Assessment of the Intrinsic Pluripotency of Mesoderm ... · obtained from four distinct niches, displays the highest intrinsic cell plasticity compared to human embryonic stem cells

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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, in-cluding brain, skin, adipose tissue, bone marrow, skeletal mus-cle, 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 differ-entiate 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 plas-ticity, human adult stem cells are today an attractive cell source for tissue engineering and for developing human-relevant alter-native 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 popu-

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 intrin-sic pluripotency of the investigated stem cell population. In the present study, an unambiguous characterization and compari-son 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 pro-files 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 hADSCPlastic 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)

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

1Dept. of Toxicology, Center for Pharmaceutical Research, Vrije Universiteit Brussel (VUB), Brussels, Belgium; 2Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Brussels, Belgium; 3Dept. of Morphology and Molecular Pathology, Katholieke Universiteit Leuven (KUL), University Hospital Leuven, Leuven, Belgium; 4Center of Physiology, Institute of Neurophysiology, University of Cologne, Cologne, Germany

SummaryDuring 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

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upon informed consent and approved by the ethical commis-sion of the UZ-Brussels. The median age of the donors was 39 years (♀/♂; range 32-49). hADSC were isolated and subcul-tivated 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) bo-vine 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 filtra-tion 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 cul-tured as a monolayer in hADSC growth medium, consisting of Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Braine-l’Alleud, Belgium) supplemented with 10% (v/v) foetal bovine serum (FBS) (Perbio Hyclone, Erembodegem, Belgium), 50 µg/ml streptomycin sulphate (Sigma-Aldrich, Bornem, Bel-gium), 7.33 IU/ml benzyl penicillin (Continental Pharma, Di-egem, 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 hBMSCBone 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, Bel-gium). 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 solu-tion (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 subse-quent passages.

Isolation and cultivation of hSKPhSKP 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 fore-skin 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 re-moved and the tissue was incubated at 37°C for another 10-20 minutes depending on the sample size. After processing the

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 Tech-nologies, Merelbeke, Belgium) supplemented with 7.33 IU/ml benzyl penicillin (Continental Pharma, Diegem, Belgium), 50 μg/ml streptomycin sulphate (Sigma-Aldrich, Bornem, Bel-gium), 2.5 μg/ml fungizone, 2% (v/v) B27 Supplement (all from Life Technologies, Merelbeke, Belgium), 40 ng/ml basic fibrob-last growth factor (FGF)-2 and 20 ng/ml epidermal growth fac-tor (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 hWJAfter informed consent from the mothers, umbilical cords (♀/♂) were collected after full-term deliveries. They were proc-essed 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 longitu-dinally and plated for 5 days in an appropriate culture medium (DMEM-LG, Lonza, Braine-l’Alleud, Belgium). After remov-ing the cord segments, the culture was pursued until subconflu-ency. Cell cultures were incubated at 37°C in a 5% (v/v) CO2, humidified atmosphere. After 48 h, non-adherent cells were re-moved 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 Mini-prep Kit (Sigma-Aldrich, Bornem, Belgium) according to the manufacturer’s instructions. The isolated RNA was quantified at 260 nm using a Nanodrop spectrophotometer (Thermo Sci-entific, Wilmington, USA). Total RNA was reverse transcribed into cDNA using iScript™ cDNA Synthesis Kit (BioRad, Naza-reth, Belgium) followed by cDNA purification with the Gen-elute PCR clean up kit (Sigma-Aldrich, Bornem, Belgium). For microarray analysis, the RNA was extracted using trizol/chloro-form 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 includ-ed 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 Univer-sal Master Mix (Applied Biosystems, Halle, Belgium), 1.25 µl 20X Assay-on-Demand Mix (Applied Biosystems, Halle, Bel-

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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: incuba-tion for 10 min at 95°C, followed by 40 cycles of 15 s denatura-tion 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 refer-ence 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).

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 ref-erence 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 analysisAll reagents and instrumentation pertaining to oligonucleotide microarrays were procured from Affymetrix (Affymetrix, San-ta 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 manufac-turer’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 (Af-fymetrix) rotating at 60 rpm at 45 ºC for 16 h. After incuba-tion, 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 Af-fymetrix Gene-Chip Scanner-3000-7G and the quality control matrices were confirmed with the Affymetrix GCOS software following the manufacturer’s guidelines. Background cor-rection, summarization, and normalization were done with Robust Multi-array Analysis (Irizarry et al., 2003). The raw dataset was normalized with the quantile normalization (Bol-stad 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 ab-sent 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 hu-man 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 differ-ential gene expression, output data files were analyzed using GeneSpring GX v11.5 software (Agilent Technologies, Wald-bronn, 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 per-formed 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).

Tab. 1: Gene expression assays used for characterization of mesoderm-derived stem cellsThe 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-3-phosphate 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 Assay-on- Amplicon Source Demand ID length (bp)

18S Hs99999901_s1 187 ABACTB Hs99999903_m1 171 ABB2M Hs99999907_m1 75 ABGAPDH Hs99999905_m1 122 ABHMBS Hs00609296_g1 69 ABKLF4 Hs00358836_m1 110 ABMYC Hs99999003_m1 65 ABNANOG Hs02387400_g1 109 ABPOU5F1 Hs00999632_g1 77 ABSFRP1 Hs00610060_m1 130 ABSFRP2 Hs00293258_m1 129 ABSOX2 Hs01053049_s1 91 ABSTAT3 Hs01047580_m1 87 ABUBC Hs00824723_m1 71 AB

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tissue,” “development of organ,” “developmental process of organism,” “differentiation of embryonic cells,” and “organo-genesis” 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 proc-ess of organism,” and “organogenesis” was found in hBMSC compared to hADSC and hWJ (Tab. 2). Furthermore, a signifi-cantly 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 functionsTo 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 organogenesisFunctional 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 dif-ferent 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).

development of embryonic tissue (416)development of organ (1623)developmental process of organism (2785)differentiation of embryonic cells (189)organogenesis (1657)




hADSC vs hBMSC -LOG ratio (%) (B-H p-value) 3.67E-01 2.64 0.00E+00 0.00 0.00E+00 0.00 3.67E-01 3.17 0.00E+00 0.00

hBMSC vs hSKP -LOG ratio (%) (B-H p-value) 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00

hSKP vs hBMSC -LOG ratio (%) (B-H p-value) 6.61E+00 17.31 3.95E+00 10.78 3.16E+00 8.87 1.68E+00 13.23 4.14E+00 10.80

hWJ vs hBMSC -LOG ratio (%) (B-H p-value) 7.97E+00 18.03 8.63E+00 12.38 1.10E+01 10.63 3.44E+00 15.87 8.63E+00 12.31

hADSC vs hSKP -LOG ratio (%) (B-H p-value) 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00

hBMSC vs hADSC -LOG ratio (%) (B-H p-value) 0.00E+00 0.00 3.58E+00 20.39 6.98E+00 18.60 1.58E+00 23.28 4.13E+00 20.58

hSKP vs hADSC -LOG ratio (%) (B-H p-value) 2.86E+00 15.14 6.16E+00 12.51 3.78E+00 9.87 0.00E+00 0.00 6.32E+00 12.49

hWJ vs hADSC -LOG ratio (%) (B-H p-value) 0.00E+00 0.00 4.77E+00 24.40 1.00E+01 22.48 2.25E+00 27.51 5.20E+00 24.50

hADSC vs hWJ -LOG ratio (%) (B-H p-value) 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00 0.00E+00 0.00

hBMSC vs hWJ -LOG ratio (%) (B-H p-value) 0.00E+00 0.00 4.13E+00 8.38 2.63E+00 6.61 0.00E+00 0.00 4.29E+00 8.39

hSKP vs hWJ -LOG ratio (%) (B-H p-value) 3.20E+00 17.55 1.06E+00 11.71 6.80E-01 9.87 0.00E+00 0.00 1.09E+00 11.71

hWJ vs hSKP -LOG ratio (%) (B-H p-value) 0.00E+00 0.00 0.00E+00 0.00 2.60E+00 26.00 0.00E+00 0.00 0.00E+00 0.00

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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 re-lated to pluripotency as compared to the other three mesoderm-derived stem cell types, resulting in a closer proximity between hBMSC, hWJ, and hADSC than hSKP, as shown by Ward’s hi-erarchical clustering (Fig. 1). Moreover, the mRNA expression profiles of hSKP showed a higher similarity with hESC, result-ing in a closer relatedness between both cell types. Indeed, the

3.2 Characteristics of mesoderm-derived stem cells determined by expression patterns of pluripotency genesExpression patterns of pluripotency genes were used to char-acterize 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 over-view of the differentially expressed transcripts known to be in-volved in pluripotency (Fig. 1). In order to validate the microar-

Fig. 1: Microarray analysis shows increased mRNA expression in hSKP of genes involved in the pluripotency of stem cellsHeat 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.

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Fig. 2: qPCR confirms increased gene expression of pluripotency genes in hSKPFold 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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was only significantly increased in hADSC. However, no dif-ference 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 alter-native 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

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 trans-membrane 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 SE-MA3A were significantly increased and decreased, respective-ly, in hBMSC. More in-depth qPCR analyses confirmed that SFRP1 was significantly increased in hSKP (Fig. 2; Tab. 3). Ad-ditionally, 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)

Tab. 3: Normalized mRNA levels of pluripotency genesFold 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 hADSC 1.000 0.980 0.498 0.507 hBMSC 1.020 1.000 0.508 0.517 hSKP 2.008 1.969 1.000 1.018 hWJ 1.973 1.934 0.983 1.000

SOX2


NANOG


MYC


KLF4


STAT3


SFRP2

hADSC hBMSC hSKP hWJ hADSC 1.000 0.028 4.028 NE hBMSC 36.345 1.000 146.393 NE hSKP 0.248 0.007 1.000 NE hWJ NE NE NE NE

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SFRP1


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contrary, Wharton’s jelly-derived mesenchymal stem cells orig-inate from extra-embryonic mesoderm (Fong et al., 2010; Jinno et al., 2010; Vodyanik et al., 2010). In the present study, the dif-ferential 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 Fig-ure 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 pluripo-tency markers SOX2 and NANOG, which could, however, be explained by the restrictive differentiation potential of hADSC due to hypermethylation of nonadipogenic lineage-specific pro-moters (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 re-ported a multipotent potential of hSKP, being capable of gener-ating 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 sig-nificant 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 in-trinsic 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.

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AcknowledgmentsThe authors thank Prof. Dr. P. Wylock (UZ-Brussels, Depart-ment of Plastic Surgery) and Dr. P. Willekens and V. Van den Borre (ATLAS kliniek) for their kind donation of human adi-pose tissue samples and Dr. V. De Boe (UZ-Brussels, Depart-ment of Urology) for donation of human foreskin tissues, upon informed consent of the involved patients, respectively parents.

Financial supportJoery 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 re-sults 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 toJoery De KockDept. ToxicologyVrije Universiteit Brussel (VUB)Laarbeeklaan 1031090 BrusselsBelgiumPhone: +32 2 477 4517 Fax.: +32 2 477 4582e-mail: [email protected]

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