transcriptional signature of human adipose tissue-derived stem cells (hascs) preconditioned for...

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Research Article

Transcriptional signature of human adipose tissue-derivedstem cells (hASCs) preconditioned for chondrogenesis inhypoxic conditions

L. Pilgaarda, P. Lunda, M. Durouxa, H. Lockstoneb, J. Taylorb, J. Emmersena, T. Finka,J. Ragoussisc, V. Zachara,⁎aLaboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg, DenmarkbBioinformatics and Statistical Genetics, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford,OX3 7BN, UKcGenomics, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford, OX3 7BN, UK

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author.E-mail address: [email protected] (V. Zachar)

0014-4827/$ – see front matter © 2009 Elseviedoi:10.1016/j.yexcr.2009.01.020

A B S T R A C T

Article Chronology:

Received 5 December 2008Revised version received19 January 2009Accepted 20 January 2009

Available online 2 February 2009

Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight intothe phenotypical changes brought about by hypoxic preconditioning prior to chondrogenicdifferentiation; we have investigated growth, colony-forming and chondrogenic capacity, andglobal transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygenconcentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-

forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a majorrole. The chondrogenic inducibility, examined by high-density pellet model, however, did notimprove on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced aspecific expression profile with 2581 significantly regulated genes and substantial functionalenrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1%oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular,leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated.In conclusion, the results of this investigation demonstrate the significance of donor demographicsand the importance of further studies into the use of regulated oxygen tension as a tool forpreparation of ASCs in order to exploit their full potential.

© 2009 Elsevier Inc. All rights reserved.

Keywords:

Adipose tissue-derived stem cellscDNA microarrayIlluminaGene expressionHypoxiaColony-forming unitChondrogenesis

Introduction

Adipose tissue is a plentiful and easily accessible source ofmesenchymal stem cells that have been shown to have multi-potent abilities regarding differentiation and possibly immunosuppressive capacity [1–3]. Adipose tissue-derived stem cells(ASCs) are therefore a promising alternative to bone marrow-derived mesenchymal stem cells (BMSCs) and have been a popular

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research subject in the field of tissue engineering world wide. Inparticular, much effort has been directed at investigating thechondrogenic potential of ASCs and their possible application inthe repair of dysfunctional cartilage [4,5], which is one of theleading causes of disability and chronic pain [6]. In horses anddogs, several studies have proven the applicability of ASCs for painrelief and tissue regeneration in the treatment of cartilage defects[7–9]. Although of similar phenotype and differentiation potential,

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http://dx.doi.org/10.1016/j.yexcr.2009.01.020

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the donor-matched BMSCs have been demonstrated to havesuperior chondrogenic capacity than ASCs [10,11]. Still, theaccessibility and numbers of ASCs advocate their application overBMSCs, as the chondrogenic potential of ASCs can be enhancedthrough the culturing conditions [12–16].

The heterogeneity of early ASC cultures and evidence of a gradualphenotypic homogenization with in vitro propagation haveprompted researchers to focus on two dimensional culturingconditions to enhance the chondrogenic potential [1,17–20]. Morespecifically, it has been shown that the fraction of ASCs expressingCD105, which is a regulatory component of the TGF-β receptorcomplex, increased during the course of four passages from 5% to70% [17]. Thus, as a result of simple in vitro culturing, the ASCs mayacquire properties that make them more susceptible to TGF-βtreatment, and consequently induction for chondrogenesis. In bothBMSC and ASC cultures, growth conditions exploiting specificseeding density, culture formats, and addition of growth factorssuch as FGF2 and BMP6 or optimized culture periods have provenbeneficial for the chondrogenic potential [12,13,15,16,21–23].Furthermore, numerous studies have identified hypoxia to beessential in differentiation processes, stem cell proliferation andsurvival, as well as in the maintenance of stem cell characteristics[24–29]. Especially in light of the fact that oxygen has a central rolein cartilage development and chondrocyte metabolism, manipula-tion of culture gaseous phase lends itself as a valuable tool to exploitstem cells to their full potential. Recently, Chen et al. demonstratedthat hypoxic exposure has the capacity to induce chondrogenesis-specific transcriptional machinery [30] but found that the full courseof chondrogenesis is better supported at ambient air oxygenconcentration [31]. In line with these findings, several recentinvestigations have adopted a hypoxic preconditioning step toimprove stem cell function and survival prior to differentiation orclinical application [32–34]. For instance, Grayson et al. [32]demonstrated that long-term cultivation of BMSCs in chronichypoxia promoted a highly proliferative phenotype with anenhanced capacity of osteogenesis and adipogenesis compared toambiently expanded cultures. In a complementary investigation,applying a short 24 h hypoxic preconditioning of BMSCs, thepotential for chondrogenesis was enhanced and likewise accom-panied by an increased proliferation [34]. Monolayer cultures ofASCs subjected to hypoxic expansion for aweek have been shown tobe enriched for chondrogenic progenitors while maintaining anundifferentiated phenotype [26]. Most frequently, the oxygenconcentration employed in these studies was around 2%. However,the applied instrumental setups, culture formats, and cultureperiods are diverse to such an extent that satisfactory inter-studycomparisons are difficult if not unfeasible [18,21,22].

The utility of global gene expression analysis through micro-arrays in stem cell research is increasing. Such approach haspreviously been instrumental, for example, in the comparativeanalysis of transcriptional fingerprints of mesenchymal stem cellsand early differentiated fetal tissue [35]. Moreover, in BMSCs, anumber of candidate genes possibly regulating stem cell self-renewal have been identified by the aid of microarrays and theelegant application of in vitro differentiation and dedifferentiationprotocols [36]. The BMSCs, along with cord blood CD133+, andfreshly isolated mononucleated cells from bone marrow have alsobeen studied with regard to hypoxic expansion [29,34]. As for theASCs, their transcriptional profile has previously been investigatedby Katz et al. [37], and compared to MSCs from other sources

[38,39]. In addition, several studies have analyzed the transcrip-tional changes in ASCs undergoing differentiation [40–42]. How-ever, the number of microarray investigations of ASCs geneexpression is limited. In particular, the effect of hypoxic cultureon ASCs has not previously been subject to a large-scale analysis. Inthe current study, we set out to investigate the effect of hypoxicmonolayer ASCs expansion on transcriptional activation at agenomic level. By exploiting a hypoxic workstation (XVivo;BioSpherix, Redfield, NY), we conducted the expansion of six ASClines at five different oxygen concentrations (ambient,15%,10%, 5%,and 1%) in parallel, enabling thus a direct comparison betweendiverse atmospheric conditions. In the present study, the hypoxia-induced transcriptional changes were related to the results fromfunctional testing of the hypoxia conditioned cultures. To ourknowledge, this is the first study applying genome-wide expres-sion profiling to obtain the molecular background for thephenotypical changes brought about by monolayer expansion ofASCs under reduced oxygen tension.

Materials and methods

Isolation of adipose tissue-derived stem cells

Allmaterials were obtained from Invitrogen (Carlsbad, Ca) or Sigma-Aldrich (Broendby, Denmark) unless otherwise stated. Protocolsinvolving human subjects were reviewed and approved by theregional Committee on Biomedical Research Ethics in NorthernJutland prior to the study. Samples of subcutaneous adipose tissuewere obtained after informed consent from six female donors (age22–44 years, mean 33.7+/−8.3 and body mass index (BMI) 20.6–34.3,mean 24.2+/−5.4) undergoing elective surgery at theGrymerPrivate Hospital, Skejby, Denmark (Supplement 1). They weredesignatedASC6,10,13, 23, and24. All tissue sampleswereharvestedusing a tumescent technique with pump-assisted aspiration using asaline solution supplemented with lidocaine and epinephrine [43].ASCs were isolated as previously described [44] with slightmodifications. In brief, the tissue was washed three times withmatching volumes of prewarmed Dulbecco's phosphate-buffered-saline (D-PBS) and then an equal volume of collagenase bufferconsisting of 0.28 Wünch U/ml crude collagenase mix (Lot. No.LTQ5230; Wako, Neuss, Germany) in PBS with 20 mg/ml bovineserum albumin (BSA) (Roche Applied Science, Hvidovre, Denmark)was added. Following digestion at 37 °Cwith gentle agitation for 1 h,the released cells were isolated by velocity sedimentation at 400 ×gfor 10min, and the pelleted cells were filtered through a 70 μmmeshcell strainer (BD Bioscience, Broendby, Denmark). Contaminatingerythrocytes were lysed and the remaining nucleated cells werefurther purified through a second round of centrifugation andfiltration. The cellswere seeded at a density corresponding to 0.15mladipose tissue/cm2 in growth medium, α-Modified Eagle Medium(αMEM) supplemented with antibiotics and 10% fetal calf serum(FCS). Twelve hours post-seeding, the medium was changed toremove the non-adherent cells.

Cell culture and colony-forming unit assay

The freshly isolated cells were expanded until subconfluent stage,at which point they were cryopreserved. For the experiment, thecell lines were thawed and cultured for one passage in growth

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medium until they reached 80% density. All normoxic cell cultureswere kept in a humidified atmosphere containing 5% CO2 bufferedwith ambient air at 37 °C, and the medium was changed twice aweek. For the hypoxic exposure, the cultures were initiated at 1000cells/cm2, and allowed to progress for 24 h at ambient airconditions. The subsequent expansion was performed simulta-neously for all four hypoxic conditions (15, 10, 5, and 1% oxygen)during the course of 14 days in the XVivo hypoxic workbench/incubator (BioSpherix, Redfield, NY). The medium was changedtwice a week, and it was independently preequilibrated to eachoxygen level prior to application.

To determine the fraction of colony-forming unit fibroblasts(CFU-F), the cells were serially diluted across the 12 rows in a 96-well culture plate to produce seeding densities of 1000 to 0.5 cellsper well. The cells were cultured for 11 days followed by formalinfixation, methylene blue staining, and analysis as describedpreviously [45].

Chondrogenic assay

For chondrogenic induction, a high glucose (4.5 g/l) Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10 ng/mlTGF-β3 (RnD Systems, Oxon, United Kingdom), 10−7 M dexa-methazone, 50 μg/ml L-ascorbic acid 2-phosphate, 40 μg/ml L-proline, antibiotics and an insulin, selenium, and transferrinsupplement (ITS; BD Bioscience, Broendby, Denmark) was used.The inducibility of the cells was assessed in a high-density pelletculture format previously described by Penick et al. [46]. Thecultures comprised 2×105 cells pelleted bycentrifugation at 500×gfor 5 min in 96-well plates with V bottom (cci3896; Corning,Schiphol-Rijk, The Netherlands). The induced as well as the controlcultures thatweremaintained in the growthmediumwere kept in ahumidified atmosphere containing 5% CO2 buffered with anambient air at 37 °C for three weeks. Media were changed threetimes a week.

After conclusion of chondrogenic induction, the pellets werefixed with 4% formaldehyde over night, washed with D-PBS, andfurther processed using standard procedures for paraffin embed-ding and sectioning. Accumulation of proteoglycans was detectedby alcian blue 8GX staining (1 g/l in 0.1 M HCl, pH 1) for 30 min atroom temperature, and Mayer's hematoxylin (Bie and Berntsen)was used as a counterstain. The stained sections were surveyedwith the aid of a Zeiss Axiovert 200M inverted microscope (Brockand Michelsen, Birkeroed, Denmark) using a 10×/0.25 A-Planobjective. The images were acquired and processed with the ZeissAxioVision software package (Brock and Michelsen).

Glycosaminoglycan quantification

The glycosaminoglycans (GAGs) were quantified according to themethod previously described by Barbosa et al. [47]. In brief, thecultures were digested with a collagenase buffer described above.The released GAGs were complexed to the dichromatic dye 1, 9dimethyl methylene blue (DMMB) at pH 3 for 1 h at 600 RPM. TheGAG–DMMB complexes were isolated by sedimentation at12,000 ×g for 5min and decomplexed at neutral pH. The absorbanceat 656 nm was measured and converted to GAG contents on thebasis of a standard, for which the chondroitin-4-sulfate was used.The concentrations were normalized to DNA content determined bya Pico Green assay according to manufactures instructions.

Isolation of RNA and microarray analysis

Total RNA was isolated using the Aurum total RNA mini kit (Bio-Rad, Copenhagen, Denmark) as recommended by the manufac-turer and the Illumina Total Prep RNA Amplification Kit (Illumina,San Diego, Ca) was used to produce in vitro transcribed and biotinlabeled cRNA. RNA integrity was assessed by a capillary electro-phoresis (Agilent 2100 Bioanalyzer; Naerum, Denmark), and thepurity and concentrationwere determined by spectrophotometery(Nanodrop; Thermo Science, Wilmington, De). The cRNA washybridized to Illumina Bead arrays, human Ref 6 chips (Illumina),which generate whole genome transcription profiles simulta-neously for six samples. The arrays were scanned using theIllumina Bead station 500GX System.

Array data processing

The background correction that was based on the average signalfrom a set of internal negative controls within individual arrayswas performed by the Illumina Beadstudio software (Illumina Inc,San Diego, CA), and the data were further processed and analysedusing R [48] and BioConductor packages [49] as well as Multi-experiment Viewer (MeV) from the TM4 suite of microarray tools[50]. The outlier samples were identified using principal compo-nent analysis (PCA), and hierarchical and k-means clustering. Suchsamples were removed prior to variance stabilisation and quantilenormalisation using algorithms implemented in the VSN package[51]. The data discussed in this publication have been deposited inNCBI's Gene Expression Omnibus [52] and are accessible throughGEO Series accession number GSE12884 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12884).

To identify significant changes in gene expression associatedwith hypoxic conditions, the linear models for microarray analysis(LIMMA) package [53] from BioConductor was used. The designallowed comparison of each of the hypoxic conditions at day 14 toambient air scenario at days 0 and 14 in a paired manner.Additionally, a comparison of adjacent oxygen concentrations wasperformed to explore a relationship between the changes intranscriptional activation and the stepwise reduction of oxygentension. Raw p-values were corrected for multiple testing using thefalse discovery rate controlling procedure of Benjamini andHochberg [54]. Genes found to be regulated more than 2-foldand with an adjusted p-value below 0.01 were consideredsignificant and included in further analysis. Genes were identifiedby Illumina Bead array specific Ids (Illumina ID) and wereannotated using the annotation file provided on the Illuminahomepage [55]. Open source David 2008 (http://david.abcc.ncifcrf.gov/) [56], Cytoscape version 2.5.2 [57], and plug-in,Bingo version 2.0 [58] were used to discover enriched functionsand form a visual presentation of the results. Over-representation(enrichment) of significantly differentially expressed genes infunctional categories was compared to the complete set of knowngenes, which was considered as background. For functionalannotation, clustering, and analysis of enriched functions inDAVID 2008, the default DAVID whole Homo sapiens genomewas used as background, whereas for Bingo, the Illuminaannotation file was applied. This way, the two approachescomplemented each other in the discovery of enriched functionsand the fraction of genes included in a functional cluster, both ofwhich are dependent on the reference background. The following

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12884

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12884

http://david.abcc.ncifcrf.gov/

http://david.abcc.ncifcrf.gov/


annotation categories were included: Gene Ontology (BiologicalFunction all and Molecular function all) and Pathway (BioCartaand KEG-PATHWAYS). Enrichment of functional categories wastested with a modified Fisher Exact p-value (EASE Score) [59], andcategories were considered significantly enriched if Benjamini-adjusted p-values were below 0.05. Only categories of minimumtwo genes were included in the analysis. Functional clustersincluding significantly enriched functional annotations and withenrichment score (negative logarithm of the geometric mean ofcluster members EASE-scores) higher than two were regarded assignificantly enriched.

Real-time RT-PCR

For each sample, 1 μg of total RNAwas used as a template for cDNAsynthesis using the iScript cDNA synthesis kit (Biorad). The primersequences were designed employing the open source programPrimer3 [60] or adapted from previous work [16,61] (Supplement2). All primers were produced by the DNA Technology (Aarhus,Denmark). Each reaction contained 2.5 pmol of each primer, exceptfor 18S, where 1.67 pmol was used, 0.125 μl of undiluted cDNAtemplate, and an iQ SYBR Green Supermix (Bio-Rad) according tostandard protocol in a total volume of 25 μl. Each sample wasanalyzed in duplicate byMy-Cycler real-time PCR system (Bio-Rad,

Fig.1 – Cultures of hASCsmaintained for 14 days at five different oxy×10 magnification. The average population doublings in days are p

Hercules, Ca). The thermocycling program consisted of an initialstep of 3min at 95 °C and 45 cycles of 15 s at 95 °C and the annealingtemperature ranging from 60 to 68 °C for 30 s Product specificitywas verified bymelting curve analysis. The relative expression levelfor each gene was calculated on the basis of a standard curvederived from the pool of all the cDNA samples. In each assay, anegative control without cDNA was included. The ribosomal RNA18S gene was used as a reference gene for normalization in theassessment of chondrogenesis. In the case of the verification ofmicroarray data, the normalization was based on the geometricmean of expression of three reference genes, the tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ),TATAA-box binding protein (TBP), and the beta-glucuronidase(GUSB) [62].

Statistical analysis

The data are presented as arithmetic mean+/−standard devia-tion (SD), unless otherwise stated. Friedman non-parametricstatistics was used to test for the differences between multiplerelated samples, and the results were complemented with post-hoc pairwise analysis by Wilcoxon signed-rank test. The correla-tion between real-time RT-PCR and microarray data was assessedusing Pearson's correlation. The statistical analysis was carried out

gen concentrations. The phase contrast imageswere acquired atresented as a mean+/−standard error of mean (SEM) (n=5).


with the aid of SPSS software package (SPSS, Chicago, Il) and thestatistical significance was assigned to the differences at p<0.05.

Results

Effect of hypoxic preconditioning on proliferation, colonyforming capacity, and chondrogenic differentiation

After two weeks of expansion, the cultures were at least 70%confluent. Morphologically, the cells maintained at low oxygenlevels appeared smaller than those at higher oxygen concentra-tions (Fig. 1). In addition, at 1% oxygen, the cultures contained

Fig. 2 – Functional characteristics of hASCs maintained for 14 daysforming unit fibroblasts (CFU-F) is expressed in proportion to the totstandard error of mean (SEM) (n=5). (B) The gene regulation was ddensity pellet cultures under ambient oxygen tension. The change incontrol cultures. The data are presented as a mean+/−standard err(C) The glycosaminoglycan contents was quantified in induced and uThedata arepresentedas amean+/−standard errorofmean (SEM) f(D) Representative images of induced high-density pellet cultures (A

numerous detached cells, as well as cells with condensed nucleiindicating apoptosis.

Interestingly, when assessing the capacity to support out-growth of the colonies, the highest yields were observed at thelowest oxygen concentration of 1%, with an average 31 CFU-Fs per100 plated cells (Fig. 2A).

The chondrogenic inducibility of the expanded cells wasinvestigated through differentiation in high-density pellet cul-tures. Corresponding uninduced control cultures were used asreferences. All control cultures displayed similar expression levelsof the chondrogenesis marker genes by semi-quantitative RT-PCRindependently of the oxygen tension. The analysis of chondrogenicinduction revealed that both the central transcription factor Sox9and the group of extracellular matrix components, including

at five different oxygen concentrations. (A) The yield of colony-al number of viable cells. The data are presented as amean+/−etermined after three weeks of chondrogenic induction in high-transcriptional expression is expressed relative to uninduced

or of mean (SEM) from five donors analyzed in duplicate (n=5).ninduced control micropellet cultures and normalized to DNA.romfivedonors analyzed in triplicate (n=5).ND:NotDetectable.SC13) stained with alcian blue are shown at ×10 magnification.


collagen I, II, and X, and aggrecan, were upregulated and their levelof expression generally showed a declining trend proportionalwith reduced oxygen concentration (Fig. 2B). Quantification ofGAGs as a measure of the accumulated extracellular matrix did not

demonstrate any significant effect of hypoxic incubation (Fig. 2C).Interestingly though, the histochemical visualization by alcian bluedemonstrated that cultures grown under ambient and 15% oxygenconditions synthesized the highest quantities of extracellular


proteoglycans (Fig. 2D). From the analysis of the chondrogenicassay it thus appears that higher oxygen concentrations provide fora more suitable environment to maintain ASCs chondrogenicpotential than those below 10%.

Effect of inter-individual variability and hypoxic exposureon the relatedness of expression patterns

The expression profiles resulting from culturing in different oxygenconditions were established by microarray analysis. On average,one third of the 47,296 probes incorporated on each of the Illuminachips identified regulated genes. Internal controls verified asuccessful sample processing and hybridization. One outlier arraywas observed for a single experimental condition (ambient air atday 0). To retain the balanced design, the single outlier array wasreplaced with average normalised intensities based on theremaining four arrays from ambient condition at day 0. The moststriking finding from the hierarchical clustering, constructed onthe basis of the neighbour-joining algorithm, was the similarity ofthe expression patterns at 1% oxygen, since these samples groupedon a distinctive branch (Fig. 3A, encircled in red). The other oxygenconditions had no particular effect on the expression patterns asthe clustering of the remaining samples was predominantly donor-dependent. Nevertheless, it appears that donor characteristics playa role since the grouping of two overweight donor cell lines, ASC6and 10, on distinctive branch suggests a higher level of similaritythan that observed between the lean donor cell lines ASC13, 23,and 24.

The 500 genes that varied most (SD>1) across all arrays wereincluded in the k-means clustering to group similar expressionpatterns. The k-means clustering was performed with theassumption of 10 gene clusters. Five expression patterns, compris-ing approximately 70% of the input genes were identified (Fig. 3B).As expected from the hierarchical clustering, the most prominentchanges were observed with a drop to 1% oxygen (clusters 1 and2). Of the 115 genes upregulated only at 1% oxygen tension, 19.3%were significantly implicated in negative regulation of biologicalprocesses (Fig. 3B table). The 96 genes, which were founddownregulated at 1% oxygen tension, were not enriched for aspecific function but included genes related to cell cycle regulation(Fig. 3B table). Clusters 3 and 4 included genes with distinctexpression levels in the ambient samples on day 0. More than halfof the genes upregulated on day 0 in cluster 3 was significantlyinvolved in multi-organism processes, which is a term coveringinteraction processes between organisms of same or differentspecies (Fig. 3B table). Likewise, the cluster 4 was enriched formulti-organism development, although not significantly. Theexpression pattern featured in cluster 5, with an incrementalupregulation with both expansion and reduced oxygen concentra-tion, was common for 61 genes. Significant enrichment was seenhere for functional annotations, such as growth factor activity and

Fig. 3 – Relatedness of transcriptional profiles by hierarchical and kof day 0 and 14 transcriptional profiles was constructed on the bassamples from 1% oxygen are encircled in red. (B) Similarity of genewas determinedwith the aid of k-means clustering based on the 500all arrays (SD>1). The profiles are presented with arbitrary relativline). In the table, listed are biological processes and molecular funindicates statistical significance (p<0.05).

developmental processes (Fig. 3B table). The 147 genes that werenot grouped into any of the five clusters were enriched in biologicalterms, such as developmental processes, biological adhesion, bloodpressure, and response to stress (Fig. 3B table).

Functional classification of transcriptional changes duringin vitro culturing and hypoxic exposure

The two-week period, during which the cells proliferated, had asignificant effect, since at ambient oxygen concentration, 26 geneswere found to be significantly regulated (Supplement 3). Of these26 genes, 17 were also significantly regulated during expansion inthe remaining hypoxic conditions. The 26 genes were enriched fordevelopmental processes, transmembrane receptor protein tyro-sine kinase signaling pathways (receptors TEK, EPHB1, and ERBB3),cell adhesion (extracellular matrix molecules DPT and HAPLN1),and prostaglandin biosynthetic processes (synthases PTGS1 andPTGIS). Relevant gene name abbreviations are available in Supple-ment 4. Taking into account exclusively hypoxic expansion, 28genes were significantly regulated across all conditions, and 75% ofthem were upregulated. It is interesting to note that BMP6, whichplays a central role in cartilage development, was found in thelatter group. Many of the upregulated genes could be found inclusters 4 and 5 from the k-means clustering (Fig. 3B).

After two weeks of expansion, only 5% and 1% oxygen weresufficiently distinct from ambient conditions to yield significantlyregulated genes. At 5% oxygen tension, 59 genes were identified, ofwhich approximately 40% were upregulated (Supplement 5). Theidentified genes were enriched for two functional clustersconcerning the response to nutrient levels/external stimulus andthe cell–cell signalling through receptor binding. The most notablegene belonging to both functional clusters was LEP with nearly 10-fold upregulation. Among the genes with a function in response tonutrients levels, three with role in oxidative stress (DDIT3, HMOX1,and TXNIP) were identified. Considering the specific biologicfunctions that were enriched, the term developmental processeswas the only one significantly enriched and contained approxi-mately 45% of the genes. The LEP transcript was also found in thedevelopmental processes group along with two other interestingtranscripts related to chondrogenesis and cell proliferation,CHRDL2 and KIT, respectively. Seven of the genes in the develop-mental processes gene list were additionally related to apoptosis(DDIT3, DDIT4, CARD9, SERPINB2, TRIB3, HMOX1, and CEBPG) andwere all downregulated. Of the 59 genes significantly regulated at5% oxygen, 38 were also significantly regulated at 1% oxygen. Themajority of the genes common to the two hypoxic conditionsdemonstrated a higher fold regulation in 1% oxygen.

A pairwise comparison of adjacent hypoxic conditions revealedthat first during lowering the oxygen concentration from 10% to 5%,a group of differentially regulated genes became evident (Table 1).From the total of 55 significantly regulated genes, 18 were

-means clusterings. (A) The unrooted hierarchical dendrogramis of Euclidean distances and neighbor-joining algorithm. Theexpression patterns by the culture day and oxygen tensiongenes exhibiting highest fluctuations in expression levels acrosse expression levels centered around the expression mean (redctions enriched in the gene clusters, where the boldface type

Table 1 – Main biological processes and molecular functions enriched in significantly regulated genes identified duringcomparison of adjacent oxygen tensions

Oxygen step Functional annotation P-value No. of genes % of genes total

10%–5% 55Upregulated genes 33Downregulated genes 67

5%–1% 936Upregulated genes 64

Apoptosis 9.4E−5 5Post-translational protein modifications 4.1E−4 8Cellular processes 4.9E−1 40Transcription 1.3E−1 11

Downregulated genes 36Primary metabolic processes 1.6E−2 44Cellular metabolic processes 1.0E−2 43Macromolecular metabolic processes 8.3E−1 36Cellular processes 1.0E0 56Transcription 1.0E0 13

5%–1% specific a 97Upregulated genes 79

Transcription regulatory activity 9.9E−1 4Protein kinase binding 1.0E0 12

Downregulated genes 21Primary metabolic processes 9.9E−1 13

a Were not found in the significantly regulated genes between day 14 ambient and 1% oxygen tensions.


upregulated, and these notably included previously mentioned LEPand CHRDL2 genes. The DNA damage inducible transcripts, DDIT3and 4, together with CARD9, were found among the downregulatedgenes. As expected, during the further step from 5% to 1% oxygen,substantially more, 936 in total hypoxia-regulated genes appeared(Table 1).

Identification of functional clusters associated with1% hypoxia

A total of 2581 genes were found significantly regulated at 1%oxygen when compared to the reference at ambient conditions onday 14. Of these genes, 46% were upregulated, and they weresignificantly enriched in several functional clusters, among whichthe most prominent were the developmental processes, apoptosis,cell proliferation, and transcriptional regulation (Fig. 4A). Thecluster of developmental processes included 254 genes. Of specialinterest, in context to expansion conditions and the intended usein cartilage engineering, were the genes involved in chondrogen-esis and bone formation (CHRD, RUNX2, CHRDL2, NOG, and ACVR1),and the genes responsive to nutrient levels as well as the hypoxicconditions (LEP, ANGPT2, ANG, PPARG, and APOE) (Fig. 4B). In theapoptosis cluster, 73 genes were included. Of these, 48 wereinvolved in the regulation of apoptosis, 19 were anti-apoptotic orsurvival genes, e.g. BCL2 and SOD2, and 14 genes had apoptosisinducing functions (e.g. PERP, HRK, and TNFRSF19). Interestingly,the apoptosis cluster also included genes involved in DNA damageand resulting growth arrest (GADD45A, GADD45G, and DDIT3). Thecell proliferation group consisted of 68 genes, of which 25 wereshared with the apoptosis cluster. The inhibitory effect on cellreplication was represented by three cyclin-dependent kinaseinhibitors, p19, p27, and p57, involved in G1 arrest and CHK1,which is implicated in checkpoint-mediated cell cycle arrest in

response to DNA damage or the presence of unreplicated DNA.Genes with a positive effect on cell replication were also found inthe cluster, such as KIT, PIM2, VEGFB, IRS2, EGFR, VEGF, CSF2, IGF2,PIM1, and FGF9. Several classes of transcription factors, includingZinc finger and forkhead box proteins, along with HOX genes, wereincluded in the cluster of 174 genes belonging to regulation oftranscription. In total, 71 genes had transcription factor activity. Inparticular, two transcription factors, ARNT and HEY1 are worthnoting, since they are part of the HIF and Notch signaling pathways,respectively.

The major significantly enriched functional clusters thatcomprised the list of downregulated genes were represented byRNA processing, organic acid metabolic process, and primarymetabolic process, (Fig. 5A). Of the 59 genes in the RNA processingcluster, 37 were involved in mRNA processing, 10 in tRNAprocessing, and 4 in rRNA processing (Fig. 5B). Distinctive for themRNA processing genes was the RNA splicing function. Theprimary metabolic process cluster constituted a variety ofbiological functions and shared gene members with both theRNA processing and organic acid metabolic processes clusters. Themost prominent group of genes was involved in the nucleobase,nucleoside, nucleotide, and nucleic acid metabolic process. CyclinsD2 and D3, together with p15, were also identified in the primarymetabolism cluster. Notably, three effectors of mitochondrialmediated apoptosis, BID, cytochrome C, and APAF1, were foundin this cluster downregulated. Aminemetabolic processes were themain function in the organic acid metabolic processes cluster.

As indicated above, the exposure to 1% oxygen significantlymodulated expression profiles when compared to the nearestassayed hypoxic condition at 5% oxygen. In Table 1, the enrichedbiological processes annotated to the gene list can be found. Notsurprisingly, biological processes such as post-translational proteinmodification, apoptosis, and regulation of biologic processes were

Fig. 4 – Gene ontology hierarchy indicating the major categories of biological processes overrepresented in upregulated genes at1% oxygen. Genes significantly regulated more than 2-fold between ambient and 1% oxygen on day 14 were enquired for GeneOntology enrichment in a hyper geometric test with Benjamini and Hochberg correction for multiple testing. (A) Significantlyenriched functions are in yellow (p<0.05), and the size of nodes corresponds to the number of genes annotated to the given geneontology term. The functions most relevant to hypoxia are highlighted in red frames. (B) Characteristics of the biological processesand molecular functions that were identified in (A) by red frames.



significantly overrepresented. The widely covering gene ontologycategories of primary, cellular, and macromolecular metabolicprocess mostly included downregulated genes. The upregulatedgenes were annotated to biological processes of apoptosis and posttranslational protein modification. Both up- and downregulated

Fig. 5 – Gene ontology hierarchy indicating the major categories of1% oxygen tension. Genes significantly regulated more than 2-foldGene Ontology enrichment in a hyper geometric test with Benjaminenriched functions are presented on a scale fromyellow (p<0.05) toof nodes corresponds to the number of genes annotated to the givenhighlighted in red frames. (B) Characteristics of the biological procframes.

genes were found in regulation of cellular processes and wereinvolved in transcription. Of the significantly regulated genes, 97were not previously identified in comparison of 1% oxygen toambient conditions at the day 14 (Table 1). Here, the 77upregulated genes were enriched in transcription regulator

biological processes overrepresented in downregulated genes atbetween ambient and 1% oxygen on day 14 were enquired fori and Hochberg correction for multiple testing. (A) Significantlydark orangewith p-value 5 orders of magnitude lower. The sizegene ontology term. The functionsmost relevant to hypoxia areesses and molecular functions that were identified in (A) by red


activity and protein kinase binding, whereas the downregulatedgenes were enriched in primary metabolical process. Two of thegenes, TRIB3 and ATF3, in the transcription regulator activity

Fig. 6 – Real-time RT-PCR analysis of molecular functions associatedpreconditioning in different hypoxic conditions. (A) Selected biolomicroarray analysis, included differentiation, cell cycling, and growof mean (SEM) from five donors analyzed in duplicate (n=5). Thecultures at the day 14, which is indicated by a horizontal line. Asteriambient condition. The table indicates correlation of the real-time1% oxygen concentration. (B) Key regulators of chondrogenesis wepresented as amean+/−standard error ofmean (SEM) from five donormalized to the respective ambient cultures at the day 14, whichdifference (p<0.05) with respect to the ambient condition.

cluster were with approximately 5-fold upregulation among thethree most regulated genes. The most regulated gene with almost7-fold upregulation was G0S2, which has been identified as a

with hypoxic responses and chondrogenesis after a 14-day longgical processes that were found significantly regulated inth arrest. The data are presented as a mean+/−standard errorhypoxic conditions were normalized to the respective ambientsks denote a significant difference (p<0.05) with respect to theRT-PCR data with those obtained by a microarray analysis forre selected to assess their hypoxic responses. The data arenors analyzed in duplicate (n=5). The hypoxic conditions wereis indicated by a horizontal line. Asterisks denote a significant


PPARγ target and associated with adipogenic differentiation aswell as growth arrest [63].

Verification of gene expression patterns by semi-quantitativeRT-PCR

Since the genes implicated in cell differentiation, cell cycle, andgrowth arrest were mostly affected by the oxygen-dependentregulation, 11 genes covering these biological processes werefurther analyzed by the semi-quantitative RT-PCR (Fig. 6A). Allgenes in the differentiation group displayed progressive upregula-tion with declining oxygen concentration. Similarly, the threetumor suppressor genes, p57, p27, and p19, from the group ofgenes involved in the regulation of cell cycle were upregulatedproportionally to the level of hypoxia, except for the Cyclin D2(CCND2) that was significantly downregulated at 1% oxygen. Incontrast to the two previous groups, the genes implicated inapoptosis and DNA damage-induced growth arrest becamediscretely upregulated exclusively at the highest level of hypoxia.Overall, the semi-quantitative RT-PCR and microarray data were ingood agreement (correlation coefficient>0.95) thus corroboratingaccuracy of the Illumina-based transcriptional analysis (Fig. 6Atable). In addition to genes readily responsive to hypoxia,differential regulation of some of the key factors implicated inchondrogenesis was explored as well (Fig. 6B). Most importantly,contrary to 5% oxygen, where the prevalent pattern appeared to beupregulation, in 1% oxygen, the gene expression seemed to bemainly suppressed.

Discussion

In the present investigation, independent ASC lineswere examinedto study the significance of low oxygen tension for the enrichmentof chondrogenic progenitor cells. The global gene expressionanalysis demonstrated distinctive transcriptional response to 1%oxygen concentration when compared to mild hypoxic expansionand, notably, highlighted the importance of biological variability.Previous studies have already shown that ASCs phenotype is notconsistent and is determined by individual donor characteristicssuch as age, gender, and BMI [64–67], but the mode of tissueharvesting has also been suggested to play a role [43,68]. Despiteattempting to minimize the donor-related variability by restrictiveinclusion criteria and standardizing the experimental approachesin our study, the hierarchical clustering demonstrated considerableinter-individual differences. It thus appears that ASC populationsdisplay unique properties dependent on the donor, and thischaracteristic may at least in part account for the discrepanciesbetween different studies [17,37,69]. Although the exact nature ofthe observed variability is not known, it is conceivable thatmicroenvironment associated with individual-specific fat distribu-tion and BMI is a contributory factor. More analysis is required inorder to provide a better understanding of the relationshipbetween donor BMI and the hypoxic response of ASCs.

Taking into account ambient conditions, in the culturesexpanded for 14 days, 26 genes were differentially expressed andthe majority of activated genes were involved in developmentalprocesses, indicating a transition from the proliferative phase.Similar pattern was previously observed with BMSCs [70]. Theeffect of exposure to lower hypoxic conditions of 5 and 1% oxygen

seemed to inhibit the culture proliferation during the 14-dayperiod but also favor preservation of clonogenic precursor cells.Selection of a more replicative cell type by long-term hypoxicexpansion has been demonstrated in previouswork [13,24,32], andthe proportions of colony-forming progenitors reported herein arewell within the range of published figures [17]. In this context, thechoice of expansion medium has to be considered. Specifically, theexpansion medium has in previous studies been proven to affectthe outcome of cell population and its differentiation potential[12,22] as well as its gene expression profile [38]. Thus it appearsthat this variable can in a potentially significant way contribute tothe discrepancies frequently seen in the literature [71,72].Traditionally, for the expansion of ASCs have been used theDulbecco's modified eagle medium (DMEM) [1,28,73–75] orDulbecco's modified eagle medium Ham's F-12 nutrient mixture(DMEM/F12) [12,17,37,69,76,77]. However, in the current investi-gation, α-MEM was chosen for expansion due to its presumablypositive effect on the chondrogenic potential and proliferation[22], and also from our own experience (manuscript in press).Consequently, a caution should be taken when comparing directlyresults from the present study with those based on ASCs culturedin the traditional media choices [18,38], let alone those using othertypes of MSCs [29,34,35]. To our knowledge, a comprehensiveinvestigation of the expression profiles of ASCs expanded in themost common growth media has not been conducted. This couldbe accomplished using the growing number of array studies thatare publicly available in databases, such as NCBI's Gene ExpressionOmnibus [52].

The transcriptional responses associated with hypoxia emergedfirst at 5% oxygen concentration but unfolded fully at 1% oxygen, asis apparent from 59 and nearly 2600 genes becoming differentiallyregulated, respectively. At 1% oxygen, the cultures were subject togrowth arrest in more phases of the cell cycle, as indicated in Fig.6A. The G1-phase arrest is known to be triggered with over-expression of members of both the Cip/Kip and the INK4 family ofcyclin dependent kinase inhibitors, represented by p57, p27, andp19 [78–80]. For p27, the induction has been proposed to be HIF-1dependent [27]. However the direct link between cyclin dependentkinase inhibitors and hypoxia regulation are still being debated, asdiscrepant results have been obtained in a number of cell types[80,81]. Growth arrest and DNA-damage-inducible transcript,GADD45G, and checkpoint, CHK1, are linked in the p53 pathwayand are involved in preventing cell cycle progression fromboth G1-and G2-phase in response to DNA damage [82,83]. DNA damage-induced activity in the p53 pathway will lead, if extensive andbeyond repair, to cell death through apoptosis [84]. The pre-apoptotic state was confirmed by upregulation of the p53apoptosis effectors, PERP and HRK. PERP is one of the intracellularregulatory proteins which functions to connect the apoptoticsignaling initiated by e.g. hypoxia or DNA damage in the p53pathway to the effectuation of cell suicide [85]. HRK inducesapoptosis via interactionwith B-cell CLL/lymphoma 2, Bcl-2 [86], asurvival-promoting protein that negatively regulates apoptosisthrough inhibition of the release of mitochondrial cytochrome C[87]. The downregulation of the three effectors of mitochondria-regulated apoptosis, the APAF1, cytochrome C, and BH3-interactingdomain death antagonist, BID, provided further evidence forapoptotic activation. It is important to note that, at the sametime, anti-apoptosis mechanisms in the form of cell cycle arrestwere activated to provide for the cell survival. In tumour cells, for


example, the super-induction of GADD45G in response to the jointeffect of DNA damage and hypoxia has been hypothesised tosupport cancer cell survival during therapy [88]. Thus the survivalas well as the enhanced colony forming ability of ASCs cultured at1% oxygen may be the result of a balance between growth arrestand apoptosis that promotes the selection of the “fittest”. Theculture confluency in combination with low oxygen tensionresembles an ischemic environment characterized by hypoxiaand nutrient deprivation. In previous studies on ischemia, BMSCswere able to survive and retain their differentiation potential forup to 72 h of the treatment [89]. Moreover, Zhu et al. [90] provedthe shortage of available serum to be the major cause of apoptosisin ischemia simulated by hypoxia and serum deprivation. In lightof these data, it is plausible that, in the current investigation, thepre-apoptotic stage of the cultures at 1% oxygenwas a reaction notto undergo a complete apoptosis by means of keeping the cellpopulation subconfluent.

It was surprising to observe that despite the cells expanded at5 and 1% oxygen were enriched for CFU-Fs, they did not exhibitan enhanced chondrogenic differentiation when induced by TGF-β3 in high-density pellet cultures. A comparison with previousstudies is challenging, since the culture format, hypoxic condi-tions, expansion periods, and the cell type vary greatly betweeninvestigations [13,24,28,32,34,91]. At the molecular level, signifi-cant upregulation of BMP6 at 5% oxygen would indicate activationof TGF-β signaling, which according to previous studies byHennig et al. [16] and Estes et al. [23] should be reflected byenhanced chondrogenesis. This, however, did not materialize,possibly as a result of upregulation of CHRDL2, NOG, and CHRD,all of which are known inhibitors of BMP signaling [92,93]. In thiscontext, further experiments based on protein analysis arewarranted. Previously, it has been suggested that hypoxia maypromote chondrogenesis through upregulation of the centraltranscription factor Sox9 in the mouse model [91]. In the currentstudy, hypoxic exposure induced Sox9 expression at the levels nothaving a statistical significance, however, most critically theexpression of Sox9 was significantly suppressed at 1% oxygen. Inview of the significance of Sox family for chondrogenesis [94,95],then the deficiency of chondrogenic differentiation in thishypoxic scenario is predictable; nevertheless, the understandingof the inefficiency of cartilage formation in milder hypoxia willneed further explanation.

Although it appears, based on the results from this investiga-tion, that the hypoxic preconditioning is not beneficial in terms ofstimulating chondrogenesis, the molecular analysis indicates thatsuch treatment may support specification into other differentia-tion pathways. One of the scenarios may involve facilitation ofosteogenesis through the action of leptin (LEP). LEP is knownmostly for its role as a key regulator of bodyweight and food intake[96], but importantly it is inducible in hypoxia by HIF-1 [97,98] andconsequently was found significantly upregulated in the currentstudy at 5 and 1% oxygen concentrations. However, from thedevelopmental point of view important is that LEP possessesangiogenic activity and also has been found to play a pivotal part inthe longitudinal growth of bone via endochondral ossification[99,100]. Other scenarios would probably be concerned withprocesses involving angiogenesis, since our cultures expanded at1% oxygen tension expressed both mitogenic (e.g. IGF2, FGF9, KIT,and CSF2) and angiogenic factors (VEGF, ANG, LEP, and ANGPT2).Hypoxic preconditioning thus could be of value for the treatment

of ischemic conditions, and first data based on BMSCs in themurine model of myocardial infarction indicate feasibility of suchapproach [33,98,101]. In light of the evidence in this and otherstudies, it is evident that the oxygen conditions during monolayerexpansion of MSCs are of great importance. The challenge is to findthe best combination of physical and biochemical parameters,along with appropriate sequence and duration of the treatmentintervals, to provide for the most suitable outcome for a givenapplication.

Acknowledgments

The authors wish to thank plastic surgeons Frants Grymer andChristian Bang, their office and nursing staff, and their patients atGrymer Privat Hospital, Aarhus, for donation of the liposuctionmaterial. The expert technical assistance of Helle Skjødt Møller andMette Bøgh Ringaard is highly appreciated. The valuable inputfrom Tim Watts from the Genomics group at The Welcome TrustCentre for Human Genetics in Oxford is also acknowledged. Theauthors greatly appreciate support from The John and BirtheMeyerand Toyota Foundations, and the Danish Medical Research Councilgrants no. 271-06-0283 and 2052-01-0045. Also, grants from theFamily Hede Nielsen Foundation, Mrs. Johanne Louise Berg bornOppermann, and Director Ditlev Berg, and the scholarship fromHorsens Statsskole are highly valued. The funding sources had noinfluence on neither study design, collection, analysis andinterpretation of data, writing of the report, nor the decision tosubmit the paper for publication.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.yexcr.2009.01.020.

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transcriptional signature of human adipose tissue-derived stem cells (hascs) preconditioned for...

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