Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1937 – 195 2

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions L. Pilgaard a , P. Lund a , M. Duroux a , H. Lockstone b , J. Taylor b , J. Emmersen a , T. Fink a , J. Ragoussis c , V. Zachara,⁎ a

Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg, Denmark Bioinformatics and Statistical Genetics, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford, OX3 7BN, UK c Genomics, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford, OX3 7BN, UK b

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

AB ST R AC T

Article Chronology:

Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into

Received 5 December 2008

the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic

Revised version received

differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and

19 January 2009

global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen

Accepted 20 January 2009

concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-

Available online 2 February 2009

forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not

Keywords:

improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-

Adipose tissue-derived stem cells

donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a

cDNA microarray

specific expression profile with 2581 significantly regulated genes and substantial functional

Illumina

enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1%

Gene expression

oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular,

Hypoxia

leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated.

Colony-forming unit

In conclusion, the results of this investigation demonstrate the significance of donor demographics

Chondrogenesis

and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential. © 2009 Elsevier Inc. All rights reserved.

Introduction Adipose tissue is a plentiful and easily accessible source of mesenchymal stem cells that have been shown to have multipotent abilities regarding differentiation and possibly immuno suppressive capacity [1–3]. Adipose tissue-derived stem cells (ASCs) are therefore a promising alternative to bone marrowderived mesenchymal stem cells (BMSCs) and have been a popular ⁎ Corresponding author. E-mail address: [email protected] (V. Zachar). 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.01.020

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

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the donor-matched BMSCs have been demonstrated to have superior chondrogenic capacity than ASCs [10,11]. Still, the accessibility and numbers of ASCs advocate their application over BMSCs, as the chondrogenic potential of ASCs can be enhanced through the culturing conditions [12–16]. The heterogeneity of early ASC cultures and evidence of a gradual phenotypic homogenization with in vitro propagation have prompted researchers to focus on two dimensional culturing conditions to enhance the chondrogenic potential [1,17–20]. More specifically, it has been shown that the fraction of ASCs expressing CD105, which is a regulatory component of the TGF-β receptor complex, increased during the course of four passages from 5% to 70% [17]. Thus, as a result of simple in vitro culturing, the ASCs may acquire properties that make them more susceptible to TGF-β treatment, and consequently induction for chondrogenesis. In both BMSC and ASC cultures, growth conditions exploiting specific seeding density, culture formats, and addition of growth factors such as FGF2 and BMP6 or optimized culture periods have proven beneficial for the chondrogenic potential [12,13,15,16,21–23]. Furthermore, numerous studies have identified hypoxia to be essential in differentiation processes, stem cell proliferation and survival, as well as in the maintenance of stem cell characteristics [24–29]. Especially in light of the fact that oxygen has a central role in cartilage development and chondrocyte metabolism, manipulation of culture gaseous phase lends itself as a valuable tool to exploit stem cells to their full potential. Recently, Chen et al. demonstrated that hypoxic exposure has the capacity to induce chondrogenesisspecific transcriptional machinery [30] but found that the full course of chondrogenesis is better supported at ambient air oxygen concentration [31]. In line with these findings, several recent investigations have adopted a hypoxic preconditioning step to improve stem cell function and survival prior to differentiation or clinical application [32–34]. For instance, Grayson et al. [32] demonstrated that long-term cultivation of BMSCs in chronic hypoxia promoted a highly proliferative phenotype with an enhanced capacity of osteogenesis and adipogenesis compared to ambiently expanded cultures. In a complementary investigation, applying a short 24 h hypoxic preconditioning of BMSCs, the potential for chondrogenesis was enhanced and likewise accompanied by an increased proliferation [34]. Monolayer cultures of ASCs subjected to hypoxic expansion for a week have been shown to be enriched for chondrogenic progenitors while maintaining an undifferentiated phenotype [26]. Most frequently, the oxygen concentration employed in these studies was around 2%. However, the applied instrumental setups, culture formats, and culture periods are diverse to such an extent that satisfactory inter-study comparisons are difficult if not unfeasible [18,21,22]. The utility of global gene expression analysis through microarrays in stem cell research is increasing. Such approach has previously been instrumental, for example, in the comparative analysis of transcriptional fingerprints of mesenchymal stem cells and early differentiated fetal tissue [35]. Moreover, in BMSCs, a number of candidate genes possibly regulating stem cell selfrenewal have been identified by the aid of microarrays and the elegant application of in vitro differentiation and dedifferentiation protocols [36]. The BMSCs, along with cord blood CD133+, and freshly isolated mononucleated cells from bone marrow have also been studied with regard to hypoxic expansion [29,34]. As for the ASCs, their transcriptional profile has previously been investigated by Katz et al. [37], and compared to MSCs from other sources

[38,39]. In addition, several studies have analyzed the transcriptional changes in ASCs undergoing differentiation [40–42]. However, the number of microarray investigations of ASCs gene expression is limited. In particular, the effect of hypoxic culture on ASCs has not previously been subject to a large-scale analysis. In the current study, we set out to investigate the effect of hypoxic monolayer ASCs expansion on transcriptional activation at a genomic level. By exploiting a hypoxic workstation (XVivo; BioSpherix, Redfield, NY), we conducted the expansion of six ASC lines at five different oxygen concentrations (ambient, 15%, 10%, 5%, and 1%) in parallel, enabling thus a direct comparison between diverse atmospheric conditions. In the present study, the hypoxiainduced transcriptional changes were related to the results from functional testing of the hypoxia conditioned cultures. To our knowledge, this is the first study applying genome-wide expression profiling to obtain the molecular background for the phenotypical changes brought about by monolayer expansion of ASCs under reduced oxygen tension.

Materials and methods Isolation of adipose tissue-derived stem cells All materials were obtained from Invitrogen (Carlsbad, Ca) or SigmaAldrich (Broendby, Denmark) unless otherwise stated. Protocols involving human subjects were reviewed and approved by the regional Committee on Biomedical Research Ethics in Northern Jutland prior to the study. Samples of subcutaneous adipose tissue were obtained after informed consent from six female donors (age 22–44 years, mean 33.7 +/− 8.3 and body mass index (BMI) 20.6– 34.3, mean 24.2 +/− 5.4) undergoing elective surgery at the Grymer Private Hospital, Skejby, Denmark (Supplement 1). They were designated ASC6,10,13, 23, and 24. All tissue samples were harvested using a tumescent technique with pump-assisted aspiration using a saline solution supplemented with lidocaine and epinephrine [43]. ASCs were isolated as previously described [44] with slight modifications. In brief, the tissue was washed three times with matching volumes of prewarmed Dulbecco's phosphate-bufferedsaline (D-PBS) and then an equal volume of collagenase buffer consisting of 0.28 Wünch U/ml crude collagenase mix (Lot. No. LTQ5230; Wako, Neuss, Germany) in PBS with 20 mg/ml bovine serum albumin (BSA) (Roche Applied Science, Hvidovre, Denmark) was added. Following digestion at 37 °C with gentle agitation for 1 h, the released cells were isolated by velocity sedimentation at 400 ×g for 10 min, and the pelleted cells were filtered through a 70 μm mesh cell strainer (BD Bioscience, Broendby, Denmark). Contaminating erythrocytes were lysed and the remaining nucleated cells were further purified through a second round of centrifugation and filtration. The cells were seeded at a density corresponding to 0.15 ml adipose 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 to remove 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, the cell lines were thawed and cultured for one passage in growth

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medium until they reached 80% density. All normoxic cell cultures were kept in a humidified atmosphere containing 5% CO2 buffered with ambient air at 37 °C, and the medium was changed twice a week. For the hypoxic exposure, the cultures were initiated at 1000 cells/cm2, and allowed to progress for 24 h at ambient air conditions. The subsequent expansion was performed simultaneously 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 changed twice a week, and it was independently preequilibrated to each oxygen 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 96well culture plate to produce seeding densities of 1000 to 0.5 cells per well. The cells were cultured for 11 days followed by formalin fixation, methylene blue staining, and analysis as described previously [45].

Chondrogenic assay For chondrogenic induction, a high glucose (4.5 g/l) Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 ng/ml TGF-β3 (RnD Systems, Oxon, United Kingdom), 10− 7 M dexamethazone, 50 μg/ml L-ascorbic acid 2-phosphate, 40 μg/ml Lproline, antibiotics and an insulin, selenium, and transferrin supplement (ITS; BD Bioscience, Broendby, Denmark) was used. The inducibility of the cells was assessed in a high-density pellet culture format previously described by Penick et al. [46]. The cultures comprised 2 × 105 cells pelleted by centrifugation at 500 ×g for 5 min in 96-well plates with V bottom (cci3896; Corning, Schiphol-Rijk, The Netherlands). The induced as well as the control cultures that were maintained in the growth medium were kept in a humidified atmosphere containing 5% CO2 buffered with an ambient air at 37 °C for three weeks. Media were changed three times a week. After conclusion of chondrogenic induction, the pellets were fixed with 4% formaldehyde over night, washed with D-PBS, and further processed using standard procedures for paraffin embedding and sectioning. Accumulation of proteoglycans was detected by alcian blue 8GX staining (1 g/l in 0.1 M HCl, pH 1) for 30 min at room temperature, and Mayer's hematoxylin (Bie and Berntsen) was used as a counterstain. The stained sections were surveyed with the aid of a Zeiss Axiovert 200M inverted microscope (Brock and Michelsen, Birkeroed, Denmark) using a 10×/0.25 A-Plan objective. The images were acquired and processed with the Zeiss AxioVision software package (Brock and Michelsen).

Glycosaminoglycan quantification The glycosaminoglycans (GAGs) were quantified according to the method previously described by Barbosa et al. [47]. In brief, the cultures were digested with a collagenase buffer described above. The released GAGs were complexed to the dichromatic dye 1, 9 dimethyl methylene blue (DMMB) at pH 3 for 1 h at 600 RPM. The GAG–DMMB complexes were isolated by sedimentation at 12,000 ×g for 5 min and decomplexed at neutral pH. The absorbance at 656 nm was measured and converted to GAG contents on the basis of a standard, for which the chondroitin-4-sulfate was used. The concentrations were normalized to DNA content determined by a Pico Green assay according to manufactures instructions.

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Isolation of RNA and microarray analysis Total RNA was isolated using the Aurum total RNA mini kit (BioRad, Copenhagen, Denmark) as recommended by the manufacturer and the Illumina Total Prep RNA Amplification Kit (Illumina, San Diego, Ca) was used to produce in vitro transcribed and biotin labeled cRNA. RNA integrity was assessed by a capillary electrophoresis (Agilent 2100 Bioanalyzer; Naerum, Denmark), and the purity and concentration were determined by spectrophotometery (Nanodrop; Thermo Science, Wilmington, De). The cRNA was hybridized to Illumina Bead arrays, human Ref 6 chips (Illumina), which generate whole genome transcription profiles simultaneously for six samples. The arrays were scanned using the Illumina Bead station 500GX System.

Array data processing The background correction that was based on the average signal from a set of internal negative controls within individual arrays was performed by the Illumina Beadstudio software (Illumina Inc, San Diego, CA), and the data were further processed and analysed using R [48] and BioConductor packages [49] as well as Multiexperiment Viewer (MeV) from the TM4 suite of microarray tools [50]. The outlier samples were identified using principal component analysis (PCA), and hierarchical and k-means clustering. Such samples were removed prior to variance stabilisation and quantile normalisation using algorithms implemented in the VSN package [51]. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [52] and are accessible through GEO Series accession number GSE12884 (http://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE12884). To identify significant changes in gene expression associated with hypoxic conditions, the linear models for microarray analysis (LIMMA) package [53] from BioConductor was used. The design allowed comparison of each of the hypoxic conditions at day 14 to ambient air scenario at days 0 and 14 in a paired manner. Additionally, a comparison of adjacent oxygen concentrations was performed to explore a relationship between the changes in transcriptional activation and the stepwise reduction of oxygen tension. Raw p-values were corrected for multiple testing using the false discovery rate controlling procedure of Benjamini and Hochberg [54]. Genes found to be regulated more than 2-fold and with an adjusted p-value below 0.01 were considered significant and included in further analysis. Genes were identified by Illumina Bead array specific Ids (Illumina ID) and were annotated using the annotation file provided on the Illumina homepage [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 functions and form a visual presentation of the results. Over-representation (enrichment) of significantly differentially expressed genes in functional categories was compared to the complete set of known genes, which was considered as background. For functional annotation, clustering, and analysis of enriched functions in DAVID 2008, the default DAVID whole Homo sapiens genome was used as background, whereas for Bingo, the Illumina annotation file was applied. This way, the two approaches complemented each other in the discovery of enriched functions and the fraction of genes included in a functional cluster, both of which are dependent on the reference background. The following

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annotation categories were included: Gene Ontology (Biological Function all and Molecular function all) and Pathway (BioCarta and KEG-PATHWAYS). Enrichment of functional categories was tested with a modified Fisher Exact p-value (EASE Score) [59], and categories were considered significantly enriched if Benjaminiadjusted p-values were below 0.05. Only categories of minimum two genes were included in the analysis. Functional clusters including significantly enriched functional annotations and with enrichment score (negative logarithm of the geometric mean of cluster members EASE-scores) higher than two were regarded as significantly enriched.

Real-time RT-PCR For each sample, 1 μg of total RNA was used as a template for cDNA synthesis using the iScript cDNA synthesis kit (Biorad). The primer sequences were designed employing the open source program Primer3 [60] or adapted from previous work [16,61] (Supplement 2). All primers were produced by the DNA Technology (Aarhus, Denmark). Each reaction contained 2.5 pmol of each primer, except for 18S, where 1.67 pmol was used, 0.125 μl of undiluted cDNA template, and an iQ SYBR Green Supermix (Bio-Rad) according to standard protocol in a total volume of 25 μl. Each sample was analyzed in duplicate by My-Cycler real-time PCR system (Bio-Rad,

Hercules, Ca). The thermocycling program consisted of an initial step of 3 min at 95 °C and 45 cycles of 15 s at 95 °C and the annealing temperature ranging from 60 to 68 °C for 30 s Product specificity was verified by melting curve analysis. The relative expression level for each gene was calculated on the basis of a standard curve derived from the pool of all the cDNA samples. In each assay, a negative control without cDNA was included. The ribosomal RNA 18S gene was used as a reference gene for normalization in the assessment of chondrogenesis. In the case of the verification of microarray data, the normalization was based on the geometric mean 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 deviation (SD), unless otherwise stated. Friedman non-parametric statistics was used to test for the differences between multiple related samples, and the results were complemented with posthoc pairwise analysis by Wilcoxon signed-rank test. The correlation between real-time RT-PCR and microarray data was assessed using Pearson's correlation. The statistical analysis was carried out

Fig. 1 – Cultures of hASCs maintained for 14 days at five different oxygen concentrations. The phase contrast images were acquired at ×10 magnification. The average population doublings in days are presented as a mean +/− standard error of mean (SEM) (n = 5).

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

Results Effect of hypoxic preconditioning on proliferation, colony forming capacity, and chondrogenic differentiation After two weeks of expansion, the cultures were at least 70% confluent. Morphologically, the cells maintained at low oxygen levels appeared smaller than those at higher oxygen concentrations (Fig. 1). In addition, at 1% oxygen, the cultures contained

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numerous detached cells, as well as cells with condensed nuclei indicating apoptosis. Interestingly, when assessing the capacity to support outgrowth of the colonies, the highest yields were observed at the lowest oxygen concentration of 1%, with an average 31 CFU-Fs per 100 plated cells (Fig. 2A). The chondrogenic inducibility of the expanded cells was investigated through differentiation in high-density pellet cultures. Corresponding uninduced control cultures were used as references. All control cultures displayed similar expression levels of the chondrogenesis marker genes by semi-quantitative RT-PCR independently of the oxygen tension. The analysis of chondrogenic induction revealed that both the central transcription factor Sox9 and the group of extracellular matrix components, including

Fig. 2 – Functional characteristics of hASCs maintained for 14 days at five different oxygen concentrations. (A) The yield of colonyforming unit fibroblasts (CFU-F) is expressed in proportion to the total number of viable cells. The data are presented as a mean +/− standard error of mean (SEM) (n = 5). (B) The gene regulation was determined after three weeks of chondrogenic induction in highdensity pellet cultures under ambient oxygen tension. The change in transcriptional expression is expressed relative to uninduced control cultures. The data are presented as a mean +/− standard error of mean (SEM) from five donors analyzed in duplicate (n = 5). (C) The glycosaminoglycan contents was quantified in induced and uninduced control micropellet cultures and normalized to DNA. The data are presented as a mean +/− standard error of mean (SEM) from five donors analyzed in triplicate (n = 5). ND: Not Detectable. (D) Representative images of induced high-density pellet cultures (ASC13) stained with alcian blue are shown at ×10 magnification.

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collagen I, II, and X, and aggrecan, were upregulated and their level of expression generally showed a declining trend proportional with reduced oxygen concentration (Fig. 2B). Quantification of GAGs 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 blue demonstrated that cultures grown under ambient and 15% oxygen conditions synthesized the highest quantities of extracellular

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proteoglycans (Fig. 2D). From the analysis of the chondrogenic assay it thus appears that higher oxygen concentrations provide for a more suitable environment to maintain ASCs chondrogenic potential than those below 10%.

developmental processes (Fig. 3B table). The 147 genes that were not grouped into any of the five clusters were enriched in biological terms, such as developmental processes, biological adhesion, blood pressure, and response to stress (Fig. 3B table).

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

Functional classification of transcriptional changes during in vitro culturing and hypoxic exposure

The expression profiles resulting from culturing in different oxygen conditions were established by microarray analysis. On average, one third of the 47,296 probes incorporated on each of the Illumina chips identified regulated genes. Internal controls verified a successful sample processing and hybridization. One outlier array was observed for a single experimental condition (ambient air at day 0). To retain the balanced design, the single outlier array was replaced with average normalised intensities based on the remaining four arrays from ambient condition at day 0. The most striking finding from the hierarchical clustering, constructed on the basis of the neighbour-joining algorithm, was the similarity of the expression patterns at 1% oxygen, since these samples grouped on a distinctive branch (Fig. 3A, encircled in red). The other oxygen conditions had no particular effect on the expression patterns as the clustering of the remaining samples was predominantly donordependent. Nevertheless, it appears that donor characteristics play a role since the grouping of two overweight donor cell lines, ASC6 and 10, on distinctive branch suggests a higher level of similarity than that observed between the lean donor cell lines ASC13, 23, and 24. The 500 genes that varied most (SD > 1) across all arrays were included in the k-means clustering to group similar expression patterns. The k-means clustering was performed with the assumption of 10 gene clusters. Five expression patterns, comprising approximately 70% of the input genes were identified (Fig. 3B). As expected from the hierarchical clustering, the most prominent changes were observed with a drop to 1% oxygen (clusters 1 and 2). Of the 115 genes upregulated only at 1% oxygen tension, 19.3% were significantly implicated in negative regulation of biological processes (Fig. 3B table). The 96 genes, which were found downregulated at 1% oxygen tension, were not enriched for a specific function but included genes related to cell cycle regulation (Fig. 3B table). Clusters 3 and 4 included genes with distinct expression levels in the ambient samples on day 0. More than half of the genes upregulated on day 0 in cluster 3 was significantly involved in multi-organism processes, which is a term covering interaction processes between organisms of same or different species (Fig. 3B table). Likewise, the cluster 4 was enriched for multi-organism development, although not significantly. The expression pattern featured in cluster 5, with an incremental upregulation with both expansion and reduced oxygen concentration, was common for 61 genes. Significant enrichment was seen here for functional annotations, such as growth factor activity and

The two-week period, during which the cells proliferated, had a significant effect, since at ambient oxygen concentration, 26 genes were found to be significantly regulated (Supplement 3). Of these 26 genes, 17 were also significantly regulated during expansion in the remaining hypoxic conditions. The 26 genes were enriched for developmental processes, transmembrane receptor protein tyrosine kinase signaling pathways (receptors TEK, EPHB1, and ERBB3), cell adhesion (extracellular matrix molecules DPT and HAPLN1), and prostaglandin biosynthetic processes (synthases PTGS1 and PTGIS). Relevant gene name abbreviations are available in Supplement 4. Taking into account exclusively hypoxic expansion, 28 genes were significantly regulated across all conditions, and 75% of them were upregulated. It is interesting to note that BMP6, which plays a central role in cartilage development, was found in the latter group. Many of the upregulated genes could be found in clusters 4 and 5 from the k-means clustering (Fig. 3B). After two weeks of expansion, only 5% and 1% oxygen were sufficiently distinct from ambient conditions to yield significantly regulated genes. At 5% oxygen tension, 59 genes were identified, of which approximately 40% were upregulated (Supplement 5). The identified genes were enriched for two functional clusters concerning the response to nutrient levels/external stimulus and the cell–cell signalling through receptor binding. The most notable gene belonging to both functional clusters was LEP with nearly 10fold upregulation. Among the genes with a function in response to nutrients levels, three with role in oxidative stress (DDIT3, HMOX1, and TXNIP) were identified. Considering the specific biologic functions that were enriched, the term developmental processes was the only one significantly enriched and contained approximately 45% of the genes. The LEP transcript was also found in the developmental processes group along with two other interesting transcripts related to chondrogenesis and cell proliferation, CHRDL2 and KIT, respectively. Seven of the genes in the developmental processes gene list were additionally related to apoptosis (DDIT3, DDIT4, CARD9, SERPINB2, TRIB3, HMOX1, and CEBPG) and were all downregulated. Of the 59 genes significantly regulated at 5% oxygen, 38 were also significantly regulated at 1% oxygen. The majority of the genes common to the two hypoxic conditions demonstrated a higher fold regulation in 1% oxygen. A pairwise comparison of adjacent hypoxic conditions revealed that 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

Fig. 3 – Relatedness of transcriptional profiles by hierarchical and k-means clusterings. (A) The unrooted hierarchical dendrogram of day 0 and 14 transcriptional profiles was constructed on the basis of Euclidean distances and neighbor-joining algorithm. The samples from 1% oxygen are encircled in red. (B) Similarity of gene expression patterns by the culture day and oxygen tension was determined with the aid of k-means clustering based on the 500 genes exhibiting highest fluctuations in expression levels across all arrays (SD > 1). The profiles are presented with arbitrary relative expression levels centered around the expression mean (red line). In the table, listed are biological processes and molecular functions enriched in the gene clusters, where the boldface type indicates statistical significance (p < 0.05).

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Table 1 – Main biological processes and molecular functions enriched in significantly regulated genes identified during comparison of adjacent oxygen tensions Oxygen step

Functional annotation

P-value

10%–5% Upregulated genes Downregulated genes

No. of genes 55

33 67

5%–1% Upregulated genes

936 Apoptosis Post-translational protein modifications Cellular processes Transcription

9.4E−5 4.1E−4 4.9E−1 1.3E−1

Primary metabolic processes Cellular metabolic processes Macromolecular metabolic processes Cellular processes Transcription

1.6E−2 1.0E−2 8.3E−1 1.0E0 1.0E0

64 5 8 40 11 36 44 43 36 56 13

Downregulated genes

5%–1% specific a Upregulated genes

97 Transcription regulatory activity Protein kinase binding

9.9E−1 1.0E0

Primary metabolic processes

9.9E−1

Downregulated genes

a

% of genes total

79 4 12 21 13

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

upregulated, and these notably included previously mentioned LEP and CHRDL2 genes. The DNA damage inducible transcripts, DDIT3 and 4, together with CARD9, were found among the downregulated genes. 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 with 1% hypoxia A total of 2581 genes were found significantly regulated at 1% oxygen when compared to the reference at ambient conditions on day 14. Of these genes, 46% were upregulated, and they were significantly enriched in several functional clusters, among which the most prominent were the developmental processes, apoptosis, cell proliferation, and transcriptional regulation (Fig. 4A). The cluster of developmental processes included 254 genes. Of special interest, in context to expansion conditions and the intended use in cartilage engineering, were the genes involved in chondrogenesis and bone formation (CHRD, RUNX2, CHRDL2, NOG, and ACVR1), and the genes responsive to nutrient levels as well as the hypoxic conditions (LEP, ANGPT2, ANG, PPARG, and APOE) (Fig. 4B). In the apoptosis cluster, 73 genes were included. Of these, 48 were involved in the regulation of apoptosis, 19 were anti-apoptotic or survival genes, e.g. BCL2 and SOD2, and 14 genes had apoptosis inducing functions (e.g. PERP, HRK, and TNFRSF19). Interestingly, the apoptosis cluster also included genes involved in DNA damage and resulting growth arrest (GADD45A, GADD45G, and DDIT3). The cell proliferation group consisted of 68 genes, of which 25 were shared with the apoptosis cluster. The inhibitory effect on cell replication was represented by three cyclin-dependent kinase inhibitors, 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 in the cluster, such as KIT, PIM2, VEGFB, IRS2, EGFR, VEGF, CSF2, IGF2, PIM1, and FGF9. Several classes of transcription factors, including Zinc finger and forkhead box proteins, along with HOX genes, were included in the cluster of 174 genes belonging to regulation of transcription. In total, 71 genes had transcription factor activity. In particular, two transcription factors, ARNT and HEY1 are worth noting, since they are part of the HIF and Notch signaling pathways, respectively. The major significantly enriched functional clusters that comprised the list of downregulated genes were represented by RNA processing, organic acid metabolic process, and primary metabolic process, (Fig. 5A). Of the 59 genes in the RNA processing cluster, 37 were involved in mRNA processing, 10 in tRNA processing, and 4 in rRNA processing (Fig. 5B). Distinctive for the mRNA processing genes was the RNA splicing function. The primary metabolic process cluster constituted a variety of biological functions and shared gene members with both the RNA processing and organic acid metabolic processes clusters. The most prominent group of genes was involved in the nucleobase, nucleoside, nucleotide, and nucleic acid metabolic process. Cyclins D2 and D3, together with p15, were also identified in the primary metabolism cluster. Notably, three effectors of mitochondrial mediated apoptosis, BID, cytochrome C, and APAF1, were found in this cluster downregulated. Amine metabolic processes were the main function in the organic acid metabolic processes cluster. As indicated above, the exposure to 1% oxygen significantly modulated expression profiles when compared to the nearest assayed hypoxic condition at 5% oxygen. In Table 1, the enriched biological processes annotated to the gene list can be found. Not surprisingly, biological processes such as post-translational protein modification, apoptosis, and regulation of biologic processes were

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

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significantly overrepresented. The widely covering gene ontology categories of primary, cellular, and macromolecular metabolic process mostly included downregulated genes. The upregulated genes were annotated to biological processes of apoptosis and post translational protein modification. Both up- and downregulated

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

Fig. 5 – Gene ontology hierarchy indicating the major categories of biological processes overrepresented in downregulated genes at 1% oxygen tension. Genes significantly regulated more than 2-fold between ambient and 1% oxygen on day 14 were enquired for Gene Ontology enrichment in a hyper geometric test with Benjamini and Hochberg correction for multiple testing. (A) Significantly enriched functions are presented on a scale from yellow (p < 0.05) to dark orange with p-value 5 orders of magnitude lower. The size of nodes corresponds to the number of genes annotated to the given gene ontology term. The functions most relevant to hypoxia are highlighted in red frames. (B) Characteristics of the biological processes and molecular functions that were identified in (A) by red frames.

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

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cluster were with approximately 5-fold upregulation among the three most regulated genes. The most regulated gene with almost 7-fold upregulation was G0S2, which has been identified as a

Fig. 6 – Real-time RT-PCR analysis of molecular functions associated with hypoxic responses and chondrogenesis after a 14-day long preconditioning in different hypoxic conditions. (A) Selected biological processes that were found significantly regulated in microarray analysis, included differentiation, cell cycling, and growth arrest. The data are presented as a mean +/− standard error of mean (SEM) from five donors analyzed in duplicate (n = 5). The hypoxic conditions were normalized to the respective ambient cultures at the day 14, which is indicated by a horizontal line. Asterisks denote a significant difference (p < 0.05) with respect to the ambient condition. The table indicates correlation of the real-time RT-PCR data with those obtained by a microarray analysis for 1% oxygen concentration. (B) Key regulators of chondrogenesis were selected to assess their hypoxic responses. The data are presented as a mean +/− standard error of mean (SEM) from five donors analyzed in duplicate (n = 5). The hypoxic conditions were normalized to the respective ambient cultures at the day 14, which is indicated by a horizontal line. Asterisks denote a significant difference (p < 0.05) with respect to the ambient condition.

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

Verification of gene expression patterns by semi-quantitative RT-PCR Since the genes implicated in cell differentiation, cell cycle, and growth arrest were mostly affected by the oxygen-dependent regulation, 11 genes covering these biological processes were further analyzed by the semi-quantitative RT-PCR (Fig. 6A). All genes in the differentiation group displayed progressive upregulation with declining oxygen concentration. Similarly, the three tumor suppressor genes, p57, p27, and p19, from the group of genes involved in the regulation of cell cycle were upregulated proportionally to the level of hypoxia, except for the Cyclin D2 (CCND2) that was significantly downregulated at 1% oxygen. In contrast to the two previous groups, the genes implicated in apoptosis and DNA damage-induced growth arrest became discretely upregulated exclusively at the highest level of hypoxia. Overall, the semi-quantitative RT-PCR and microarray data were in good agreement (correlation coefficient > 0.95) thus corroborating accuracy of the Illumina-based transcriptional analysis (Fig. 6A table). In addition to genes readily responsive to hypoxia, differential regulation of some of the key factors implicated in chondrogenesis was explored as well (Fig. 6B). Most importantly, contrary to 5% oxygen, where the prevalent pattern appeared to be upregulation, in 1% oxygen, the gene expression seemed to be mainly suppressed.

Discussion In the present investigation, independent ASC lines were examined to study the significance of low oxygen tension for the enrichment of chondrogenic progenitor cells. The global gene expression analysis demonstrated distinctive transcriptional response to 1% oxygen concentration when compared to mild hypoxic expansion and, notably, highlighted the importance of biological variability. Previous studies have already shown that ASCs phenotype is not consistent and is determined by individual donor characteristics such as age, gender, and BMI [64–67], but the mode of tissue harvesting has also been suggested to play a role [43,68]. Despite attempting to minimize the donor-related variability by restrictive inclusion criteria and standardizing the experimental approaches in our study, the hierarchical clustering demonstrated considerable inter-individual differences. It thus appears that ASC populations display unique properties dependent on the donor, and this characteristic may at least in part account for the discrepancies between different studies [17,37,69]. Although the exact nature of the observed variability is not known, it is conceivable that microenvironment associated with individual-specific fat distribution and BMI is a contributory factor. More analysis is required in order to provide a better understanding of the relationship between donor BMI and the hypoxic response of ASCs. Taking into account ambient conditions, in the cultures expanded for 14 days, 26 genes were differentially expressed and the majority of activated genes were involved in developmental processes, indicating a transition from the proliferative phase. Similar pattern was previously observed with BMSCs [70]. The effect of exposure to lower hypoxic conditions of 5 and 1% oxygen

seemed to inhibit the culture proliferation during the 14-day period but also favor preservation of clonogenic precursor cells. Selection of a more replicative cell type by long-term hypoxic expansion has been demonstrated in previous work [13,24,32], and the proportions of colony-forming progenitors reported herein are well within the range of published figures [17]. In this context, the choice of expansion medium has to be considered. Specifically, the expansion medium has in previous studies been proven to affect the outcome of cell population and its differentiation potential [12,22] as well as its gene expression profile [38]. Thus it appears that this variable can in a potentially significant way contribute to the discrepancies frequently seen in the literature [71,72]. Traditionally, for the expansion of ASCs have been used the Dulbecco's modified eagle medium (DMEM) [1,28,73–75] or Dulbecco's modified eagle medium Ham's F-12 nutrient mixture (DMEM/F12) [12,17,37,69,76,77]. However, in the current investigation, α-MEM was chosen for expansion due to its presumably positive 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 directly results from the present study with those based on ASCs cultured in the traditional media choices [18,38], let alone those using other types of MSCs [29,34,35]. To our knowledge, a comprehensive investigation of the expression profiles of ASCs expanded in the most common growth media has not been conducted. This could be accomplished using the growing number of array studies that are publicly available in databases, such as NCBI's Gene Expression Omnibus [52]. The transcriptional responses associated with hypoxia emerged first at 5% oxygen concentration but unfolded fully at 1% oxygen, as is apparent from 59 and nearly 2600 genes becoming differentially regulated, respectively. At 1% oxygen, the cultures were subject to growth arrest in more phases of the cell cycle, as indicated in Fig. 6A. The G1-phase arrest is known to be triggered with overexpression of members of both the Cip/Kip and the INK4 family of cyclin dependent kinase inhibitors, represented by p57, p27, and p19 [78–80]. For p27, the induction has been proposed to be HIF-1 dependent [27]. However the direct link between cyclin dependent kinase inhibitors and hypoxia regulation are still being debated, as discrepant 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 pathway and are involved in preventing cell cycle progression from both G1and G2-phase in response to DNA damage [82,83]. DNA damageinduced activity in the p53 pathway will lead, if extensive and beyond repair, to cell death through apoptosis [84]. The preapoptotic state was confirmed by upregulation of the p53 apoptosis effectors, PERP and HRK. PERP is one of the intracellular regulatory proteins which functions to connect the apoptotic signaling initiated by e.g. hypoxia or DNA damage in the p53 pathway to the effectuation of cell suicide [85]. HRK induces apoptosis via interaction with B-cell CLL/lymphoma 2, Bcl-2 [86], a survival-promoting protein that negatively regulates apoptosis through inhibition of the release of mitochondrial cytochrome C [87]. The downregulation of the three effectors of mitochondriaregulated apoptosis, the APAF1, cytochrome C, and BH3-interacting domain death antagonist, BID, provided further evidence for apoptotic activation. It is important to note that, at the same time, anti-apoptosis mechanisms in the form of cell cycle arrest were activated to provide for the cell survival. In tumour cells, for

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example, the super-induction of GADD45G in response to the joint effect of DNA damage and hypoxia has been hypothesised to support cancer cell survival during therapy [88]. Thus the survival as well as the enhanced colony forming ability of ASCs cultured at 1% oxygen may be the result of a balance between growth arrest and apoptosis that promotes the selection of the “fittest”. The culture confluency in combination with low oxygen tension resembles an ischemic environment characterized by hypoxia and nutrient deprivation. In previous studies on ischemia, BMSCs were able to survive and retain their differentiation potential for up to 72 h of the treatment [89]. Moreover, Zhu et al. [90] proved the shortage of available serum to be the major cause of apoptosis in ischemia simulated by hypoxia and serum deprivation. In light of these data, it is plausible that, in the current investigation, the pre-apoptotic stage of the cultures at 1% oxygen was a reaction not to undergo a complete apoptosis by means of keeping the cell population subconfluent. It was surprising to observe that despite the cells expanded at 5 and 1% oxygen were enriched for CFU-Fs, they did not exhibit an enhanced chondrogenic differentiation when induced by TGFβ3 in high-density pellet cultures. A comparison with previous studies is challenging, since the culture format, hypoxic conditions, expansion periods, and the cell type vary greatly between investigations [13,24,28,32,34,91]. At the molecular level, significant upregulation of BMP6 at 5% oxygen would indicate activation of TGF-β signaling, which according to previous studies by Hennig et al. [16] and Estes et al. [23] should be reflected by enhanced 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 this context, further experiments based on protein analysis are warranted. Previously, it has been suggested that hypoxia may promote chondrogenesis through upregulation of the central transcription factor Sox9 in the mouse model [91]. In the current study, hypoxic exposure induced Sox9 expression at the levels not having a statistical significance, however, most critically the expression of Sox9 was significantly suppressed at 1% oxygen. In view of the significance of Sox family for chondrogenesis [94,95], then the deficiency of chondrogenic differentiation in this hypoxic scenario is predictable; nevertheless, the understanding of the inefficiency of cartilage formation in milder hypoxia will need further explanation. Although it appears, based on the results from this investigation, that the hypoxic preconditioning is not beneficial in terms of stimulating chondrogenesis, the molecular analysis indicates that such treatment may support specification into other differentiation pathways. One of the scenarios may involve facilitation of osteogenesis through the action of leptin (LEP). LEP is known mostly for its role as a key regulator of body weight and food intake [96], but importantly it is inducible in hypoxia by HIF-1 [97,98] and consequently was found significantly upregulated in the current study at 5 and 1% oxygen concentrations. However, from the developmental point of view important is that LEP possesses angiogenic activity and also has been found to play a pivotal part in the longitudinal growth of bone via endochondral ossification [99,100]. Other scenarios would probably be concerned with processes involving angiogenesis, since our cultures expanded at 1% 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

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of ischemic conditions, and first data based on BMSCs in the murine model of myocardial infarction indicate feasibility of such approach [33,98,101]. In light of the evidence in this and other studies, it is evident that the oxygen conditions during monolayer expansion of MSCs are of great importance. The challenge is to find the best combination of physical and biochemical parameters, along with appropriate sequence and duration of the treatment intervals, to provide for the most suitable outcome for a given application.

Acknowledgments The authors wish to thank plastic surgeons Frants Grymer and Christian Bang, their office and nursing staff, and their patients at Grymer Privat Hospital, Aarhus, for donation of the liposuction material. The expert technical assistance of Helle Skjødt Møller and Mette Bøgh Ringaard is highly appreciated. The valuable input from Tim Watts from the Genomics group at The Welcome Trust Centre for Human Genetics in Oxford is also acknowledged. The authors greatly appreciate support from The John and Birthe Meyer and Toyota Foundations, and the Danish Medical Research Council grants no. 271-06-0283 and 2052-01-0045. Also, grants from the Family Hede Nielsen Foundation, Mrs. Johanne Louise Berg born Oppermann, and Director Ditlev Berg, and the scholarship from Horsens Statsskole are highly valued. The funding sources had no influence on neither study design, collection, analysis and interpretation of data, writing of the report, nor the decision to submit the paper for publication.

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

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