Assessment of the Intrinsic Pluripotency of
Mesoderm-Derived Stem Cells from Different Niches
Joery De Kock 1, Mehdi Najar 2, Jennifer Bolleyn 1, Feras Al Battah 1, Gordana Raicevic 2,
Olivier Govaere 3, Steven Branson 1, Smita Jagtap 4, John Antonydas Gaspar 4, Tania Roskams 3,
Agapios Sachinidis 4, Laurence Lagneaux 2, Tamara Vanhaecke 1, and Vera Rogiers 1
1 Dept.
of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel (VUB), Brussels, Belgium; 2 Laboratory of
Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Brussels, Belgium; 3 Dept. of Morphology
and Molecular Pathology, Katholieke Universiteit Leuven (KUL), University Hospital Leuven, Leuven, Belgium; 4 Center of
Physiology, Institute of Neurophysiology, University of Cologne, Cologne, Germany
Summary
During the last decade, human adult stem cells have become an attractive cell source for tissue engineering
and for the development of human-relevant alternative in vitro toxicity models. However, distinct stem cell
populations show differences in function and differentiation potential. Whether those differences are the result
of cell culture, donor variability, or intrinsic properties remains unclear. Therefore, comparative transcriptome
analyses were used to determine which of the commonly used human mesoderm-derived stem cell populations,
obtained from four distinct niches, displays the highest intrinsic cell plasticity compared to human
embryonic stem cells (hESC): adipose-tissue derived stromal cells (hADSC), bone marrow-derived stromal
cells (hBMSC), skin-derived precursor cells (hSKP), or Wharton’s jelly-derived mesenchymal stem cells (hWJ).
Our data suggest that, compared to hESC gene expression profiles, the intrinsic cell plasticity, defined by
the expression of pluripotency genes and enrichment of biological functions that are involved in embryogenesis
and organogenesis, is least prominent in hADSC and most prominent in hSKP, clearly indicating the high
multipotent character of the latter.
Keywords: bone marrow stromal cells, skin-derived precursor cells, adipose tissue-derived stromal cells,
Wharton’s jelly, pluripotency
1 Introduction
For more than a decade, human tissue-specific adult stem cells
were known for their capacity to differentiate along their lineage
of origin. However, over the recent years, numerous reports in
the field of stem cell biology demonstrated that adult stem cells
possess greater plasticity than what was previously dictated by
established paradigms of embryonic development (De Kock et
al., 2009, 2011; Al Battah et al., 2011). Indeed, “multipotent
adult stem cells” have been isolated from various sources, including brain, skin, adipose tissue, bone marrow, skeletal muscle, and umbilical cord blood (De Kock et al., 2008, 2009; Al
Battah et al., 2011; Najar et al., 2010; De Bruyn et al., 2011). In
addition, several methodologies have been developed to differentiate these adult stem cells in vitro across germinal boundaries,
a process commonly referred to as “transdifferentiation” (De
Kock et al., 2009, 2011; Al Battah et al., 2011). Due to their plasticity, human adult stem cells are today an attractive cell source
for tissue engineering and for developing human-relevant alternative in vitro models for toxicity studies normally performed
on animals, thereby leading to the reduction, refinement, and/or
replacement of currently used animal-based models (Lee et al.,
2011; Scanu et al., 2011). However, differences in function and
differentiation potential exist between distinct stem cell popuAltex Proceedings, 1/12, Proceedings of WC8
lations. Whether those differences are due to donor variation,
cell culture, or intrinsic properties remains unclear (Shafiee et
al., 2011). Therefore, a first step in the process of generating
the human target cell of interest is the evaluation of the intrinsic pluripotency of the investigated stem cell population. In the
present study, an unambiguous characterization and comparison of four human mesodermal-derived stem cell populations
are presented. Briefly, transcriptome analyses are performed on
human bone marrow-derived stromal cells (hBMSC), adipose
tissue-derived stromal cells (hADSC), skin-derived precursor
cells (hSKP), and Wharton’s jelly-derived mesenchymal cells
(hWJ), and the results are compared to gene expression profiles of human embryonic stem cells (hESC). Special attention
is paid to their differential expression of pluripotency genes and
the identification of enriched developmental functions that are
involved in embryogenesis and organogenesis.
2 Materials and methods
Isolation and cultivation of hADSC
Plastic surgical “waste material” (i.e., abdominal fat; ♀) was
obtained in cooperation with the Department Plastic Surgery of
the UZ-Brussels (Belgium) and the ATLAS Kliniek (Belgium)
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De Kock et al.
upon informed consent and approved by the ethical commission of the UZ-Brussels. The median age of the donors was
39 years (♀/♂; range 32-49). hADSC were isolated and subcultivated as previously described (Al Battah et al., 2011). Briefly,
±125 g of processed adipose tissue was incubated for 90 minutes
at 37°C in dissociation medium (1:1) consisting of 1% (v/v) bovine serum albumin (BSA, Sigma-Aldrich, Bornem, Belgium)
and 1 mg/ml collagenase A (Roche Applied Science, Vilvoorde,
Belgium) in phosphate buffered saline (PBS). After two filtration steps, the filtrate was carefully brought on top of 15 ml of
Histopaque®-1077 (Sigma-Aldrich, Bornem, Belgium). Upon
centrifugation for 20 minutes at 1000 g (4°C), the top layer was
removed and the hADSC were collected in 50 ml PBS/BSA
(1%). Typically 5-20 x 107 viable cells were obtained per 250 g
of processed adipose tissue. The isolated hADSC were then cultured as a monolayer in hADSC growth medium, consisting of
Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Brainel’Alleud, Belgium) supplemented with 10% (v/v) foetal bovine
serum (FBS) (Perbio Hyclone, Erembodegem, Belgium), 50
µg/ml streptomycin sulphate (Sigma-Aldrich, Bornem, Belgium), 7.33 IU/ml benzyl penicillin (Continental Pharma, Diegem, Belgium), and 2.5 µg/ml fungizone (Life Technologies,
Merelbeke, Belgium). Cell cultures were incubated at 37°C in
a 5% (v/v) CO2, humidified atmosphere. Growth media was
changed every 3 days.
Isolation and cultivation of hBMSC
Bone marrow was aspirated by sternal puncture in healthy
volunteers or obtained by needle aspiration from iliac crest of
bone marrow transplant donors as previously described (Najar
et al., 2010). The median age of the donors was 26 years (♀/♂;
range 3-57). Informed consent was obtained from all donors.
The ethic committee of the Institut Jules Bordet approved the use
of the tissue material for this study. Briefly, mononuclear cells
(MNC) were isolated from bone marrow aspirates by density
gradient centrifugation (Linfosep, Biomedics, Madrid, Spain)
and washed in HBSS medium (Lonza, Braine-l’Alleud, Belgium). MNC were seeded at a cell density of 2 × 104 cells/cm2
in low glucose DMEM (DMEM-LG, Lonza, Braine-l’Alleud,
Belgium) supplemented with 15% (v/v) heat-inactivated FBS,
2 mM L-glutamine and 0.5% (v/v) antibiotic/antimycotic solution (all from Life Technologies, Merelbeke, Belgium). Cells
were incubated at 37°C in a 5% (v/v) CO2-enriched, humidified
atmosphere, cultured up to 90% confluency, trypsinized (Tryple
Select solution, Lonza, Braine-l’Alleud, Belgium), centrifuged,
and subcultured at lower density (5000 cells/cm2) for all subsequent passages.
Isolation and cultivation of hSKP
hSKP were isolated and subcultivated as previously described
(De Kock et al., 2011). The median age of the donors was
3 years (♂; range 1-3). Briefly, freshly collected human foreskin samples were incubated with 25 ml of 0.2 mg/ml Liberase
DH solution (Roche Applied Science, Vilvoorde, Belgium)
and incubated for 20 h at 4°C. Next, the epidermis was removed and the tissue was incubated at 37°C for another 1020 minutes depending on the sample size. After processing the
532
samples, typically 5-15 x 106 viable cells were obtained per
5-8 cm2 foreskin. For cultivation, a cell density of 20 000 cells/
cm2 was applied. Growth medium for hSKP consisted of DMEM
+ GLUTAMAX/F12 Nutrient Mixture (3:1) (all from Life Technologies, Merelbeke, Belgium) supplemented with 7.33 IU/
ml benzyl penicillin (Continental Pharma, Diegem, Belgium),
50 μg/ml streptomycin sulphate (Sigma-Aldrich, Bornem, Belgium), 2.5 μg/ml fungizone, 2% (v/v) B27 Supplement (all from
Life Technologies, Merelbeke, Belgium), 40 ng/ml basic fibroblast growth factor (FGF)-2 and 20 ng/ml epidermal growth factor (EGF) (both from Promega, Leiden, The Netherlands). Cell
cultures were incubated at 37°C in a 5% (v/v) CO2, humidified
atmosphere. Growth media was refreshed every 2-3 days.
Isolation and cultivation of hWJ
After informed consent from the mothers, umbilical cords
(♀/♂) were collected after full-term deliveries. They were processed according to the protocol of De Bruyn et al. (2011). More
specifically, MSC were isolated from Wharton’s jelly (WJ)
without enzyme digestion or dissection. The procedure is based
only on the migratory and plastic adhesive properties of MSC.
Briefly, umbilical cord segments of 5-10 cm were cut longitudinally and plated for 5 days in an appropriate culture medium
(DMEM-LG, Lonza, Braine-l’Alleud, Belgium). After removing the cord segments, the culture was pursued until subconfluency. Cell cultures were incubated at 37°C in a 5% (v/v) CO2,
humidified atmosphere. After 48 h, non-adherent cells were removed by washing, and the medium was changed twice a week.
When subconfluence (80-90%) was achieved, adherent cells
were harvested after detachment by 10 min incubation with
TrypLE Select solution (Lonza, Braine-l’Alleud, Belgium) and
expanded by replating at a lower density (1,000 cells/cm2).
Isolation of RNA and reverse transcriptase-polymerase
chain reaction (PCR)
For qPCR analysis, total RNA was extracted from all samples
using the GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma-Aldrich, Bornem, Belgium) according to the
manufacturer’s instructions. The isolated RNA was quantified
at 260 nm using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, USA). Total RNA was reverse transcribed
into cDNA using iScript™ cDNA Synthesis Kit (BioRad, Nazareth, Belgium) followed by cDNA purification with the Genelute PCR clean up kit (Sigma-Aldrich, Bornem, Belgium). For
microarray analysis, the RNA was extracted using trizol/chloroform and purified with RNeasy mini columns as recommended
by the manufacturer’s instruction (Qiagen, Hilden, Germany).
Quantitative real-time PCR (qPCR)
cDNA products were used for quantitative amplification of
the target genes. The primers used in this study were listed in
Tab. 1. All samples were done in duplicate and each run included two negative controls (NTC) and a serial dilution of a pooled
cDNA mix from all samples to estimate the qPCR efficiency.
The qPCR reaction mix consisted of 12.5 µl TaqMan Universal Master Mix (Applied Biosystems, Halle, Belgium), 1.25 µl
20X Assay-on-Demand Mix (Applied Biosystems, Halle, BelAltex Proceedings, 1/12, Proceedings of WC8
De Kock et al.
gium) and 2 µl of cDNA in a 25 µl volume adjusted with DNase/
RNase-free water. qPCR conditions, using the iQ5™ Bio-Rad
system (BioRad, Nazareth, Belgium), were as follows: incubation for 10 min at 95°C, followed by 40 cycles of 15 s denaturation at 95°C, annealing for 1 min at 60°C (BioRad, Nazareth,
Belgium).
qPCR data analysis
qPCR efficiency was estimated by the iQ5™ Optical System
Software (Version 2), and the data were only used when the
calculated PCR efficiency ranged from 0.85 to 1.15. Moreover,
for selecting reliable reference genes to normalize the qPCR
data, we first evaluated the expression stability of six candidate
reference genes: glyceraldehyde 3-phosphate dehydrogenase
(GAPDH), beta-2-microglobulin (B2M), hydroxy-methylbilane
synthase (HMBS), 18S, beta-actin (ACTB) and ubiquitin C
(UBC). According to geNorm®, the optimal number of reference targets to be used in this experiment was 5 (V<0.15). As
such, B2M, UBC, 18S, HMBS and GAPDH were selected as the
most stable reference genes in all 4 stem cell populations using
qbasePLUS® software (geNorm®, Biogazelle, Gent, Belgium).
Tab. 1: Gene expression assays used for characterization of
mesoderm-derived stem cells
The listed gene expression assays are used to determine
the most stable reference genes and to investigate the intrinsic
pluripotency of mesoderm-derived stem cells.
Abbreviations: Applied Biosystems (AB); beta-2-microglobulin
(B2M); beta-actin (ACTB); base pair (bp); glyceraldehyde-3phosphate dehydrogenase (GAPDH); hydroxy-methylbilane
synthase (HMBS); Kruppel-like factor 4 (KLF4); Nanog
homeobox (NANOG); POU class 5 homeobox 1 (POU5F1);
ribosomal RNA S18 (18S); secreted frizzled-related
protein 1 (SFRP1); secreted frizzled-related protein 2 (SFRP2);
signal transducer and activator of transcription 3 (STAT3);
SRY (sex determining region Y)-box 2 (SOX2); ubiquitin C (UBC);
v-myc myelocytomatosis viral oncogene homolog (MYC).
Gene
18S
Assay-on-
Demand ID
Amplicon
length (bp)
Source
Hs99999901_s1
187
AB
B2M
Hs99999907_m1
75
AB
HMBS
Hs00609296_g1
69
AB
ACTB
GAPDH
Hs99999903_m1
171
Hs99999905_m1
122
Hs00358836_m1
110
Hs02387400_g1
109
SFRP1
Hs00610060_m1
130
SOX2
Hs01053049_s1
91
KLF4
MYC
Hs99999003_m1
POU5F1
Hs00999632_g1
NANOG
SFRP2
STAT3
UBC
Hs00293258_m1
Hs01047580_m1
Hs00824723_m1
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65
77
129
87
71
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
Thereafter, to compare the relative mRNA expression levels
of the target genes (Tab. 1), results were expressed as the fold
changes normalized against the geometric means of all 5 reference gene mRNAs using qbasePLUS® software (Biogazelle,
Gent, Belgium). Statistical analyses were performed using a
one-way ANOVA and Student’s t-test. The significance level
was set at 0.05.
Microarray data analysis
All reagents and instrumentation pertaining to oligonucleotide
microarrays were procured from Affymetrix (Affymetrix, Santa Clara, CA, USA; http://www.affymetrix.com). Total RNA
(100 ng) was used for amplification and in vitro transcription
using the Genechip 3’ IVT Express Kit as per the manufacturer’s instructions (Affymetrix). The amplified RNA (aRNA)
was purified with magnetic beads and 15 μg Biotin-aRNA was
fragmented with fragmentation reagent. 12.5 μg fragmented
aRNA was hybridized to Affymetrix Human Genome U133
plus 2.0 arrays along with a hybridization cocktail solution
and then placed in a Genechip Hybridization Oven-645 (Affymetrix) rotating at 60 rpm at 45 ºC for 16 h. After incubation, arrays were washed on a Genechip Fluidics Station-450
(Affymetrix) and stained with the Affymetrix HWS kit as per
manufacturer’s protocols. The chips were scanned with an Affymetrix Gene-Chip Scanner-3000-7G and the quality control
matrices were confirmed with the Affymetrix GCOS software
following the manufacturer’s guidelines. Background correction, summarization, and normalization were done with
Robust Multi-array Analysis (Irizarry et al., 2003). The raw
dataset was normalized with the quantile normalization (Bolstad et al., 2003) method execuTab. with R (Affy)-package
(Gautier et al., 2004) carried out at probe feature level. Probe
sets that were detected to be present were selected; those absent were eliminated. MAS5 Expression Summary (Pepper et
al., 2007) was used to detect present calls. Publicly available
datasets were obtained from the Gene Expression Omnibus
(GEO) database. The microarray data “Mesenchymal Stromal
Cells of Different Donor Age”, “Transcriptome analysis of human Wharton’s jelly stem cells” and “Efficient Generation of
Transgene-Free Induced Pluripotent Stem Cells from Normal
and Neoplastic Bone Marrow and Cord Blood Mononuclear
Cells” are accessible through GEO series accession numbers
GSE12274, GSE20126 and GSE26672, respectively. These
datasets were used for comparative gene expression analysis
with own hSKP and hADSC data. For determination of differential gene expression, output data files were analyzed using
GeneSpring GX v11.5 software (Agilent Technologies, Waldbronn, Germany). Genes with a fold change >2 and p-value
<0.05 were selected as putative candidate genes and further
used for functional analysis and hierarchical clustering by
Ward’s method (Ward, 1963). Functional analyses were performed using Ingenuity Pathways Analysis (IPA, version SEP
2011; Ingenuity Systems) using Benjamini-Hochberg (B-H)
multiple testing corrected p-values to identify enriched basic
functional developmental annotations (fold change >2; B-H
p-value <0.05).
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De Kock et al.
tissue,” “development of organ,” “developmental process of
organism,” “differentiation of embryonic cells,” and “organogenesis” was observed for hSKP and hWJ but not for hADSC
(Tab. 2). In contrast, a significant enrichment of increased genes
involved in the “development of organ,” “developmental process of organism,” and “organogenesis” was found in hBMSC
compared to hADSC and hWJ (Tab. 2). Furthermore, a significantly increased gene expression was observed in hBMSC for
the “differentiation of embryonic cells” compared to hADSC
(Tab. 2).
3 Results
3.1 Mesoderm-derived stem cells
differ significantly in enrichment of basic
developmental functions
To identify basic developmental functions that are significantly
enriched, transcriptome profiles of hADSC, hBMSC, hSKP,
and hWJ were mutually compared. In comparison to hBMSC,
a significantly (B-H p-value <0.05; fold change >2) increased
expression of genes involved in the “development of embryonic
Tab. 2: hSKP show a significant enrichment of basic developmental functions that are involved in embryogenesis and
organogenesis
Functional analyses are performed using Ingenuity Pathways Analysis (IPA, version SEP 2011; Ingenuity Systems) using Benjamini-Hochberg
(B-H) multiple testing corrected p-values (B-H p-value <0.05) to identify enriched basic functional developmental annotations. Only significantly different expressed genes are used to identify the pathways (fold change >2; p-value <0.05). Significantly enriched biological functions (B-H p-value
<0.05) are marked by a dark grey color. The total number of genes involved in the function are displayed between brackets. The ratio represents
the number of genes increased in the function divided by the total number of genes involved in the function (%).
Abbreviations: Benjamini-Hochberg multiple testing corrected p-values (B-H p-value); human adipose-derived stromal cell (hADSC); human bone
marrow-derived stromal cell (hBMSC); human skin-derived precursor cell (hSKP); human Wharton's jelly-derived mesenchymal stem cell (hWJ).
hADSC vs hBMSC
development of embryonic tissue (416)
development of organ (1623)
developmental process of organism (2785)
differentiation of embryonic cells (189)
organogenesis (1657)
-LOG
(B-H p-value)
3.67E-01
0.00E+00
0.00E+00
3.67E-01
0.00E+00
development of embryonic tissue (416)
development of organ (1623)
developmental process of organism (2785)
differentiation of embryonic cells (189)
organogenesis (1657)
-LOG
(B-H p-value)
0.00E+00
0.00E+00
0.00E+00
0.00E+00
0.00E+00
development of embryonic tissue (416)
development of organ (1623)
developmental process of organism (2785)
differentiation of embryonic cells (189)
organogenesis (1657)
-LOG
(B-H p-value)
6.61E+00
3.95E+00
3.16E+00
1.68E+00
4.14E+00
development of embryonic tissue (416)
development of organ (1623)
developmental process of organism (2785)
differentiation of embryonic cells (189)
organogenesis (1657)
-LOG
(B-H p-value)
7.97E+00
8.63E+00
1.10E+01
3.44E+00
8.63E+00
ratio (%)
2.64
0.00
0.00
3.17
0.00
hBMSC vs hSKP
ratio (%)
0.00
0.00
0.00
0.00
0.00
hSKP vs hBMSC
ratio (%)
17.31
10.78
8.87
13.23
10.80
hWJ vs hBMSC
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ratio (%)
18.03
12.38
10.63
15.87
12.31
hADSC vs hSKP
-LOG
(B-H p-value)
0.00E+00
0.00E+00
0.00E+00
0.00E+00
0.00E+00
ratio (%)
0.00
0.00
0.00
0.00
0.00
hBMSC vs hADSC
-LOG
(B-H p-value)
0.00E+00
3.58E+00
6.98E+00
1.58E+00
4.13E+00
ratio (%)
0.00
20.39
18.60
23.28
20.58
hSKP vs hADSC
-LOG
(B-H p-value)
2.86E+00
6.16E+00
3.78E+00
0.00E+00
6.32E+00
ratio (%)
15.14
12.51
9.87
0.00
12.49
hWJ vs hADSC
-LOG
(B-H p-value)
0.00E+00
4.77E+00
1.00E+01
2.25E+00
5.20E+00
ratio (%)
0.00
24.40
22.48
27.51
24.50
hADSC vs hWJ
-LOG
(B-H p-value)
0.00E+00
0.00E+00
0.00E+00
0.00E+00
0.00E+00
ratio (%)
0.00
0.00
0.00
0.00
0.00
hBMSC vs hWJ
-LOG
(B-H p-value)
0.00E+00
4.13E+00
2.63E+00
0.00E+00
4.29E+00
ratio (%)
0.00
8.38
6.61
0.00
8.39
hSKP vs hWJ
-LOG
(B-H p-value)
3.20E+00
1.06E+00
6.80E-01
0.00E+00
1.09E+00
ratio (%)
17.55
11.71
9.87
0.00
11.71
hWJ vs hSKP
-LOG
(B-H p-value)
0.00E+00
0.00E+00
2.60E+00
0.00E+00
0.00E+00
ratio (%)
0.00
0.00
26.00
0.00
0.00
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De Kock et al.
Fig. 1: Microarray analysis shows increased mRNA expression in hSKP of genes involved in the pluripotency of stem cells
Heat map showing the relative expression levels of 47 pluripotency genes in 3 independent hESC, 3 independent hADSC, 7 independent
hSKP, 7 independent hBMSC, and 6 independent hWJ cell isolates. Relative expression levels are color-coded as per color key. Ward’s
hierarchical clustering shows a closer proximity between hBMSC, hWJ and hADSC whereas a closer similarity is observed for hSKP and
hESC gene expression profiles.
3.2 Characteristics of mesoderm-derived
stem cells determined by expression patterns
of pluripotency genes
Expression patterns of pluripotency genes were used to characterize the four mesoderm-derived stem cell populations and
to evaluate their intrinsic “stemness” properties on a molecular
level. Hierarchical clustering was carried out to give an overview of the differentially expressed transcripts known to be involved in pluripotency (Fig. 1). In order to validate the microarAltex Proceedings, 1/12, Proceedings of WC8
ray results, more accurate qPCR analyses were performed (Fig.
2; Tab. 3). More specifically, microarray analyses showed a
significantly increased mRNA expression in hSKP for genes related to pluripotency as compared to the other three mesodermderived stem cell types, resulting in a closer proximity between
hBMSC, hWJ, and hADSC than hSKP, as shown by Ward’s hierarchical clustering (Fig. 1). Moreover, the mRNA expression
profiles of hSKP showed a higher similarity with hESC, resulting in a closer relatedness between both cell types. Indeed, the
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De Kock et al.
Fig. 2: qPCR confirms increased gene expression of pluripotency genes in hSKP
Fold changes of genes involved in the pluripotency of stem cells are determined for all four mesoderm-derived stem cell types.
*Significantly increased or decreased mRNA expression between mutually compared stem cell types (fold change >2; p-value <0.05)
Abbreviations: Kruppel-like factor 4 (KLF4); Nanog homeobox (NANOG); POU class 5 homeobox 1 (POU5F1); secreted frizzled-related
protein 1 (SFRP1); secreted frizzled-related protein 2 (SFRP2); signal transducer and activator of transcription 3 (STAT3); SRY (sex
determining region Y)-box 2 (SOX2); v-myc myelocytomatosis viral oncogene homolog (MYC).
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pluripotency genes secreted frizzled related protein 2 (SFRP2),
deleted in azoospermia-like (DAZL), SFRP1, semaphorin 3A
(SEMA3A), nuclear receptor subfamily 6, group A, member
1 (NR6A1), growth factor receptor-bound protein 7 (GRB7),
NR5A2, telomerase reverse transcriptase (TERT), transcription
factor CP2-like 1 (TFCP2L1), FGF4, interferon induced transmembrane protein 1 (IFITM1), and left-right determination
factor 2 (LEFTY2) were found to be significantly (fold change
>2, p-value <0.05) increased in hSKP (Fig. 1). SFRP2 was, in
addition, significantly increased in hADSC, whereas frizzled
5 (FZD5), and interleukin 6 signal transducer (IL6ST) and SEMA3A were significantly increased and decreased, respectively, in hBMSC. More in-depth qPCR analyses confirmed that
SFRP1 was significantly increased in hSKP (Fig. 2; Tab. 3). Additionally, a significantly higher gene expression of SRY (sex
determining region Y)-box 2 (SOX2) and SFRP2 was found in
both hADSC and hSKP, whereas Nanog homeobox (NANOG)
was only significantly increased in hADSC. However, no difference in gene expression could be observed for POU class 5
homeobox 1 (POU5F1), Kruppel-like factor 4 (KLF4), signal
transducer and activator of transcription 3 (STAT3), and v-myc
myelocytomatosis viral oncogene homolog (MYC) in all four
stem cell populations (p>0.05; Fig. 2; Tab. 3).
4 Discussion
Human adult stem cells are an attractive cell source for tissue
engineering and for the development of human-relevant alternative in vitro models (Lee et al., 2011; Scanu et al., 2011). The
most widely used adult stem cell populations all originate from
the mesoderm. Indeed, this is true also for bone marrow- and
adipose-derived stromal cells, as skin-derived precursor cells
derived from trunk skin trace back to the mesoderm. On the
Tab. 3: Normalized mRNA levels of pluripotency genes
Fold changes of genes involved in the pluripotency of stem cells are determined for all four mesoderm-derived stem cell types.
Significantly increased and decreased mRNA expression are marked by dark and light grey, respectively.
Abbreviations: Kruppel-like factor 4 (KLF4); Nanog homeobox (NANOG); POU class 5 homeobox 1 (POU5F1); secreted frizzled-related
protein 1 (SFRP1); secreted frizzled-related protein 2 (SFRP2); signal transducer and activator of transcription 3 (STAT3); SRY (sex
determining region Y)-box 2 (SOX2); v-myc myelocytomatosis viral oncogene homolog (MYC).
POU5F1
hADSC
hBMSC
hSKP
hWJ
SOX2
hADSC
1.000
1.020
2.008
1.973
hBMSC
0.980
1.000
1.969
1.934
hSKP
0.498
0.508
1.000
0.983
hWJ
0.507
0.517
1.018
1.000
NANOG
hADSC
hBMSC
hSKP
hWJ
hADSC
1.000
18.614
15.245
17.898
hBMSC
0.054
1.000
0.819
0.962
hSKP
0.066
1.221
1.000
1.174
hWJ
0.056
1.040
0.852
1.000
hBMSC
0.021
1.000
0.035
2.101
hSKP
0.608
28.774
1.000
60.450
hWJ
0.010
0.476
0.017
1.000
hADSC
hBMSC
hSKP
hWJ
hADSC
1.000
0.995
0.875
1.117
hBMSC
1.005
1.000
0.880
1.122
hSKP
1.143
1.137
1.000
1.276
hWJ
0.895
0.891
0.784
1.000
hADSC
1.000
0.655
1.211
1.000
hBMSC
1.527
1.000
1.848
1.527
hSKP
0.826
0.541
1.000
0.826
hWJ
1.000
0.655
1.211
1.000
hADSC
hBMSC
hSKP
hWJ
STAT3
hADSC
1.000
0.863
2.007
5.394
hBMSC
1.159
1.000
2.326
6.250
hSKP
0.498
0.430
1.000
2.688
hWJ
0.185
0.160
0.372
1.000
hADSC
1.000
1.275
0.063
3.887
hBMSC
0.784
1.000
0.050
3.049
hSKP
15.755
20.087
1.000
61.241
hWJ
0.257
0.328
0.016
1.000
down
equal
up
SFRP1
hADSC
hBMSC
hSKP
hWJ
hADSC
1.000
47.292
1.644
99.353
MYC
KLF4
hADSC
hBMSC
hSKP
hWJ
hADSC
hBMSC
hSKP
hWJ
hADSC
hBMSC
hSKP
hWJ
SFRP2
Legend
Altex Proceedings, 1/12, Proceedings of WC8
hADSC
hBMSC
hSKP
hWJ
1.000
36.345
0.248
NE
0.028
1.000
0.007
NE
4.028
146.393
1.000
NE
NE
NE
NE
NE
537
De Kock et al.
contrary, Wharton’s jelly-derived mesenchymal stem cells originate from extra-embryonic mesoderm (Fong et al., 2010; Jinno
et al., 2010; Vodyanik et al., 2010). In the present study, the differential expression of pluripotency genes and the identification
of enriched basic developmental functions that are involved in
embryogenesis and organogenesis were investigated by in-depth
comparative transcriptome analyses and qPCR measurements.
To first assess relatedness between the stem cell populations,
Ward’s hierarchical clustering was performed on a defined set
of genes relating to stem cell pluripotency. As shown in Figure 1, the independent samples of each stem cell type grouped
together, with hBMSC being more closely related to hWJ for
the expression of pluripotency genes. hADSC and hSKP were
more distinct. In addition, hSKP shared a closer proximity with
hESC, the gold standard of pluripotent stem cells. Furthermore,
functional analyses showed that no significant enrichment of
basic developmental functions could be observed in hADSC.
hADSC displayed an increased gene expression of the pluripotency markers SOX2 and NANOG, which could, however, be
explained by the restrictive differentiation potential of hADSC
due to hypermethylation of nonadipogenic lineage-specific promoters (Boquest et al., 2006). Indeed, several reports showed
that epigenetic modification of hADSC is required to cross these
lineage restrictions (Aurich et al., 2009; Choi et al., 2010). In
contrast, hSKP showed a significant enrichment of functions that
are involved in embryogenesis and organogenesis, suggesting a
highly multipotent character of hSKP. This is supported by the
increased expression of the embryonic stem cell markers FGF4
(Shi et al., 2011), GRB7, IFITM1, LEFTY2 (Barroso-delJesus et
al., 2011), NR5A2 (Gabut et al., 2011), NR6A1 (Akamatsu et al.,
2009), SEMA3A (Tamariz et al., 2010), SFRP1 (Katoh, Y. and
Katoh, M., 2005), SFRP2 (Katoh, M. and Katoh, M., 2005),
SOX2, TFCP2L1 (To et al., 2010), and TERT (Gourronc and
Klingelhutz, 2011), and the primordial germ cell marker DAZL
(Linher et al., 2009). In addition, several other studies have reported a multipotent potential of hSKP, being capable of generating neuronal, glial, mesodermal, endodermal and primordial
germ cell progeny (Toma et al., 2005; Fernandes et al., 2006;
Biernaskie et al., 2006, 2007; McKenzie et al., 2006; Lavoie
et al., 2009; De Kock et al., 2009, 2011; Linher et al., 2009).
Finally, minor differences could be observed between hBMSC
and hWJ. Indeed, only FZD5 is increased in hBMSC, whereas
IL6ST and SEMA3A are decreased compared to hWJ. No significant differences could be observed for general pluripotency
markers such as POU5F1, NANOG, SOX2, MYC and KLF4, as
further indicated by their close relatedness. In conclusion, these
data suggest that, amongst the different mesoderm-derived
stem cells tested (hADSC, hBMSC, hSKP, and hWJ), the intrinsic cell plasticity, defined by the expression of pluripotency
genes and enrichment of biological functions that are involved
in embryogenesis and organogenesis, is the most prominent in
hSKP.
References
Akamatsu, W., DeVeale, B., Okano, H., et al. (2009). Suppression of Oct4 by germ cell nuclear factor restricts pluripotency
538
and promotes neural stem cell development in the early neural
lineage. J. Neurosci. 29, 2113-2124.
Al Battah, F., De Kock, J., Ramboer, E., et al. (2011). Evaluation
of the multipotent character of human adipose tissue-derived
stem cells isolated by Ficoll gradient centrifugation and red
blood cell lysis treatment. Toxicol. In Vitro 25, 1224-1230.
Aurich, H., Sgodda, M., Kaltwasser, P. et al. (2009). Hepatocyte differentiation of mesenchymal stem cells from human
adipose tissue in vitro promotes hepatic integration in vivo.
Gut 58, 570-581.
Barroso-delJesus, A., Lucena-Aguilar, G., Sanchez, L., et al.
(2011). The Nodal inhibitor Lefty is negatively modulated
by the microRNA miR-302 in human embryonic stem cells.
FASEB J. 25, 1497-1508.
Biernaskie, J. A., McKenzie, I. A., Toma, J. G., et al. (2006).
Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny. Nat. Protoc. 1, 2803-2812.
Biernaskie, J. A., Sparling, J. S., Liu, J., et al. (2007). Skinderived precursors generate myelinating Schwann cells that
promote remyelination and functional recovery after contusion spinal cord injury. J. Neurosci. 27, 9545-9559.
Bolstad, B. M., Irizarry, R. A., Astrand, M., and Speed, T. P.
(2003). A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.
Bioinformatics 19, 185-193.
Boquest, A. C., Noer, A., and Collas, P. (2006). Epigenetic programming of mesenchymal stem cells from human adipose
tissue. Stem Cell Rev. 2, 319-329.
Choi, Y. S., Dusting, G. J., Stubbs, S., et al. (2010). Differentiation of human adipose-derived stem cells into beating cardiomyocytes. J. Cell Mol. Med. 14, 878-889.
De Bruyn, C., Najar, M., Raicevic, G., et al. (2011). A rapid,
simple, and reproducible method for the isolation of mesenchymal stromal cells from Wharton’s jelly without enzymatic
treatment. Stem Cells Dev. 20, 547-557.
De Kock, J., Vanhaecke, T., Rogiers, V., and Snykers, S. (2008).
Chromatin remodeling, a novel strategy to expedite the hepatic differentiation of adult bone marrow stem cells in vitro.
AATEX 14, 605-611.
De Kock, J., Vanhaecke, T., Biernaskie J. A., et al. (2009).
Characterization and hepatic differentiation of skin-derived
precursors from adult foreskin by sequential exposure to
hepatogenic cytokines and growth factors reflecting liver development. Toxicol. In Vitro 23, 1522-1527.
De Kock, J., Snykers, S., Ramboer, E., et al. (2011). Evaluation
of the multipotent character of human foreskin-derived precursor cells. Toxicol. In Vitro 25, 1191-1202.
Fernandes, K. J., Kobayashi, N. R., Gallagher, C. J., et al.
(2006). Analysis of the neurogenic potential of multipotent
skin-derived precursors. Exp. Neurol. 201, 32-48.
Fong, C. Y., Subramanian, A., Biswas, A., et al. (2010). Derivation efficiency, cell proliferation, freeze-thaw survival, stemcell properties and differentiation of human Wharton’s jelly
stem cells. Reprod. Biomed. Online 21, 391-401.
Gabut, M., Samavarchi-Tehrani, P., Wang, X., et al. (2011).
An alternative splicing switch regulates embryonic stem cell
Altex Proceedings, 1/12, Proceedings of WC8
De Kock et al.
pluripotency and reprogramming. Cell, in press.
Gautier, L., Cope, L., Bolstad, B. M., and Irizarry, R. A. (2004).
affy – analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 307-315.
Gourronc, F. A. and Klingelhutz, A. J. (2011). Therapeutic opportunities: Telomere maintenance in inducible pluripotent
stem cells. Mutat. Res., in press.
Irizarry, R. A., Bolstad, B. M., Collin, F., et al. (2003). Summaries of Affymetrix GeneChip probe level data. Nucleic Acids
Res. 31, e15.
Jinno, H., Morozova, O., Jones, K. L., et al. (2010). Convergent
genesis of an adult neural crest-like dermal stem cell from
distinct developmental origins. Stem Cells 28, 2027-2040.
Katoh, M. and Katoh, M. (2005). Comparative genomics on
SFRP2 orthologs. Oncol. Rep. 14, 783-787.
Katoh, Y. and Katoh, M. (2005). Comparative genomics on
SFRP1 orthologs. Int. J. Oncol. 27, 861-865.
Lavoie, J. F., Biernaskie, J. A., Chen, Y., et al. (2009). Skinderived precursors differentiate into skeletogenic cell types
and contribute to bone repair. Stem Cells Dev. 18, 893-906.
Lee, H. J., Cha, K. E., Hwang, S. G., et al. (2011). In vitro screening system for hepatotoxicity: comparison of bone-marrowderived mesenchymal stem cells and Placenta-derived stem
cells. J. Cell Biochem. 112, 49-58.
Linher, K., Dyce, P., and Li, J. (2009). Primordial germ celllike cells differentiated in vitro from skin-derived stem cells.
PLoS One 4, e8263.
McKenzie, I. A., Biernaskie, J. A., Toma, J. G., et al. (2006).
Skin-derived precursors generate myelinating Schwann cells
for the injured and dysmyelinated nervous system. J. Neurosci. 26, 6651-6660.
Najar, M., Raicevic, G., Id Boufker, H., et al. (2010). Modulated
expression of adhesion molecules and galectin-1: role during
mesenchymal stromal cell immunoregulatory functions. Exp.
Hematol. 38, 922-932.
Pepper, S. D., Saunders, E. K., Edwards, L. E., et al. (2007).
The utility of MAS5 expression summary and detection call
algorithms. BMC Bioinformatics 8, 273.
Scanu, M., Mancuso, L., and Cao, G. (2011). Evaluation of the
use of human Mesenchymal Stem Cells for acute toxicity
tests. Toxicol. In Vitro, in press.
Shafiee, A., Seyedjafari, E., Soleimani, M., et al. (2011). A
comparison between osteogenic differentiation of human
unrestricted somatic stem cells and mesenchymal stem cells
from bone marrow and adipose tissue. Biotechnol. Lett. 33,
1257-1264.
Shi, G., Gao, F., and Jin, Y. (2011). The regulatory role of histone deacetylase inhibitors in Fgf4 expression is dependent
Altex Proceedings, 1/12, Proceedings of WC8
on the differentiation state of pluripotent stem cells. J. Cell
Physiol., in press.
Tamariz, E., Díaz-Martínez, N. E., Díaz, N. F., et al. (2010).
Axon responses of embryonic stem cell-derived dopaminergic neurons to semaphorins 3A and 3C. J. Neurosci. Res. 88,
971-980.
To, S., Rodda, S. J., Rathjen, P. D., and Keough, R. A. (2010).
Modulation of CP2 family transcriptional activity by CRTR-1
and sumoylation. PLoS One 5, e11702.
Toma, J. G., McKenzie, I. A., Bagli, D., and Miller, F. D. (2005).
Isolation and characterization of multipotent skin-derived
precursors from human skin. Stem Cells 23, 727-737.
Vodyanik, M. A., Yu, J., Zhang, X., et al. (2010). A mesodermderived precursor for mesenchymal stem and endothelial
cells. Cell Stem Cell 7, 718-729.
Ward, J. (1963). Hierarchical grouping to optimize an objective
function. J. Am. Stat. Assoc. 58, 236-244.
Acknowledgments
The authors thank Prof. Dr. P. Wylock (UZ-Brussels, Department of Plastic Surgery) and Dr. P. Willekens and V. Van den
Borre (ATLAS kliniek) for their kind donation of human adipose tissue samples and Dr. V. De Boe (UZ-Brussels, Department of Urology) for donation of human foreskin tissues, upon
informed consent of the involved patients, respectively parents.
Financial support
Joery De Kock is a doctoral research fellow of the Institute for
the Promotion of Innovation through Science and Technology
in Flanders (IWT-Vlaanderen). The research leading to these results has also received funding from the European Community’s
Seventh Framework Programme (FP7/2007-2013) under grant
agreement n°20161 (ESNATS) and from ISRIB (Brustem) and
BELSPO (IAP).
Correspondence to
Joery De Kock
Dept. Toxicology
Vrije Universiteit Brussel (VUB)
Laarbeeklaan 103
1090 Brussels
Belgium
Phone: +32 2 477 4517
Fax.: +32 2 477 4582
e-mail: jdekock@vub.ac.be
539