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Running Title: MePR: a model of mesenchymal stem cells.
MePR: a novel human mesenchymal progenitor model
with characteristics of pluripotency.
Marco Miceli1,2
, Gianluigi Franci1,2,3
, Carmela Dell’Aversana2, Francesca Ricciardiello
1,2,
Francesca Petraglia1, Annamaria Carissimo
1, Lucia Perone
4, Giuseppe Maria Maruotti
5,
Marco Savarese1, Pasquale Martinelli
5, Massimo Cancemi
6, Lucia Altucci
1,2,*.
1 Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università di Napoli,
Vico L. De Crecchio 7, 80138, Napoli, IT; 2 Institute of Genetics and Biophysics Adriano
Buzzati-Traverso, IGB-CNR, via P. Castellino111, 80131, Napoli, IT; 3 present address:
Department of Molecular Biology, Radboud University, Nijmegen Center for Molecular Life
Sciences, Nijmegen, NL; 4 Telethon Institute of Genetics and Medicine (TIGEM), Via P.
Castellino 111, 80131 Naples, IT; 5 High-Risk Pregnancy and Prenatal Diagnosis Centre
Department of Gynaecology and Obstetrics, Federico II University, Naples, IT; 6 Center
‘Ricerche e Diagnosi Genetiche’, C.so V. Emanuele, Naples, IT.
*Correspondence should be addressed to Prof. Lucia Altucci, Dipartimento di Biochimica,
Biofisica e Patologia Generale, Seconda Università di Napoli, Vico L. De Crecchio 7, 80138,
Napoli, Italy; Tel: +390815667569; Fax:+39081450169; [email protected]
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Abstract
Human embryo stem cells or adult tissues are excellent models for discovery and
characterization of differentiation processes. The aims of regenerative medicine are to define
molecular and physiological mechanisms that govern stem cells and differentiation. Human
Mesenchymal Stem Cells (hMSCs) are multipotent adult stem cells able to differentiate into a
variety of cell types under controlled conditions both in vivo and in vitro, and have the
remarkable ability of self-renewal. hMSCs derived from amniotic fluid and characterized by
the expression of Oct-4 and Nanog, typical markers of pluripotent cells, represent an excellent
model for studies on stemness. Unfortunately, the limited amount of cells available from each
donation and, above all, the limited number of replications do not allow for detailed studies.
Here, we report on the immortalization and characterization of novel Mesenchymal
Progenitor (MePR) cell lines from amniotic fluid-derived hMSCs, whose biological properties
are similar to primary amniocytes. Our data indicate that MePR cells display the multipotency
potential and differentiation rates of hMSCs, thus representing a useful model to study both
mechanisms of differentiation and pharmacological approaches to induce selective
differentiation. In particular, MePR-2B cells, which carry a bona fide normal karyotype,
might be used in basic stem cell research leading to the development of new approaches for
stem cell therapy and tissue engineering.
Keyword: Stem cells, differentiation, regenerative medicine, multipotency, pluripotency.
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Introduction
Human stem cell engineering and its application in human diseases is a hot issue in current
research. The fact that human Embryonic Stem Cells (hESCs) can only be derived from the
Inner Cell Mass (ICM) during embryonic development raises a number of ethical questions
(1,2), severely limiting their use. hESCs are pluripotent cells, able to generate all possible
tissues of an adult organism. Currently, hESCs cannot be used in regenerative surgery as it is
not yet possible to avoid teratoma formation upon differentiation (3,4). Thus, the optimization
of differentiation protocols, together with the creation of novel hESC models, represents a key
objective of stem cell research. Adult human stem cells are currently being investigated and
exploited as alternatives to ESCs (5-7).
Human Mesenchymal Stem Cells (hMSCs) are multipotent stem cells, retaining good self-
renewal properties. These cells differentiate in vivo and in vitro into a wide range of tissues,
such as neurons, glia, chondrocytes, adipocytes, cardiomiocytes, osteoblasts, etc. (8-10).
hMSCs can be isolated from several adult tissues (including peripheral blood, periosteum,
muscle, adipose and connective tissues, skin, bone marrow, brain, etc.), as well as from
embryonic appendages such as placenta, umbilical cord blood and amniotic fluid (11-14).
hMSCs derived from adult tissues are an important source for the regeneration of damaged
tissues and the maintenance of homeostasis in tissues in which they are located (adult stem
cells) (7,15-21). Although hMSCs display multipotent capability and self-renewal, these cells
do not pose major ethical issues when used in research (8-10,22-24). hMSCs include a broad
range of cells with different morphology, physiology and surface expression markers (25-27);
therefore sorting and collection of amniotic hMSC sub-populations depends on their ability to
attach to a plastic surface. To date, most studies on the molecular mechanism(s) and
characterization of hMSCs have been carried out using Bone Marrow (BM) cells. While
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surface markers from BM are CD44, CD105 (SH2; endoglin), CD106 (vascular cell adhesion
molecule; VCAM-1), CD166, CD29, CD73 (SH3 and SH4), CD90 (Thy-1), CD117, STRO-1
e Sca-1 (28-32), αυβ3 and αυβ5, LFA-3 and L-selectin (22,29,30,33-35), other markers,
typical of hematopoietic and epidermal cells (CD11b, CD14, CD31, CD33, CD34, CD133,
and CD45), are absent (22). Pittenger et al. showed that only 0.01% to 0.001% of
mononuclear cells isolated on density gradient (Ficoll/Percoll) give rise to plastic-adherent
fibroblast-like colonies (22,36-38). One of the main problems in the use of BM-derived
hMSCs is their extremely low concentration. Moreover, the number of hMSCs seems to
decrease with age (37) and infirmity (38). An additional problem is represented by
senescence, which occurs after relatively few duplication cycles (40-50 PDL = Population
Doubling Level) (18,19,21).
hMSCs from cord blood, placenta and amniotic fluid offer a number of advantages over adult
BM-derived hMSCs: i) easy availability with lower risk (collection of amniotic fluid is a
routine test carried out between the16th
and 18th
week of pregnancy, with low risk for the fetus
<0.1%) (39); the umbilical cord and placenta are removed at childbirth after informed
consent; ii) less stringent criteria for donor-recipient HLA matching, allowing the use of
umbilical cord blood, placental and amniotic samples for transplants between unrelated or
partially compatible patients (the reduced risk is correlated to the lower expression of HLA
class II antigens) (40); iii) reduced risk of Graft-Versus-Host-Disease (GVHD) due to
incomplete development of the infant's immune system (and therefore the relative immaturity
of T cells), even when donor/recipient compatibility is not perfect (40); iv) low risk of
infection, for example caused by CytoMegaloVirus (CMV) (<1% of infants contract the virus
in the womb) (41).
Although the growth potential in long-term cultures of hMSCs derived from umbilical cord,
placenta and amniotic fluid is superior to that of BM cells, they are used exclusively for
transplantation in pediatric patients due to the limited amount of cells derived from donations
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(40). Even a small amount (about 2 mL) of amniotic fluid taken during the second trimester of
pregnancy is able to generate typical MSC-expressing markers (22,29,30,33-35). Their ability
to differentiate into multiple cell lines after cultivation in specific differentiating media has
also been demonstrated (42). Amniocytes deriving from the epithelium, skin, uro-genital
apparatus, respiratory and gastrointestinal systems of the fetus have been described in the
literature (43-47). Early classifications of these cells were mainly based on morphological
criteria, and are thus inadequate. Very limited biochemical data on these cells, their
morphology and growth characteristics exist to classify these human amniotic fluid cells into
epithelium cells, amniotic fluid-specific cells and fibroblastic cells (44,45). Different origins
have been suggested for all three cell types (1,43-49). The very recent discovery of the
existence of a population of adult stem cells expressing Oct-4 in human amniotic fluid is a
promising source of stem cells (50), which can be harvested without the ethical controversies
associated with hESCs (4-6,43-49). Finally, amniotic fluid stem cells are not able to form
tumors in immune-deficient mice (51-58), thus increasing their potential use in the treatment
of human diseases. Human amniotic fluid stem cells express markers of adult stem cells
together with typical markers of ESCs, indicating that these cells might be considered as
having some features of both embryonic and adult stem cells. Whether these cells present the
advantages of both types of stem cells remains to be established (1,50).
Here, we describe the creation of Mesenchymal Progenitor (MePR) cells, immortalized cell
lines derived from amniotic fluid cells whose biological properties are very similar to primary
hMSCs. Normal hMSCs have a limited replicative potential with a PDL of up to 40-50
duplications (18,19,21). The novel MePR cell lines replicate indefinitely, enabling the
complete biological and molecular characterization of these currently little known cells.
Therefore, despite not suitable for clinical use, MePR cells may help to study the properties
and therapeutic potential of hMSCs.
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Materials and methods
Cell collection, culture and infection.
Amniotic fluid samples were obtained after informed consent from pregnant women (aged 20-
42 years) between the 16th
and 18th
week of gestation through ultrasound-guided
transabdominal puncture. Samples carrying an abnormal karyotype were excluded. Collection
of amniotic fluid samples (20mL) is a routine medical procedure used in prenatal diagnosis
(with low risk for the fetus <0.1%) (39), and only 2mL of amniotic fluid was donated for our
experiments.
Cells were centrifuged and re-suspended in 7mL RPMI 1640 medium 4.5g/L glucose
(Euroclone, UK) supplemented with 20% Fetal Bovine Serum (FBS) (Euroclone, UK),
100U/ml pen-strep (Lonza, Belgium), 2mM L-Glutamine (Lonza, Belgium) at 37°C and 5%
CO2 in a fully humidified atmosphere. Cells were first grown for 10 days until the appearance
of Colony-Forming Cells (CFC). After a first splitting, amniocytes were grown to confluence
and co-infected with HPV16-E6/E7 and HPV16-hTERT lentiviral vectors (infection #1, Fig.
1A). After a week the cells were split and infected again (infection #2, Fig.1A). After a
second week the cells were split and infected again with HPV16-E6/E7 or hTERT (infection
#3, Fig. 1A). After a further week a fourth infection was carried out in the same way as
described above (infection #4, Fig. 1A). At the end of the multi-infection, eight cell lines were
obtained and cultured for one month. Samples were observed and photographed with DMI
6000 inverted microscope (Leica Microsystems) using Leica LAS Image Analysis software
(Leica Microsystems) (Fig. 2A).
hPV16-E6/E7 and -hTERT lentiviral production.
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HIV-1-based SIN lentiviral vectors were derived from SINF-MU3-W-S vector backbone (59).
hPV16-E6/E7 was inserted upstream of a gene cassette containing an encephalomyocarditis
virus internal ribosome entry site (IRES) and yellow fluorescent protein (YFP) gene into
SINF-MU3-W-S to generate SINF-MU3-E6E7-IRES-YFPW-S. SINF-MU3-hTERT-IRES-
GFPW-S was generated by inserting hTERT cDNA upstream of a gene cassette containing an
IRES and green fluorescent protein (GFP) gene into SINF-MU3-W-S. VSV-G-pseudotyped
lentiviral vectors were generated in 150mm tissue culture dishes by transient co-transfection
with i) 66μg VSV-G-expressing construct pCMV-VSV-G (Invitrogen, USA), ii) 48μg
packaging construct pCMVΔR8.2 (Addgene), and iii) 66μg lentiviral vector plasmids (pSin
hTERT or Psin E6/E7) into sub-confluent HEK 293FT cells (Invitrogen) by calcium
phosphate precipitation (Clontech, Calphos Mammalian Transfection Kit) (60). The
supernatant containing the virus was produced in HEK 293FT, collected, filtered and used to
infect primary amniocytes.
Calculation of population doublings.
Calculation of population doublings was carried out at each cell passage, assuming
exponential growth of cells, according to the following formula (61):
Nx = N0 * 2X
X = ln (NX/N0) * (1/0,6931)
where N0 is the number of cells at the time of plating in culture dishes (beginning of growth
period), Nx is the number of cells at the time of harvest (end of growth period), and X is the
number of population doublings between N0 and Nx. To calculate the population doubling,
200,000 cells were seeded in the dishes (100mm) (N0). After one week cells were harvested,
centrifuged and re-suspended in 1mL of medium. Cells were counted using Trypan blue assay
(Sigma) (Nx). The procedure was subsequently repeated weekly over a period of 23 weeks,
recording the number of population doublings each week.
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Cell cycle analysis.
Cells were re-suspended in the staining solution containing RNAse A, propidium iodide
(50μg/ml), sodium citrate (0.1%), NP40 (0.1%) in PBS 1X for 30 min in the dark. Cell cycle
distribution was assessed with a FACScalibur flow cytometer (Becton Dickinson), and 10,000
cells were analyzed by ModFit version 3 Technology (Verity) and Cell Quest (Becton
Dickinson) (62).
RNA extraction, RT-PCR and Real-Time PCR.
Total RNA was extracted using TRIZOL (Life Technologies) and reverse transcription was
carried out using SuperScript® VILO™ cDNA Kit (Invitrogen) according to the
manufacturer’s protocol. Converted cDNA was amplified using AmplyTaq Gold™
(Roche).
Amplified DNA fragments were loaded on 1.0% agarose gel and photographed on a Gel
Logic 200 Imaging system UV transilluminator (Kodak). Real-Time PCR was performed
using iQ™
SYBR® Green Supermix (Bio-Rad) in a DNA Engine Opticon2 thermal cycler
(MJ Research Incorporated). Primers for amplification and experimental conditions are shown
in Tables 1 and 2.
Western blot analysis.
Forty micrograms of total protein extract was separated on 10% polyacrylamide gel and
blotted as previously described (62). Western blot of Col2A1 (1:1000; Santa Cruz) was
performed and extracellular-signal-regulated kinases (ERKs) (1:1000; Santa Cruz) were used
for equal loading.
Differentiation assays.
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Myogenic differentiation (63,64).
To induce myogenic differentiation, amniocytes (control) and the three cell lines were grown
in the following differentiating medium: RPMI 1640 4.5g/L glucose supplemented with 2%
FBS, 10ng/ml Epidermal Growth Factor (EGF), 10ng/ml Platelet-Derived Growth Factor
(PDGF-BB) (both by Peprotech) and 3μM 5-azacytidine (Sigma). After 24h of treatment, the
myogenic medium was replaced without adding 5-azacytidine. The cells were also cultured in
a commercial skeletal muscle cell growth medium (PromoCell). The medium was replaced
weekly and the cultures were observed for the presence of multinucleated cells (myotubes).
After 14 days of culture, Real-Time PCR analysis was performed to analyze changes in the
expression of myogenic markers (Myogenin; MyoD).
Adipogenic differentiation (63,64).
To induce adipogenic differentiation, amniocytes (control) and the three cell lines were
cultured for 2-3 weeks in RPMI 1640 4.5g/L glucose supplemented with 10% FBS, 0.5mM
isobutyl-methylxanthine, 200μM indomethacin, 10-6
M dexamethasone and 10μg/ml insulin,
(all by Sigma). The medium was replaced weekly. After 3 weeks of culture, Real-Time PCR
analysis was performed to analyze changes in the expression of adipogenic marker PPARγ2,
and PLIN2, a marker of lipid accumulation in diverse cell types (65,66).
Osteogenic differentiation (63,64).
Osteogenic differentiation was performed by culturing the cells with RPMI 1640 4.5g/L
glucose supplemented with 10% FBS, 10-8
M dexamethasone, 0.2mM ascorbic acid, and
10mM ß-glycerol phosphate (all by Sigma) for 2-3 weeks. The medium was replaced weekly.
Real-Time PCR analysis was also performed using osteopontin- and osteocalcin-specific
primers.
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Chondrogenic differentiation (67).
Chondrogenic differentiation was performed by culturing the cells with serum-free RPMI
1640 4.5g/L glucose supplemented with 10ng/mL TGF-ß3 (Sigma) for 2 weeks. The medium
was replaced weekly. Real-Time PCR analysis was also performed using specific primers
(Sox9, Colxa1 and Col2a1).
Neuro-glial differentiation (63,64).
For differentiation of neural cells, amniocytes were incubated with RPMI 1640 supplemented
with 20% FBS, 1mM/l βmercapto-ethanol, 5ng/ml bFGF (Sigma) for 24h, and then treated
with serum depletion for 5h. Immunocytochemical staining and Real-Time PCR was also
performed with neuronal-specific marker, βIII Tubulin (TuJ-1); glial marker, GFAP, was used
to assess the capacity of neural differentiation.
Detection of neuronal differentiation by immunocytochemical analysis.
Cells were grown in Lab Tech tissue culture chamber slides (NalgeNunc International, USA).
Ten thousand cells were plated and cultured for 24h before starting differentiation. Treated
and untreated cells (see differentiation methods) were then washed three times with PBS and
fixed with 4% paraformaldehyde (PFA) in PBS 1X at room temperature for 30 min. After
washing, cells were incubated with 10% Normal Goat Serum in 0.1% Triton X-100/1X PBS
for 15 min at room temperature. The samples were incubated with primary antibody (mouse
anti-βIII-tubulin 1:400 (Sigma-Aldrich), and Rabbit anti-GFAP (Dako; Glostrup), 1:300) in
10% Normal Goat Serum/1X PBS for 1h at room temperature. Fluorophore-conjugated
secondary antibodies were used for visualization: 1:400 DyLightTM 488-conjucated (Green)
AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch) and 1:400 DyLightTM 594-
conjucated (Red) AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch), in 10%
Normal Goat Serum/1X PBS for 30 min at room temperature in the dark. Cells were then
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incubated with Hoechst 33342 (Thermo Scientific) 1μg/mL in 1X PBS for 5 min at room
temperature. After washing, PBS residuals were carefully removed. Cells were observed and
photographed with DM 6000/B Upright microscope (Leica Microsystem) using Leica LAS
Image Analysis software (Leica Microsystem) (Fig. 8A).
Cell staining.
Staining experiments were performed after differentiation (adipogenic, osteogenic and
chondrogenic) to detect accumulation of the final products characteristic of differentiation.
Ten thousand cells per well were plated and cultured in Lab Tech tissue culture chamber
slides (Nalge Nunc International, USA).
Adipocyte detection (intracellular lipid vesicles).
Oil Red O (0.3%) was dissolved in isopropanol and stored in the dark. Cells were washed
with PBS, fixed with PFA (4%) and incubated at room temperature for at least 30 min. Three
parts of the Oil Red O stock solution were diluted with 2 parts of distilled water, and the
mixture was filtered with a syringe filter. The fixation buffer was removed and cell monolayer
was washed. After removing water, cell monolayer was covered with 60% isopropanol and
incubated at room temperature for 5 min. Isopropanol was removed, and the cell monolayer
was covered with Oil Red O staining solution and incubated at room temperature for 15 min.
The cell monolayer was then washed several times until the water became clear.
Osteoblast detection (calcium deposits).
2g Alizarin Red S was dissolved in 100 ml of distilled water, and 0.1% NH4OH was added
until pH was between 4.1 and 4.3. The solution was filtered and stored in the dark. Cells were
washed with PBS, fixed with PFA (4%) and incubated at room temperature for at least 30
min. The fixation buffer was removed and cell monolayer was washed. After, the cellular
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his
artic
le h
as b
een
peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
ion,
but
has
yet
to u
nder
go c
opye
ditin
g an
d pr
oof
corr
ectio
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he f
inal
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monolayer was covered with Alizarin Red S staining solution and incubated at room
temperature in the dark for 45 min. Later, cells were washed four times with distilled water
and once with PBS.
Chondroblast detection (extracellular matrix).
60 ml ethanol (98-100%) was mixed with 40 ml acetic acid (98-100%). 10 mg Alcian blue 8
GX was dissolved in this solution. 120 ml ethanol was mixed with 80 ml acetic acid to obtain
the destaining solution. The chamber slides were washed twice with PBS 1X, covered with
PFA (4%) and incubated at room temperature for 60 min. PFA was aspirated and cells were
washed twice. The Alcian staining solution was added to cover the cells. Chamber slides were
incubated overnight at room temperature in the dark. Alcian staining solution was removed
and cells were washed with the destaining solution for 20 min. Washing step was repeated
twice. The destaining solution was removed and PBS added. Cells were observed and
photographed with DM 6000/B Upright microscope (Leica Microsystem) using Leica LAS
Image Analysis software (Leica-Microsystem).
Measurement of chromosome number and aberrations.
Cells were prepared from exponentially growing cells at 80 PDL. Chromosomal analysis was
performed according to standard methods (68). Chromosomes were counted and examined
through a Nikon Eclipse-1000 epi-fluorescent microscope (Nikon Instruments), equipped with
Genikon System V.3.8.5. (Nikon). To examine statistically significant chromosome numbers,
±1 deviation was allowed and 50-100 metaphase spreads were scored for each assay.
CGH Array.
Molecular karyotyping was performed using a 4X180K Agilent microarray. Genomic DNA
was extracted according to the manufacturer’s protocol. Labelling, hybridization and post-
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hara
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s of
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ripo
tenc
y (d
oi: 1
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cd.2
012.
0498
)T
his
artic
le h
as b
een
peer
-rev
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ed a
nd a
ccep
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for
publ
icat
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but
has
yet
to u
nder
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d pr
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corr
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he f
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washing were performed according to the manufacturer’s specifications (Agilent
Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol, version 6.1; Agilent
Technologies, USA). Array slides were analyzed with an Agilent G2505 scanner. Scanned
image analysis was carried out with Feature Extraction software (version 10.5.1.1; Agilent
Technologies, USA). For identifying duplications and deletions, the standard set-up of the
Aberration Detection, Method 2 (ADM-2) algorithm for the data that passed QC metrics
testing was used. All copy number changes observed were compared to Copy Number
Variants (CNVs) reported in previous studies of normal populations documented on the
Database of Genomic Variants (DGV).
Trascriptome analysis.
RNA concentration and integrity were determined by NanoDrop spectrophotometer
(Nanodrop Technologies), Agilent 2100 Bioanalyzer (RNA 6000 Nano Chip kit Agilent) and
agarose gel electrophoresis. Gene expression profiles were analyzed by Whole Human
Genome Two-Color Microarray (Agilent Technologies no. G4112F), following the
manufacturer’s protocol.
Gene expression microarray data processing.
Microarray quality control reports generated by Agilent Feature Extraction software were
used to detect hybridization artifacts. Probe level raw intensity was processed using
R/BioConductor (69) and Limma package (70). Background correction was performed using
normexp Limma method and data normalization was carried out in two steps: loess
normalization within array to correct systematic dye bias and quantile normalization between
arrays to detect systematic non-biological bias. Ratios representing the relative target mRNA
intensities compared to control RNA probe signals were derived from normalized data. In
order to detect the statistical significance of differential expression among the four different
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hara
cter
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s of
plu
ripo
tenc
y (d
oi: 1
0.10
89/s
cd.2
012.
0498
)T
his
artic
le h
as b
een
peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
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but
has
yet
to u
nder
go c
opye
ditin
g an
d pr
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corr
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he f
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cell types, a one-way ANOVA and Tukey multiple comparison test as Post-ANOVA was
performed. For each p-value, the Benjamini-Hochberg procedure was used to calculate the
False Discovery Rate (FDR) in order to avoid the problem of multiple testing (71). The
selected gene lists were obtained using the following thresholds: FDR<0.01 and abs(ratio)>2.
The relative abundance of GeneOntology Biological Process (BP) terms in each of the
selected lists was analyzed using the Database for Annotation, Visualization and Integrated
Discovery (DAVID) Functional Annotation Clustering tool (72).
Results
Immortalization of MePR cells.
hESCs escape cellular senescence through the expression of human Telomerase Reverse
Transcriptase (hTERT) (73-77). The ectopic expression of hTERT has been reported to
extend the life span of hMSCs and progenitor cells of human neurons (76,77). The use of
hTERT alone is not sufficient to immortalize hMSCs, but requires the combinatorial
expression of human papillomavirus type16 genes (HPV16) E6 and E7 (17), which accelerate
degradation of p53 and pRb, respectively (78). E7 is also able to bind and inactivate the
cyclin-dependent kinase inhibitors p21 and p27 (79). After morphological selection, the three
cell populations (MePR-3, MePR-2, MePR-0) were infected with HPV16-E6/E7 and hTERT,
using lentiviral vectors expressing pSin hTERT and pSin E6/E7 (80).
To overcome the difficulties in infecting human amniotic cells (81), we developed a "Multi-
Infection Program" as outlined in figure 1A. This approach was applied to all MePR cell
types. At the end of the procedure, some clones died while others survived in all cell lines. In
MePR-0A cells, for example, eight clones were obtained, but only six survived and were
tested for the presence of hTERT and E6-E7 transcripts (Fig. 1B). Based on RT-PCR data, we
chose a clone having a high level of hTERT transcript, since E6/E7 expression level is always
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hara
cter
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s of
plu
ripo
tenc
y (d
oi: 1
0.10
89/s
cd.2
012.
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)T
his
artic
le h
as b
een
peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
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but
has
yet
to u
nder
go c
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ditin
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d pr
oof
corr
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he f
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similar (Fig. 1B). The procedure was repeated twice to obtain MePR-2B and MePR-3A cells
(Fig. 2B). NB4 Acute Promyelocytic Leukemia (APL) cells were added as positive control for
the expression of hTERT (Fig. 2B).
Identification of MePR-0, MePR-2 and MePR-3 as epithelial and fibroblastic cell lines.
During our studies, a much lower number of cuboidal cells in primary fibroblastic amniotic
culture (Fig. 2A top left) was observed. These cells are completely different in terms of
morphology. Fibroblastic cells account for over 99% of cell populations, displaying a
fusiform shape similar to small fibroblasts (type I or fibroblastic). Less than 1% of cell
populations is made up of cuboidal cells having a more abundant cytoplasm and an epithelial
cuboidal shape (Fig. 2A top left asterisk). From primary amniocytes (Fig 2A top left and
right), we obtained three cell lines after infection: epithelial-type (MePR-0A) (Fig. 2A middle
left) and fibroblastic-type (MePR-2B and MePR-3A) (Fig. 2A middle right and bottom left,
respectively). While in MePR-3A cells (Fig. 2A bottom left) both morphologies
(fibroblastic/epithelial) are detectable, in MePR-2B clone epithelial-like cells are absent (Fig.
2A middle right).
Cell cycle and population doubling analysis of MePR cells.
To assess whether the three immortalized cell lines were able to grow indefinitely without
activation of senescence pathways, we calculated the population doubling at each passage
(61). While the primary cells stopped growing after the tenth week of culture (ten
duplications), the three MePR cell lines replicated for an extended period, with a constant
doubling time (Fig. 3A). The lower duplication number (about ten duplications) compared to
the 40-50 duplications reported in the literature (18,19,21) is due to the limited number of
CFCs derived from the samples.
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plu
ripo
tenc
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oi: 1
0.10
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cd.2
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0498
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his
artic
le h
as b
een
peer
-rev
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ed a
nd a
ccep
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for
publ
icat
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but
has
yet
to u
nder
go c
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d pr
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As shown in Fig. 3A, MePR-2B cells duplicated faster with an index of duplication nearly
twice that of MePR-0A. MePR-3A cells displayed an intermediate growth index. As
expected, primary amniocytes were slower to duplicate than MePR cell lines.
When the cell cycle was assessed in the three MePR cell lines, the percentage of G1, S and
G2 phases did not undergo major changes at the various passages, unlike primary amniocytes
in which G1 phase progressively increased up to 100% at the tenth week (Fig. 3B). Thus,
MePR cell lines are able to duplicate in culture for an extended period.
MePR karyotype analysis through G-banding and CGH array.
The immortalization of cultured cells frequently induces an abnormal number of
chromosomes (aneuploidy) or chromosome aberrations (78,82,83), especially in long-term
cell cultures. MePR-0A, MePR-2B and MePR-3A cells were therefore analyzed for their
chromosomal content and stability. None of the MePR cell lines observed in a PDL of 80
duplications (excluding the number of duplications before immortalization) displayed changes
in chromosome number. In particular, G-banding showed that MePR-2B cell line has a
normal karyotype (Fig. 4B). In contrast, MePR-3A displayed 100% metaphases carrying
karyotype 46, XX, add (19) (p13.3) (Fig. 4C). One chromosomal aberration was detected in
MePR-0A: 100% of the cells carried karyotype 46, XY, add (21), (Fig. 4A). Comparative
Genomic Hybridization (CGH Array) showed results similar to those obtained with G-
banding. While MePR-2B did not display chromosomal abnormalities (SI Fig. 1), MePR-3A
cells carried a deletion in the short arm telomeric region of chromosome 19 and duplication in
the sub-telomeric region of the same chromosome (p13.3-p.13.2) (Fig. 4C). MePR-0A
showed a duplication of the full chromosome 20 (about 4000 duplicated probes) (Fig. 4A),
suggesting that the whole duplicated chromosome 20 is translocated on chromosome 21. A
deletion of the short arm telomeric region of chromosome 17 (q21.3-q23.2) of about 13Mb
(797 deleted probes) (not shown in the G-banging experiment, Fig. 4A) was also detected.
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tenc
y (d
oi: 1
0.10
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his
artic
le h
as b
een
peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
ion,
but
has
yet
to u
nder
go c
opye
ditin
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d pr
oof
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Gene expression patterns in MePR cell lines.
To determine whether immortalization of MePR cells altered their gene expression profile, we
generated an array profile of each of the three MePR cell lines and compared them to the gene
expression profile of primary amniocytes (78,82,83), using Agilent Chip Two-color
MicroArray-Based Gene Expression Analysis. Patterson correlation was used on normalized
data for each of the three MePR cell lines (Fig. 5A). The correlation was similar for both
MePR-2B (0.80) and MePR-3A (0.81), but was lower for MePR-0A (0.55) (Fig.5A).
By applying a fold-change of Log2 ±2 to 41,000 genes, derived from array experiments, one
thousand genes are regulated (up-down regulated) in all three immortalized cell lines (SI
Table 3). Of these genes, 804 are common to all three MePR cell lines (234 up-regulated, 487
down-regulated) (Fig. 5C), while 83 genes belong to the so-called list of mixed genes,
common to only two of the three MePR cell lines (SI Table 4). Gene Ontology analysis of the
804 genes led to the identification of 12 clusters (divided into subgroups) (Fig. 5B and SI
Table 5). Only two clusters were statistically significant with 9.11x10-17
p-value for cell cycle
and 3.54x10-10
p-value for multicellular organismal development (SI Table 5). The free MeV
platform (Multiple Array Viewer) was used to obtain hierarchical clustering of the three
MePR cell lines compared to primary amniocytes. When heat maps were generated, MePR-
2B genes always cluster with MePR-3A. Conversely, MePR-0A forms a separate cluster (Fig.
6). These data have been deposited in NCBI's Gene Expression Omnibus (GEO) (84) and are
accessible through GEO Series accession number GSE37615
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37615).
Differentiative potential of MePR cell lines.
To investigate whether the MePR cell lines retain multipotency, we assessed the
differentiation potential for the two germ layers: mesoderm and ectoderm. MePR cells were
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hara
cter
istic
s of
plu
ripo
tenc
y (d
oi: 1
0.10
89/s
cd.2
012.
0498
)T
his
artic
le h
as b
een
peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
ion,
but
has
yet
to u
nder
go c
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d pr
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he f
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induced to form myocytes, osteocytes, chondrocytes and adipocytes (mesoderm) as well as
neural cells (ectoderm).
To assess neural differentiation, MePR cells were incubated in differentiation medium
(85,86). Primary amniocytes were used as control. Real-Time PCR and immunohistochemical
assays for βIII Tubulin (neuronal marker) and GFAP (glial marker) (52,86) were used to
analyze differentiation. In these settings, all three MePR cell lines were able to differentiate
into neural fate, with a clear increase in expression of βIII Tubulin and GFAP, consistent with
the modulation observed in primary amniocytes (Fig. 7 and 8A).
To test whether MePR cells were also able to differentiate into adipocytes, the cells were
incubated for 3 weeks in differentiating medium (85,86). The presence of PPARγ2, marker of
mature adipocytes, and PLIN2, marker of lipid accumulation (86,87), was tested by RT-PCR
showing the potential of MePR-2B and MePR-3A cell lines to undergo adipocytic
differentiation (Fig. 7). Moreover, Oil Red O staining assays for intracellular lipid vesicles
performed in MePR-2B cells fully confirmed qPCR results, as shown in Fig. 8B.
To verify the ability of MePR cells to differentiate into myocytes (mesoderm), cells were
incubated for three weeks with myogenic differentiating media (85,86). MyoD and Myogenin
(88-91) expression was analyzed by RT-PCR (Fig. 7). A higher increase of MyoD and
Myogenin was detectable in primary amniocytes, whereas a lower expression level was
observed for all MePR cell lines (Fig. 7). Furthermore, we tested the ability of MePR cells
and primary amniocytes to differentiate into osteoblasts and chondrocytes (mesoderm). To
test osteogenic differentiation, cells were incubated for three weeks with osteogenic
differentiating media (85,86). Osteocalcin and osteopontin (86) were used as markers for
differentiation. While all MePR cells and primary amniocytes were able to undergo
osteogenic differentiation, primary amniocytes and MePR-0A showed slightly stronger
expression of osteopontin compared to MePR-2B and MePR-3A (Fig. 7). Alizarin Red
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oi: 1
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his
artic
le h
as b
een
peer
-rev
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nd a
ccep
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for
publ
icat
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but
has
yet
to u
nder
go c
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staining assay confirmed the ability of MePR-2B to differentiate into osteocytes, showing
extracellular phosphate calcium deposits (Fig. 8B).
Finally, to demonstrate chondrogenic differentiation, cells were incubated for two weeks with
specific medium (67). Sox9, ColIIa1, ColXa1 (by Real-Time PCR) and Col2A1 (by Western
blot) were used as markers for differentiation (Fig. 7 and SI Fig. 2). Data shown confirm that
the ability of MePR-2B/MePR-3A to differentiate is greater than that of MePR-0A, compared
to primary amniocytes (Fig. 7 and SI Fig. 2). Alcian blue staining assay corroborated the
capability of MePR-2B to differentiate into chondrocytes by revealing extracellular collagen
fibers (Fig. 8B).
Analysis of hMSC- specific markers.
To evaluate at molecular level the multipotency potential of the novel MePR cell lines, the
expression level of typical hMSC markers was assessed by RT-PCR. In addition, two
important markers of pluripotent stem cells, Oct-4 and Nanog (1,92,93), were tested to show
the pluripotency of MePR cells.
As shown in Fig. 8C, all three MePR cell lines express the main markers of hMSCs (CD29,
CD44, CD73, CD90, CD105 and CD166) at a level comparable to that of primary amniocytes
(28-32). Furthermore, MePR cells do not express markers of hematopoietic cells such as
CD34+ (22). These results clearly suggest that MePR cells are similar to hMSCs. In addition,
Oct-4 and Nanog (1,92,93) were expressed in MePR-2B and MePR-3A as in primary
amniocytes (Fig. 8C). MePR-0A cells express Oct-4 in the same way as primary amniocytes,
but are not positive to Nanog (Fig. 8C).
Discussion
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oi: 1
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his
artic
le h
as b
een
peer
-rev
iew
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nd a
ccep
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for
publ
icat
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but
has
yet
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nder
go c
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d pr
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Currently, BM is the main source of hMSCs. However, BM aspiration is a painful and
invasive procedure. Moreover, the frequency and differentiation potential of BM-derived
hMSCs decreases significantly with age (94,95) and disease (38). The search for alternative
sources of hMSCs is therefore of paramount importance. Various tissues (96,97) have been
reported as potential sources for hMSC isolation. Among these, amniotic fluid, umbilical cord
or placenta cells offer key advantages for their accessibility, painless acquisition and low risk
of viral contamination. Furthermore, their ‘young’ biological age makes them particularly
appealing.
The embryonic cells of three germ layers were identified in amniotic fluid many years ago
(44,45,98). Though speculated for decades (99,100), the presence of mesenchymal cells in
amniotic fluid has only recently been demonstrated (42,101,102).
Amniotic fluid is known to contain a heterogeneous population of progenitor cells, including
mesenchymal, epithelial, hematopoietic, and trophoblast cells as well as embryonic-like stem
cells (46). However, the relatively low number of donations, the limited number of cell
duplications before senescence, and the variability of amniotic fluid cells have made it
difficult to analyze and compare data from different laboratories.
To standardize and obtain comparable data, we established three different cell lines from
amniotic fluid cells: MePR-0A (epithelial-like), MePR-2B (fibroblastic-like) and MePR-3A,
which contains both fibroblastic and epithelial cell types in a proportion similar to that of
primary cultures (>99:1, respectively). These novel MePR cell lines replicate exponentially
without obvious alteration of cell cycle progression, unlike primary amniocytes, which enter
senescence after ten weeks (about 10 duplications) (Fig. 3A).
Genetic alterations such as translocation, inversion, etc. (7,78,82,83) frequently occur during
immortalization. Analysis of MePR cell line karyotypes shows similar results, using both G-
banding and CGH Array: no chromosomal aberrations in MePR-2B cells, a single aberration
in MePR-3A cells, and two aberrations in MePR-0A cells (Fig.4 A, B and C). Thus, our
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his
artic
le h
as b
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ccep
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for
publ
icat
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but
has
yet
to u
nder
go c
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results indicate that MePR cell lines (and MePR-2B in particular) can be used in the place of
primary cells in different research settings.
Expression profile analysis likewise shows that the majority of genes are not modified
compared to primary cells, suggesting that any modification in gene expression is mainly due
to the reactivation of cell cycle progression. In accordance with this hypothesis,
GeneOntology evaluation shows 12 principal clusters (Fig. 5B and SI Table 5), only two of
which (cell cycle and multicellular organismal development, SI Table 5) are statistically
significant. The fact that the ‘cell cycle’ cluster is the most significant, strongly corroborates
the impact of the immortalization process in conformity with the data shown in Fig. 3A-B.
When analyzing the Heat Map image of all MePR cell lines, unlike primary amniocytes,
MePR-2B always clusters with MePR-3A (fibroblastic-like cells), (Fig.6). Particularly, the
Heat Map of mixed genes suggests that some of the regulated genes (84 genes) may be linked
to the different morphology of all three cell lines. For example, four members of the collagen
family (COL12A1, COL1A2, COL3A1 and COL4A6, SI Table 4) are down-regulated in
MePR-0A and up-regulated in MePR-2B/MePR-3A. Although these mixed regulations
remain to be better mined, it is tempting to speculate on their correlation with morphological
differences characterizing the different MePR cell lines.
MePR (0A-2B-3A) cell positivity to typical hMSC markers (CD29, CD44. CD73, CD90,
CD105 and CD166) (Fig. 8C), together with the expression of Oct-4 and Nanog, suggests that
MePR cells represent a novel human mesenchymal progenitor model (Multipotent Stem
Cells) with characteristics of pluripotency.
Of key importance is the ability of MePR cells to differentiate into tissues derived from
embryonic layers (endoderm, mesoderm, ectoderm). Although MePR-0A cells show a weaker
potential to differentiate (Fig. 7) and do not express Nanog (Fig. 8C), all MePR cell lines
display significant differentiation potential. The fact that MePR-0A cells carry two different
chromosome abnormalities might influence and account for their minor (but observed)
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differentiation potential. While MePR-2B and MePR-3A display similar neural, osteogenic,
chondrogenic and adipogenic differentiation potential compared to primary amniocytes,
myogenic differentiation is slightly reduced (Fig. 7). Myogenic differentiation requires cell
cycle block in G0 and this may account for the lower (but observed) capability of MePR cell
lines to differentiate as a result of the reactivation of cell cycle progression (103-106).
Moreover, given that primary amniocytes are a non-homogenous cell population, some of the
differences observed in the transcriptome experiments (Fig. 5B and SI Table 5) might also be
due to intrinsic differences between the primary cells from patient samples.
In summary, our data indicate that MePR cells display the multipotency potential and
differentiation rates of hMSCs, and thus represent a useful model to study both mechanisms
of differentiation and possible pharmacological approaches to induce selective differentiation.
In particular, despite not for clinical use, MePR-2B cells, which carry a bona fide normal
karyotype, might be used in basic stem cell research leading to the development of new
approaches for stem cell therapy and tissue engineering.
Acknowledgments.
We thank the ‘Cell Culture and Cytogenetics’ TIGEM Core, and the ‘Integrated Microscopy
Facilities’ of IGB-CNR, Naples, Italy, for support. This work was supported by EU: APO-
SYS (contract no. 200767), Blueprint (contract no. 282510), ATLAS (contract no. 221952);
Epigenomics Flagship Project ‘EPIGEN’ (MIUR-CNR); the Italian Association for Cancer
Research (AIRC no. 11812); Italian Ministry of University and Research
(PRIN_2009PX2T2E_004); PON002782; PON0101227. We thank C. Fisher for editing the
manuscript and Dr G. Minchiotti helpful suggestions.
Disclosure of potential conflicts of interest
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The authors declare that there is no conflict of interest
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tenc
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ccep
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Titles and legends to figures.
Figure 1: Collection and “Multi-Infection Program”.
(A) Schematic representation of the collection of amniotic fluid through an ultrasound-guided
transabdominal puncture for prenatal diagnosis; the resulting cells underwent a “Multi-
Infection Program”. (B) RT-PCR for hTERT, E6-E7 as indicated. GAPDH represents equal
loading.
Figure 2: Morphological analysis.
(A) Upper left: inverted microscope photograph of primary fibroblastoid amniocytes
(presence of an interspersed epitheloid cell between fibroblastic cells (see*)); (second images
on top left) primary epitheloid amniocytes; (top right) MePR-0A; (bottom left), MePR-2B;
(bottom right) MePR-3A (see*). (B) RT-PCR for hTERT, E6-E7 as indicated. GAPDH
represents equal loading.
Figure 3: Calculation of population doublings and cell cycle analysis.
(A) Calculation of population doublings and cell cycle analysis of the three cell lines
compared to primary amniocytes. (B) Cell cycle of the three immortalized cell lines compared
to primary amniocytes.
Figure 4: Chromosomal analysis.
G-banding and CGH Array experiments: (A) MePR-0A; (B) MePR-2B; (C) MePR-3A.
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Figure 5: Gene expression patterns.
(A): Scatter plot (Patterson Correlation) of MePR cell lines compared to primary amniocytes.
(B): GeneOntology of the total list of 804 genes divided into 12 clusters with similar
biological functions. (C) Venn diagrams of commonly regulated genes in MePR cells
compared to primary amniocytes (fold-change +/-2).
Figure 6: Hierarchical clustering.
Hierarchical cluster of MePR cell lines compared to primary amniocytes, using the MEV
platform.
Figure 7: Analysis of differentiation markers.
Real-Time PCR for neural, myogenic, adipogenic, osteogenic and chondrogenic
differentiation markers.
Figure 8: Differentiative potential and analysis of specific markers.
(A) Immunohistochemistry with anti-GFAP and anti-βIII Tubulin after neural differentiation.
(B) Staining assay for MePR-2B cell line. (C) RT-PCR analysis of typical hMSC and stem
cells markers (Oct-4 and Nanog). GAPDH represents equal loading.
Supplementary Fig. 1: CGH Array experiments: MePR-2B.
Supplementary Fig. 2: Western blot analysis of Col2A1 levels.
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Table 1: Primers RT-PCR
Gene Primers Annealing temp. °C No. cycles
hGAPD Forward caccatcttccaggagcgag
Reverse tcacgccacagtttcccgga
58 25
CD29 (ITGB1)
Forward gtagcaaaggaacagcagagaag
Reverse ctgaagtccgaagtaatcctcct
58 27
CD44 (Indian blood group) Forward cagggagaaaggggtagtgatac
Reverse tccaagtgagggactacaacag
58 27
CD73 (NT5E)
Forward ggaagaacaggactccaggac
Reverse gaaagaggacagaggcagagc 60 27
CD90 (THY1)
Foward gtgactgtgtatagtgccaccac
Reverse gagaagtcagggaagaggaagag
60 31
CD105 (ENG) Forward gggtctcaagaccaggaagtc
Reverse gtaccagagtgcagcagtgag
60 31
CD166 (Alcam)
Forward gtgtgcatgctagtaactgagg
Reverse gccatctggataactgtcttctg 58 27
Oct-4 (POU5F1) Forward gagaaggatgtggtccgagtg
Reverse gaaagggaccgaggagtacag
60 31
Nanog
Forward cagccccgattcttccaccagtccc
Reverse cggaagattcccagtcgggttcacc 62 31
CD34+
Forward cagacctttcaaccactagcac
Reverse ctcccctgtccttcttaaactc
62 31
hTERT Forward aggagctgacgtggaagatga
Reverse ttgcaacttgctccagacact
60 27
E6-E7
Forward ccaaagccactgtgtcctgaa
Reverse catcctcctcctctgagctgt 60 27
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d pr
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ectio
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inal
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44
Table 2: Real-Time PCR Primers
Gene Primers Marker
hGAPD Forward caccatcttccaggagcgag
Reverse tcacgccacagtttcccgga
Housekeeping Gene
Osteocalcin
Forward tgcagagtccagcaaaggtg
Reverse gatgtggtcagccaactcgtc Ostiogenic differentiation
PPARgamma2 Forward gctgaatccagagtccgctg
Reverse gcaaactcaaacttgggctcc
Adipogenic differentiation
MyoD
Forward agcactacagcggcgact
Reverse gcgactcagaaggcacgtc Myogenic differentiation
Myogenin
Forward cagcgaatgcagctctcaca
Reverse agttgggcatggtttcatctg
Myogenic differentiation
βIII Tubulin Forward agatgtacgaagacgacgaggag
Reverse gtatccccgaaaatataaacacaaa
Neurogenic differentiation
GFAP
Forward gtgactcatcctcttgaagatgc
Reverse acagatcccaccagtctgctcac Neuroglia differentiation
Page 44 of 51
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uman
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ench
ymal
pro
geni
tor
mod
el w
ith c
hara
cter
istic
s of
plu
ripo
tenc
y (d
oi: 1
0.10
89/s
cd.2
012.
0498
)T
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artic
le h
as b
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peer
-rev
iew
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nd a
ccep
ted
for
publ
icat
ion,
but
has
yet
to u
nder
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d pr
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Table 3: Patterson Correlations and Fold-change.
R2 Patterson
Correlation
No. genes
(Fold-change
+/-2)
% genes
(Fold-change
+/-0.5)
% genes
(Fold-change
+/-1)
% genes
(Fold-change
+/-2)
Amniocytes/MePR-0A 0.55 1128 genes 68.66% 91.72% 98.61%
Amniocytes/MePR-2B 0.80 1613 genes 61.62% 84.22% 95.25%
Amniocytes/MePR-3A 0.81 1620 genes 62.01% 84.25% 95.07%
Page 45 of 51
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mes
ench
ymal
pro
geni
tor
mod
el w
ith c
hara
cter
istic
s of
plu
ripo
tenc
y (d
oi: 1
0.10
89/s
cd.2
012.
0498
)T
his
artic
le h
as b
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peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
ion,
but
has
yet
to u
nder
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d pr
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Table 4: Gene mix
GeneSymbol GeneName
Genbank
Accession
UniGeneID
ABCB1 ATP-binding cassette, sub-family B (MDR/TAP), member 1 NM_000927 Hs.489033
BCHE Butyrylcholinesterase NM_000055 Hs.420483
BTBD11 BTB (POZ) domain containing 11 NM_152322 Hs.271272
C1orf186 chromosome 1 open reading frame 186 NM_001007544 Hs.514375
C1orf54 chromosome 1 open reading frame 54 NM_024579 Hs.91283
C2orf27 chromosome 2 open reading frame 27 NM_013310 Hs.469971
C9orf58 chromosome 9 open reading frame 58 NM_001002260 Hs.4944
CLEC4E C-type lectin domain family 4, member E NM_014358 Hs.236516
CLSTN2 Calsyntenin 2 NM_022131 Hs.158529
COL12A1 collagen, type XII, alpha 1 NM_004370 Hs.101302
COL1A2 collagen, type I, alpha 2 NM_000089 Hs.489142
COL3A1 collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant) NM_000090 Hs.443625
COL4A6 collagen, type IV, alpha 6 NM_033641 Hs.145586
CRLF1 cytokine receptor-like factor 1 NM_004750 Hs.114948
CTAGE5 CTAGE family, member 5 NM_203356 Hs.540038
DKFZP686A01247 hypothetical protein NM_014988 Hs.335163
DOPEY2 dopey family member 2 NM_005128 Hs.204575
ELOVL7 ELOVL family member 7, elongation of long chain fatty acids (yeast) NM_024930 Hs.274256
EPS8L2 EPS8-like 2 NM_022772 Hs.55016
FAM101A family with sequence similarity 101, member A NM_181709 Hs.432901
FKBP11 FK506 binding protein 11, 19 kDa NM_016594 Hs.119177
FLJ21986 hypothetical protein FLJ21986 NM_024913 Hs.189652
FLJ46266 FLJ46266 protein NM_207430 Hs.411600
FOLR1 folate receptor 1 (adult) NM_016725 Hs.73769
FOXA1 forkhead box A1 NM_004496 Hs.163484
FOXA3 forkhead box A3 NM_004497 Hs.36137
GAD1 glutamate decarboxylase 1 (brain, 67kDa) NM_013445 Hs.420036
GPC4 glypican 4 NM_001448 Hs.58367
GPR68 G protein-coupled receptor 68 NM_003485 Hs.8882
GPRIN2 G protein regulated inducer of neurite outgrowth 2 AB011086 Hs.447449
HAS3 Hyaluronan synthase 3 NM_005329 Hs.592069
Page 46 of 51
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uman
mes
ench
ymal
pro
geni
tor
mod
el w
ith c
hara
cter
istic
s of
plu
ripo
tenc
y (d
oi: 1
0.10
89/s
cd.2
012.
0498
)T
his
artic
le h
as b
een
peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
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but
has
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to u
nder
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HGD homogentisate 1,2-dioxygenase (homogentisate oxidase) NM_000187 Hs.616526
HOP homeodomain-only protein NM_139211 Hs.121443
HOXD1 homeobox D1 NM_024501 Hs.83465
HOXD10 homeobox D10 NM_002148 Hs.123070
HS6ST2 heparan sulfate 6-O-sulfotransferase 2 NM_001077188 Hs.385956
IBRDC2 IBR domain containing 2 NM_182757 Hs.148741
KMO Kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) NM_003679 Hs.409081
KRT7 keratin 7 NM_005556 Hs.411501
LAMA3 laminin, alpha 3 NM_198129 Hs.436367
LGR4 leucine-rich repeat-containing G protein-coupled receptor 4 NM_018490 Hs.502176
LINCR
likely ortholog of mouse lung-inducible Neutralized-related C3HC4 RING domain
protein BC012317 Hs.149219
LOC441774 similar to 40S ribosomal protein S4, Y isoform 1 XR_018247 Hs.647382
LOC652524 similar to Keratin, type II cytoskeletal 8 (Cytokeratin-8) (CK-8) (Keraton-8) (K8) XR_019369 Hs.647670
LQK1 LQK1 hypothetical protein short isoform AY030238 Hs.552649
MAL Mal, T-cell differentiation protein NM_002371 Hs.80395
MEGF6 multiple EGF-like-domains 6 NM_001409 Hs.593645
MGC16291 hypothetical protein MGC16291 BC007394 Hs.55977
MXRA5 matrix-remodelling associated 5 NM_015419 Hs.369422
MYBPC2 myosin binding protein C, fast type NM_004533 Hs.85937
NMNAT3 nicotinamide nucleotide adenylyltransferase 3 NM_178177 Hs.208673
NR2F1 nuclear receptor subfamily 2, group F, member 1 NM_005654 Hs.519445
PDLIM5 PDZ and LIM domain 5 NM_006457 Hs.480311
PHLDA1 pleckstrin homology-like domain, family A, member 1 NM_007350 Hs.602085
PKP3 Plakophilin 3 NM_007183 Hs.534395
PXDNL Peroxidasin homolog-like (Drosophila) NM_144651 Hs.444882
QPRT
Quinolinate phosphoribosyltransferase (nicotinate-nucleotide pyrophosphorylase
(carboxylating))
NM_014298 Hs.513484
RAB31 RAB31, member RAS oncogene family NM_006868 Hs.99528
RASGRP1 RAS guanyl releasing protein 1 (calcium and DAG-regulated) NM_005739 Hs.591127
RNASET2 ribonuclease T2 NM_003730 Hs.529989
SERTAD4 SERTA domain containing 4 AK021425 Hs.600545
SOCS2 Suppressor of cytokine signaling 2 NM_003877 Hs.485572
SPECC1 sperm antigen with calponin homology and coiled-coil domains 1 NM_152904 Hs.431045
Page 47 of 51
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nov
el h
uman
mes
ench
ymal
pro
geni
tor
mod
el w
ith c
hara
cter
istic
s of
plu
ripo
tenc
y (d
oi: 1
0.10
89/s
cd.2
012.
0498
)T
his
artic
le h
as b
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peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
ion,
but
has
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to u
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STYK1 serine/threonine/tyrosine kinase 1 NM_018423 Hs.24979
TNFSF4
tumor necrosis factor (ligand) superfamily, member 4 (tax-transcriptionally activated
glycoprotein 1, 34kDa)
NM_003326 Hs.181097
TNNI3 troponin I type 3 (cardiac) NM_000363 Hs.644596
TRIM58 tripartite motif-containing 58 NM_015431 Hs.323858
TSPAN2 Tetraspanin 2 NM_005725 Hs.310458
VAMP5 vesicle-associated membrane protein 5 (myobrevin) NM_006634 Hs.172684
XIST X (inactive)-specific transcript NR_001564 Hs.529901
Page 48 of 51
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nov
el h
uman
mes
ench
ymal
pro
geni
tor
mod
el w
ith c
hara
cter
istic
s of
plu
ripo
tenc
y (d
oi: 1
0.10
89/s
cd.2
012.
0498
)T
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artic
le h
as b
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peer
-rev
iew
ed a
nd a
ccep
ted
for
publ
icat
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but
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Table 5: GeneOntology
GeneOntology Term Number
of Gene
% p-Value
Cell Cycle: Enrichment Score: 11.141697182442861
GO:0007049~cell cycle 74 12.29 9.11E-17
GO:0022402~cell cycle process 61 10.13 3.90E-16
GO:0051301~cell division 34 5.64 6.15E-10
GO:0006996~organelle organization 69 11.46 1.24E-04
Multicellular Organismal Development: Enrichment Score: 7.819837969750425
GO:0048856~anatomical structure development 137 22.75 3.54E-10
GO:0007275~multicellular organismal development 142 23.58 6.35E-08
GO:0048869~cellular developmental process 95 15.78 1.54E-07
Positive Regulation of Cellular Process: Enrichment Score: 4.249349200444362
GO:0048522~positive regulation of cellular process 98 16.27 8.94E-07
GO:0048518~positive regulation of biological process 100 16.61 1.82E-05
GO:0009893~positive regulation of metabolic process 44 7.30 0.01096111
Negative Regulation of Cellular Process: Enrichment Score: 2.6634279914650625
GO:0048519~negative regulation of biological process 87 14.45 1.82E-04
GO:0048523~negative regulation of cellular process 79 13.12 5.25E-04
GO:0009892~negative regulation of metabolic process 33 5.48 0.107123714
Transport: Enrichment Score: 1.3989250015664927
GO:0051051~negative regulation of transport 11 1.82 0.012999239
GO:0032879~regulation of localization 29 4.81 0.04336031
GO:0051050~positive regulation of transport 12 1.99 0.112775484
Cell Activation: Enrichment Score: 1.231343365680732
GO:0001775~cell activation 17 2.82 0.0263634
GO:0045321~leukocyte activation 15 2.49 0.027689508
GO:0002682~regulation of immune system process 16 2.65 0.277110672
Page 49 of 51
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pro
geni
tor
mod
el w
ith c
hara
cter
istic
s of
plu
ripo
tenc
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oi: 1
0.10
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cd.2
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-rev
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Cellular Component Assembly: Enrichment Score: 0.7623714251719184
GO:0022607~cellular component assembly 38 6.31 0.073980001
GO:0043933~macromolecular complex subunit organization 28 4.65 0.234396804
GO:0070271~protein complex biogenesis 20 3.32 0.297728016
Cell Motion: Enrichment Score: 0.7489186453041561
GO:0050900~leukocyte migration 6 0.99 0.037814689
GO:0006928~cell motion 19 3.15 0.290159471
GO:0051674~localization of cell 13 2.15 0.303398944
GO:0048870~cell motility 13 2.15 0.303398944
Reproductive Process: Enrichment Score: 0.5711049265228876
GO:0022414~reproductive process 34 5.64 0.058502169
GO:0007276~gamete generation 16 2.65 0.310086335
GO:0048609~reproductive process in a multicellular organism 18 2.99 0.424319439
GO:0032504~multicellular organism reproduction 18 2.99 0.424319439
GO:0019953~sexual reproduction 17 2.82 0.427006922
Regulation of Biological Process: Enrichment Score: 0.5090101518246354
GO:0050789~regulation of biological process 242 40.19 0.204953949
GO:0050794~regulation of cellular process 232 38.53 0.222521091
GO:0019222~regulation of metabolic process 116 19.26 0.651541487
Regulation of Response to Stimulus: Enrichment Score: 0.299209320641796
GO:0002682~regulation of immune system process 16 2.65 0.277110672
GO:0002684~positive regulation of immune system process 9 1.49 0.524547969
GO:0048583~regulation of response to stimulus 16 2.65 0.555836332
GO:0048584~positive regulation of response to stimulus 7 1.16 0.786642353
Cellular Metabolic Process: Enrichment Score: 0.06469501372334059
GO:0043170~macromolecule metabolic process 183 30.39 0.67858115
GO:0044238~primary metabolic process 215 35.71 0.881158533
GO:0044237~cellular metabolic process 205 34.05 0.893104052
GO:0009058~biosynthetic process 105 17.44 0.907965455
GO:0006807~nitrogen compound metabolic process 106 17.60 0.979259297
Page 50 of 51
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nov
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mes
ench
ymal
pro
geni
tor
mod
el w
ith c
hara
cter
istic
s of
plu
ripo
tenc
y (d
oi: 1
0.10
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cd.2
012.
0498
)T
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artic
le h
as b
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peer
-rev
iew
ed a
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ccep
ted
for
publ
icat
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has
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Page 51 of 51
Stem
Cel
ls a
nd D
evel
opm
ent
MeP
R: a
nov
el h
uman
mes
ench
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pro
geni
tor
mod
el w
ith c
hara
cter
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s of
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ripo
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cd.2
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0498
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-rev
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om th
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.
