
transcription factors are known to act in concert as the core components of a network that promotes pluripotency in stem cells
[29]. In addition, Sox2 have been found to directly regulate Nanog
expression [51]; thus, it is expected that lower levels of Sox2
would result in lower levels of Nanog expression [51]. The decrease in the expression levels of Sox2, Oct4, and Nanog after
miR-21 induction in SS-AF-MSCs was followed by a decrease in
the clonogenic potential and proliferation rate of these cells,
but there was no evidence for an apoptotic effect. Similarly, recent studies demonstrated that miR-21 regulates the proliferation rate of adipose tissue-derived MSCs (AT-MSCs) and also
that miR-21 overexpression decreased clonogenic potential cell
proliferation in white adipose tissue in a mouse model with induced obesity from a high-fat diet [52]. More importantly, Sox2
downregulation resulted in a statistically significant reduction
of the clonogenic potential and the proliferation rate of SS-AFMSCs, similar to the phenotype observed after miR-21 induction.
These results indicated that miR-21 acts as a regulator of clonogenic potential and proliferation of SS-AF-MSCs by targeting
Sox2. In addition, G0/G1 cell cycle arrest was observed after
miR-21 induction and sequential Sox2 suppression in SS-AFMSCs. This effect was mainly demonstrated although there were
decreased levels of cyclin D1, cyclin E1, and cyclin A. In agreement
with our data, Sox2 has previously been reported to promote proliferation by facilitating the G1/S transition and by transcriptional
regulation of the CCND1 gene [53]. More importantly, in human
MSCs, sh-Sox2 treatment resulted in a decrease in the proportion
of cells in S phase and thus in a reduced proliferation rate [54]. We
next analyzed the expression levels of the cell cycle inhibitors
p27 and p18, which bind to cyclin-CDK complexes and inhibit
their catalytic activity, resulting in suppression of G1 phase
[55, 56]. As expected, p27 and p18 were upregulated after
miR-21 induction in SS-AF-MSCs. Additionally, recent studies
have shown that miR-21 negatively regulates the cell division cycle 25A protein (Cdc25A), resulting in cell cycle arrest in colon
cancer cells [57]. Similarly, in this study, SS-AF-MSCs expressing
miR-21 exhibited decreased expression of Cdc25A compared
with EF1-transduced cells. This may explain the cell cycle arrest
observed after miR-21 induction, as Cdc25A positively regulates
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67

G1/S transition [57]. In addition, we demonstrated relatively

high SA b-gal activity of SS-AF-MSCs expressing miR-21 compared with EF1-transduced cells, which may be the result of cell
cycle arrest [58, 59].
To further study the functional role of miR-21 and explain the
robust expression of this miRNA in RS-AF-MSCs, we performed
miR-21 inhibition experiments using a specific antagonist. The
suppression of miR-21 in RS-AF-MSCs rescued the low proliferation rate and increased the expression levels of Sox2, Oct4, and
Nanog. Although miR-21 appears to regulate the proliferation
rate of the two AF-MSC subpopulations, no alterations in RSAF-MSC morphology were observed after miR-21 inhibition. Interestingly, the inhibition of miR-21 in BM-MSCs had similar
effects. Notably, the proliferation rate of BM-MSCs was lower
than that of SS-AF-MSCs, as described in our previous studies
[5]. In agreement, rescue experiments in RS-AF-MSCs and BMMSCs with sh-Sox2 reduced the increased expression of Sox2,
which initially resulted from the miR-21 antagonist.
In this study, the induction miR-21 resulted in enhanced
osteogenesis and inhibited adipogenesis and chondrogenesis
in SS-AF-MSCs. However, Sox2 inhibition resulted in opposite
effects, inducing adipogenesis and chondrogenesis and reducing
osteogenesis. These observations suggest that a different mechanism has been activated during the differentiation process in shSox2-transduced SS-AF-MSCs that is likely independent of the
miR-21 pathway.

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
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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.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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