culture of human embryonic stem cells - Future Medicine

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Transcriptome alterations due to physiological normoxic (2% O2) culture of human embryonic stem cells Human embryonic stem cells (hESCs) hold great promise therapeutically. In order to deliver on this promise the correct defined conditions for long-term propagation must first be established. Researchers have now provided reports describing the benefits of culturing hESCs in physiologically approximate levels of oxygen. These physiological values fall in the range of 2 to 5% O2. Benefits include reduced spontaneous differentiation, enhanced chromosomal stability and increased clonality. Aims: The aim of our study was to examine the transcriptional consequences of culturing hESCs in physiological normoxia (2% O2) using microarray technology. Methods: Three karyoptically normal hESC lines (H1, H9 and RH1) were examined. At the initiation of this experiment, established hESC lines were redesignated as passage (p) 0 in 21% O2, then bifurcated into 21% O2 and 2% O2, and maintained for a further ten passages at which time samples were again collected. RNA was extracted from all sample points and subjected to microarray analysis using the Affymetrix U133 Plus 2.0 platform. Bioinformatic analysis was performed using dChip and GoStat. Results: We performed grouped analyses of gene expression of early (p0) versus late (p10) air-cultured cells. This revealed relative stability with six (air p0 baseline vs p10 experimental) and one (air p10 baseline vs p0 experimental) gene(s) displaying both greater than twofold and statistically significant upregulation. Conversely, we identified 302 gene upregulations and 56 downregulations when comparing 21% O2 (p0p10) with 2% O2 (p10). These significantly upregulated changes clustered into 82 over-represented and 9 underrepresented ontology terms. These terms were indicative of signaling pathways, developmental potential and metabolism. Hierarchical clustering indicated a trend for 2% O2 cultured cells to cluster collectively with reduced heterogeneity when compared with 21% O2 cultured cells. Conclusions: The gene changes associated with 2% O2 culture may be predictive of novel cellular requirements for stable self-renewal, maintenance of pluripotency, and a reduction of hESC-line heterogeneity. KEYWORDS: embryonic stem cells, gene transcription, hypoxia, microarray

The conditions required for long-term stable propagation of human embryonic stem cells (hESCs) have not yet been determined. While the bulk of hESC cultures are apparently normal, there is a tendency for specific chromosomal alterations to occur with repeated passage [1,2] . To overcome this problem valuable studies have been performed to establish standard profi les for pluripotent hESCs on which others can be benchmarked [3] . Microarray ana lysis has been used by several groups questioning the existence of a pluripotency ‘fingerprint’ [4–6] . There is very little consensus between these studies, with a reported seven commonly upregulated genes in undifferentiated versus differentiated analyses (SEPHS1, PSIP2, SLC16A1, CRABP1, GJA1, RAMP and RPL24) [4–7] . The fi ngerprints differ greatly, although the use of alternative ana lysis methods expands this list to 111 genes [7] . A further study identified a core ‘fingerprint’ of 186 genes from seven individual hESC lines [8] . Direct comparisons of these 186 genes with the previously

grouped 111 reveal a shared consensus of five genes (CD24, NASP, POU5F1, SEMA6A and SLC16A1). The clear message from the aforementioned studies is that complex heterogeneity exists between different hESC lines, which is probably further amplified through variations in culture methodology [9] . The pre-implantation blastocyst receives metabolites and O2 from the uterine fluid. Uterine fluid itself has an approximate 4% dissolved O2 concentration [9,10] . There is a strong rationale that mimicry of physiological conditions will provide optimal conditions for hESC propagation. Several groups have now investigated the consequences of using physiologic (2–5%) O2 for hESC culture [11–13] . These groups have reported reductions in spontaneous differentiation, enhanced clonality, reduced spontaneous chromosomal aberrations and smaller, less complex cells. None of these prior reports have investigated the transcriptional consequences of culturing hESCs in physiologic O2 . The purpose of this study was to

10.2217/17460751.3.6.817 © 2008 Future Medicine Ltd

Regen. Med. (2008) 3(6), 817–833

Nicholas R Forsyth1†, Alasdair Kay1, Karen Hampson1, Alison Downing2, Richard Talbot2 & Jim McWhir2 †

Author for correspondence: The Guy Hilton Research Centre, Keele University Medical School, Stoke-on-Trent, ST4 7QB, UK Tel.: +44 017 8255 5261; Fax: +44 017 8274 7319; E-mail: [email protected] 2 Gene Func on and Development, Roslin Ins tute, Roslin, EH25 9PS, UK 1

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RESEARCH ARTICLE

Forsyth, Hampson, Kay, Downing, Talbot & McWhir

identify consensus transcriptional changes across three well-characterized hESC lines in response to 2% O2 [11,14–17] . Using applied stringency inclusion measures of greater than or equal to twofold upregulation, relative expression values greater than or equal to 10, and p ≤ 0.05 we identified 302 genes that were upregulated in grouped ana lysis of 2% O2 hESCs versus 21% O2 hESCs. Grouped ana lysis of late-passage room O2 cells revealed six gene upregulations when compared with early-passage room O2. Reverse ana lysis identified a solitary gene, ELOVL2, which was significantly downregulated in late-passage hESCs. The ana lysis of microarray data is complex and tools such as the Gene Ontology (GO) databases have provided a means to interpret these complex data in terms of functional association [18] . The 302 genes upregulated in 2% O2 grouped into 82 overrepresented ontology groups concerned with signaling, proliferation, behavior, metabolism, differentiation and O2 response. The nine underrepresented ontology groups were largely concerned with a reduction in intracellular organelle structure in support of our previous descriptive findings [11] . Materials & methods

Cell culture ESCs (H1, RH1 and H9) were maintained in culture using a modification of Xu et al., described in detail elsewhere [11,19] . Starting passage numbers for H1, H9 and RH1 were 72, 68 and 72, respectively. Cells were passaged enzymatically using trypsin/ethylene glycol tetra-acetic acid and as reported elsewhere, no karyotypic abnormalities were detected during the course of our experiment [11] . All cultures were maintained in either a standard humidified incubator at 37°C in 5% CO2/air or in a modular O2 controlled system (RS Biotech) with constant control maintained at 2% O2/5% CO2/ 93% N2. Oxygen levels

RNA collection points

p0

p1

p10

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Figure 1. Schematic of cell culture and RNA extraction points. The schematic illustrates the progression of the cell culture component of the experiment and the precise points at which RNA was extracted from samples. The red circles indicate isolation points while ‘p’ indicates passage number.

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RNA extraction & quality control RNA was extracted from cell pellets archived in a -80°C freezer. For extraction the 12 samples from each sample grouping (4 × p0 air O2, 4 × p10 air O2, 4 × p10 2%O2) were processed simultaneously (FIGURE 1) . RNA extraction was performed using the RNeasy ® Kit (Qiagen) following manufacturers guidelines. RNA quality was determined with the Eukaryote Total RNA Nano Series II chip on a Bioanalyzer (Agilent Technologies). Samples with a RNA Integrity Number (RIN) of 7.0 or greater were used in our study. For the most part (24/32) all samples achieved a RIN value of 10.0. Quantification of RNA was performed on a ND1000 Spectrophotometer (NanoDrop®).

Microarray probe preparation & hybridization Biotin-labeled cRNA was synthesized with a standard one-cycle target labeling assay protocol (Affymetrix). Brief ly, the first-strand cDNA was generated from 5 µg total RNA, and was used as a template to synthesize the second-strand cDNA. Labeled cRNA was created from the cDNA by in vitro transcription in the presence of biotinylated ribonucleotides. The biotin-labeled cRNA was fragmented and spike controls added to 10 µg of fragmented cRNA before overnight hybridization with the Human Genome U133 Plus 2.0 GeneChip® Array (Affymetrix). Arrays were then washed and stained with streptavidin–phycoerythrin (Invitrogen), before being scanned on the Affymetrix GeneChip 7G scanner. Following scanning, array images were assessed by eye to confirm the grid alignment and the absence of significant bubbles or scratches. When scaled to a target intensity of 100 (using Affymetrix MAS 5.0 array ana lysis software), scaling factors and background Q values were checked and found within acceptable limits. The 3´/5´ ratios for glyceraldehydes-3´-phosphate dehydrogenase (GAPDH) and β-actin were confirmed to be within acceptable limits from the quality control report generated in the GCOS software. Bioinformatic analysis Primary ana lysis of raw cell data files was performed using the dChip 2006 (build date 16th February 2006) platform [20] . Sample groupings were established and invariant set normalized using median probe intensity as baseline array. Model-based expression was calculated using the Perfect Match/Mismatch difference future science group

Normoxic (2% O2) culture of human embryonic stem cells

model method with no expression value truncations after computation. Gene arrays were then fi ltered to less than 10,000 transcripts where variation across samples (after pooling replicate arrays) with x < standard deviation/ mean < 1000. The values attributable to x were generally in the range of 0.5 to 0.6. Filtered gene lists of less than 10,000 were clustered by sample and gene using default parameters. Multivariate ana lysis was performed using the fi ltered gene lists couple to the Compare Samples tool, where mean Experimental/ Base more than 2, p-value for paired T-Test less than 0.05, and where samples where permuted 50 times to assess the false discovery rate. Sample comparison fi le output was then sorted by gene name, relative expression values of less than 10 removed, and multiple probe hits deleted after generating averaged values. Ontological ana lysis was performed using the GoStat [101] freeware platform [18] . Searches were performed using Affymetrix Tags identified from gene lists generated through sample comparisons using the Affymetrix HG-U133_ Plus2 gene collection against MAS5 input. Minimal length of considered GO paths was 3, maximal p-value was 0.01, no GO clustering

was applied, and Benjamini False Discovery Testing used to correct for multiple testing. All values were correct as of 15th April 2008. Results

Hierarchical clustering of differentially expressed probes Filtering of all U133 Plus 2.0 probe sets of all samples revealed a distinct set of 7205 differentially expressed probes (SUPPLEMENTARY TABLE 1) (see online www.futuremedicine.com/toc/rme/3/6). Hierarchical clustering of these differentially expressed probes illustrated a strong tendency for 2% O2 -cultured hESC cells to cluster together as a distinct and separate population to the 21% O2-cultured hESC cells (FIGURE 2) . The 2% O2-cultured hESC cells bore a greater resemblance to each other than to their parental 21% O2 cells. Within the 21% O2-cultured cells there appeared to be a greater cell-line dependant identity where all early- and late-passage 21% O2-cultured H9 and RH1 hESCs clustered together with unique regions of differentially expressed probes apparent. Interestingly, earlyand late-passage 21% O2-cultured H1 hESCs did not cluster as a group. The early-passage H1 21% O2 samples (p0 ) clustered together, as did

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Figure 2. Hierarchical clustering of human embryonic stem cell 2% O2 versus 21% O2-filtered genes. The hierarchical clustering tool was used to generate a samples/genes heatmap of all human embryonic stem cell samples using a filtered gene list (< 10,000 transcripts, standard deviation/mean < 0.5). The bulk of 2% O2 samples were noted to cluster together excepting a solitary sample (344_ RH1_2_p9_3) while 21% O2 samples clustered according to their specific lineage. This indicates a reduction of heterogeneity in 2% O2-cultured hESCs. Red indicates upregulated while blue indicates downregulated

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Table 1. Genes upregulated (>fivefold) in late-passage 21% O2 cells versus early-passage 21% O2 cells. Gene name (official symbol)

21% p0 mean 21% p10 mean Fold change

p-value

(A) H1 Homo sapiens, clone IMAGE:5170503, mRNA ZFP42 ID3 EIF1AY Nasopharyngeal carcinoma-associated antigen (NPC-A)-5 DDX3Y SCFD1 IGFBP5 PDGFB GDF15 FOXC2 Hypothetical locus LOC388666 DUSP6 DACT1 D21S2088E QPCT ARRB1 Transcribed locus, weakly similar to XP_527234.1 similar to FRD envelope protein (Pan troglodytes) SOHLH2 CEBPZ HES1 PUS7L C1orf76

0.06 37.36 34.08 11.73 67.04 17.51 2.17 4.36 13.08 48.93 5.56 258.85 125.85 115.73 8.23 8.45 24.93 3.03

12.94 710.75 576.78 178.21 762 184.21 21.49 40.36 119.53 640.78 46.14 2135.93 930.73 893.17 61.91 56.98 119.61 19.17

211.95 19.02 16.92 15.19 11.5 10.52 9.89 9.27 9.14 8.78 8.3 8.25 7.78 7.72 7.53 6.74 6.41 6.32

0.014831 0.003452 0.027453 0.011154 0.018432 0.030083 0.011666 0.000357 0.000562 0.002582 0.012366 0.031553 0.001169 0.000136 0.000501 0.000354 0.013721 0.035202

20.5 27.65 57.82 8.54 2.1

114.77 173.35 295.23 43.47 10.55

5.6 5.53 5.11 5.09 5.03

0.002718 0.019156 0.029667 0.017077 0.046275

1.61

10.02

6.23

0.036627

3.08 2.98

22.13 16.4

7.18 5.5

0.010558 0.005762

(B) H9 RUNX2 (C) RH1 cDNA clone IMAGE:5301169 GJA5

Table 2. Genes upregulated (>fivefold) in early-passage 21% O2 cells versus late-passage 21% O2 cells. Gene name (official symbol)

21% p0 mean 21% p10 mean Fold change

p-value

(A) H1 CREBBP SOSTDC1 CXCL13 Transcribed locus, strongly similar to XP_530274.1 LOC458133 [Pan troglodytes] LILRB2/LILRB3 PCDHB18 Hypothetical protein LOC153469 INVS Hypothetical protein LOC254848 gb:BC042086.1 (Homo sapiens, clone IMAGE:5756137, mRNA)

1 3.56 1.68 1.57

12.36 25.74 10.98 10.09

12.36 7.23 6.54 6.41

0.020607 0.014793 0.020901 0.018297

2.85 1.99 5.61 1.91 2.58 2.24

17.56 12.07 32.78 10.39 13.51 11.34

6.16 6.06 5.84 5.44 5.24 5.05

0.030706 0.026067 0.001115 0.022723 0.026209 0.024625

0.75 1.6

10.17 11.68

13.52 7.28

0.047661 0.021008

(B) H9 TPM4 DNAJC15

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the late-passage H1 21% O2 samples (p10 ) but they were separated by the H9 21% O2 samples (FIGURE 1) . The late passage H1 21% O2 cells were grossly karyotypically normal and this may be indicative of the development of a precursor to abnormality that had occurred below the level of microscopic ana lysis.

Stability of expression: early- versus late-room O2 We next sought to determine the relative geneexpression changes that could be identified through comparison of hESC lines at p0 versus p10 in 21% O2. Two of the three lines used in this study (H1 and H9) had previously been demonstrated as retaining karyotypic normalcy from passage points before and after the conclusion of this study. The third line (RH1) came from karyotypically normal stocks and was not checked at the study end. Gene over-representation ana lysis suggested no significant structural alterations had occurred over the duration of this study (data not shown). Comparison of early- versus late-passage room O2 hESC cells revealed gains in expression in H1 (117 genes [146 probes]), H9 (33 genes [52 probes]) and RH1 (24 genes [36 probes]) in late-passage room O2 hESC (SUPPLEMENTARY TABLES 2, 3 & 4) . Downregulation was also observed in H1 (60 genes [78 probes]), H9 (29 genes [42 probes]) and RH1 (11 genes [33 probes]) (SUPPLEMENTARY TABLES 2, 3 & 4) . To establish if any consensus transcriptional alterations were occurring over the experimental process we performed grouped early- versus late-passage ana lysis. Grouped ana lysis revealed six genes (17 probes) upregulated in late-passage room O2 cells while a solitary gene (18 probes) was downregulated (SUPPLEMENTARY TABLE 5) . For ontological ana lysis (described later) we included all genes identified through sample comparison with greater than twofold expression change, significance of less than 0.05, and a relative experimental expression greater than ten. We also wished to identify those genes that had undergone substantial change (fivefold) in 2% O2 versus 21% O2 grouped. Gene name (official symbol)

21% p0p10 mean 2% p10 mean

Fold change p-value

5.8 10.31 33.33 7.88 43.42 23.52 8.72 17.53 7.1 20.43 95.92

166.14 209.93 807.77 153.93 745.89 355.66 121.98 236.04 94.11 244.43 972.64

28.64 20.36 19.98 19.54 17.18 15.12 13.98 13.47 13.26 11.97 11.68

0.003654 0.001548 0.0075633 0.000002 0.008447 0.000849 0.005097 0.003737 0.003661 0.000138 0.0011277

52.34 41.79 8.7 14.62 240.6 13.4 4.73 37.98 59.97 6.4 79.9 29.02 35.83 28.92 215.54 12.94 12.07 5.32 2.2 10.68 587.97 518.8 7.98 47.73 4.45 4.34 21.32 19.6 252.4 67.63 11.19 207.6 83.53 733.32 14.52 12.03 9.05 32.54

697.85 464.79 94.53 156.31 2208.76 95.78 41.93 334.83 527.88 55.46 687.12 247.71 287.91 218.32 1623.48 92.42 87.49 36.95 15.25 77.02 2329.4 3374.19 52.21 308.92 28.69 27.23 133.45 122.52 1572.02 442.9 69.39 1276.05 511.68 4459.74 91.9 71.22 53.41 184.25

11.3 11.12 10.87 10.69 9.75 9.18 8.86 8.82 8.8 8.66 8.6 8.54 8.03 7.55 7.53 7.35 7.25 6.95 6.92 6.81 6.65 6.56 6.54 6.47 6.44 6.28 6.26 6.25 6.23 6.2 6.2 6.15 6.14 6.08 6 5.92 5.9 5.66

0.0001815 0.000076 0.000315 0 0.0000635 0.002669 0.001925 0.000005 0.000017 0.034082 0.000116 0.000688 0.000188 0.003436 0.000003 0.001168 0.020082 0.006491 0.001123 0.0034585 0 0.0000225 0.033316 0.005566 0.01599 0.008966 0.002838 0.000175 0.000022 0.0045287 0.017399 0.000148

(A) H1 NRN1 CA3 SLC16A3 TJP2 CH25H FZD10 gb:AA625683 (Hs.143659) SPAG4 APOLD1 Transcribed locus, weakly similar to NP_062553.1 Homo sapiens, Similar to otoconin 90, clone IMAGE:4285317, mRNA BHLHB2 ANKRD37 Hypothetical protein LOC441054 Hypothetical protein LOC154092 SERPINE1 CD300A NOG KISS1R LEPREL1 PPFIA4 C1orf61 Transcribed locus (AI761207) NPPB NDUFA4L2 MSTP150 CDNA FLJ41489 fis BHLHB3 SOX14 SPN BCAN SLC2A1 BNIP3 SGCG NDRG1 CDNA FLJ23869 fis, SLC8A3 gb:AA741300 (weakly similar to ALU8) FOXD1 PFKFB3 STC1 ADCY8 GBE1 TXNIP ADM FOSL2 CDNA FLJ26031 fis Transcribed locus, strongly similar to NP_000914.1 CCL26

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0 0.003103 0.000973 0.003805 0.000021



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Table 3. Genes upregulated (>fivefold) in 2% O2 versus 21% O2 grouped (cont.). LY6K EGLN1 LOX IGFBP2 AHNAK PFKFB4 PDK1 TRIB3 Anti-HIV-1 gp120 V3 loop antibody DO142–10 light chain variable region TXNRD2 SP5 ERRFI1 gb:AB020690.1 (KIAA0883, Hs.7782.0) PPP1R3C PTPRB IGFBP5

16.6 67.92 16.53 759.7 62.62 110.23 237.83 270.87 3.16

93.66 393.31 99.56 4227 192.04 665.28 1132.46 1438.49 16.71

5.64 5.63 5.62 5.56 5.39 5.37 5.37 5.31 5.3

0.003756 0.003169

0.0013685 0 0.034844

134.96 58.88 444.56 185.8 14.94 13.81 19.81

690.54 301.1 2257.85 940.83 75.42 69.33 99.35

5.12 5.11 5.08 5.06 5.05 5.04 5.01

0.000488 0.000208 0.000008 0.000562 0.000237 0.0003865 0.000053

0.03 17 19.01 50.07 27.13 86.02

143.32 605.22 408.29 855.04 434.9 1199.07

4339.49 31.1 21.48 17.08 16.03 15.72

0.026885 0.041121 0.03351 0.043547 0.03222 0.002941

7.29 32.43 17.77 24.73 10.38 10.32 68.9 145.08 393.92 5.09 427.3 51.63 181.17 672.63 8.43 12.55 7.11 439.94 104.3 30.81 56.85 194.61 15.12 138.38 15.63 14.8 14.46

73.6 414.44 213.17 279.08 96.34 86.01 565.19 1154.6 2860.51 35.01 2821.62 367.88 1171.6 4258.22 52.83 76.92 43 1727.44 613.66 177.99 388.33 1005.45 85.71 768.54 85.1 80.53 77.84

14.5 12.19 12 11.28 9.28 8.34 8.2 7.96 7.3 6.88 6.6 6.52 6.47 6.33 6.27 6.13 6.05 5.98 5.88 5.78 5.76 5.74 5.67 5.55 5.45 5.44 5.38

0.034703 0.024042 0.018985 0.039309 0.022261 0.034863 0.034189 0.046587 0.030498 0.048818 0.008208 0.035159 0.028816 0.028168 0.012901 0.017833 0.03935 0.006343 0.032267 0.038278 0.029882 0.034367 0.020584 0.040518 0.034768 0.009261 0.047697

0.000718 0.0119025

(B) H9 Hypothetical protein LOC154092 SLC16A3 ANKRD37 LEPREL1 Hypothetical protein LOC153469 Homo sapiens, similar to otoconin 90, clone IMAGE:4285317, mRNA cDNA FLJ41489 fis STC1 LDHC PFKFB4 TJP2 WDR5B TXNIP MSTP150 BNIP3 NCR1 ADM EGLN1 PFKFB3 IGFBP2 S100A1 Similar to HSPC323 PPFIA4 SLC2A1 CH25H NPAS4 BHLHB2 PDK1 Transcribed locus, weakly similar to NP_062553.1 Hypothetical protein MGC33846 gb:AI803010 (Hs.126877) SERPINB3 COL7A1



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Table 3. Genes upregulated (>fivefold) in 2% O2 versus 21% O2 grouped (cont.). EGR1 INSIG2 CCL26 HES1 C22orf35 CLN6

282.63 85.65 43.03 50.84 45.38 34.22

1520.23 457.74 226.59 281.3 234.32 173.53

5.38 5.34 5.27 5.23 5.16 5.09

0.035671 0.034422 0.043091 0.02339 0.046693 0.031213

5.84 33.55 14.29 27.23 29 37.51

62.37 248.17 94.92 169.86 424.86 200.7

10.67 7.4 6.64 6.24 14.65 5.35

0.001264 0.037097 0.00395 0.008722 0.004882 0.037209

14.65 5.45 23.1 8.65 56.1

323.47 110.13 490 115.89 685

22.08 20.2 17.8 13.39 12.3

0.019446 0.001462 0.001161 0.000858 0.00027733

13.97 47.73 5.73 49.73 15.35 32.87 150 9.54 43.3 10.99 397.8 10.24 24.58 16.53

141.21 481.8 55.66 452.63 130.94 275.06 1178 57.3 384 78.71 2819.21 72.12 172.45 113.11

10.11 10.09 9.71 9.1 8.53 8.37 8.3 7.45 7.33 7.16 7.09 7.04 7.01 6.84

0.002215 0.000864 0.002273 0.00008 0.002966 0.000133 0.0001235 0.000639 0.0001555 0.001959 0.000005 0.011005 0.003175 0.000599

44.9 27.24 27.77 52.75 151.86 2.15

321 180.45 181.7 335.4 905.86 11.62

6.75 6.62 6.54 6.36 5.97 5.41

0.00003775 0.009514 0.005848 0.000728 0.000985 0.000643

438 184 11.42 603.98 19.5 10.2 32.17 15.4 26.94 75.49

2340 985.81 60.61 3167.13 119 51 166.04 72.7 136.78 382.85

5.41 5.36 5.31 5.24 5.2 5.17 5.16 5.15 5.08 5.07

0.0000815 0.000046 0.02305 0.000037 0.0022705 0.000483 0.000376 0.00078367 0.000143 0.000038

(C) RH1 QPCT NOX4 Hypothetical gene supported by AF086204 C19orf33 gb:J05008 (endothelin 1) Hypothetical protein LOC153469 (D) Grouped NDUFA4L2 NRN1 SLC16A3 CA3 Homo sapiens, similar to otoconin 90, clone IMAGE:4285317, mRNA SPAG4 LEPREL1 Hypothetical protein LOC441054 CH25H UPK1A ANKRD37 SERPINE1 CD300A BHLHB2 gb:AA625683 (Hs.143659) ADM BHLHB3 NPPB Transcribed locus, weakly similar to NP_062553.1 Hypothetical protein FLJ11267 (Homo sapiens) STC1 Hypothetical protein LOC153469 CSRP1 C1orf61 MSTP150 Anti-HIV-1 gp120 V3 loop antibody DO142–10 light-chain variable region BNIP3 PFKFB3 A2ML1 IGFBP2 PFKFB4 CDNA FLJ41489 fis, clone BRTHA2004582 KISS1R LOX LDHC TXNRD2

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Table 4. Genes upregulated (>fivefold) in 21% O2 grouped versus 2% O2. Gene name (official symbol)

2% p10 mean

21% p0p10 mean Fold change

p-value

0.45 0.74 1.48 1.27 3.99 3.6 2.01 4.65 4.02 4.04 1.54 2.13 24.78 45.85 28.94 35.54 2.32 12.51 3.17 1.96 153.23 184.64

193.16 21.72 15.57 12.97 35.34 27.45 14.62 32.66 27.78 26.51 10.02 13.5 115.32 281.92 174.78 196.4 13.04 69.75 17.4 10.63 830.27 844.1

427.97 29.16 10.49 10.18 8.86 7.62 7.26 7.03 6.91 6.56 6.5 6.32 6.23 6.15 6.04 6.02 5.62 5.58 5.49 5.43 5.42 5.0

0.000501 0.020739 0.023829 0.004562 0.004487 0.003527 0.019887 0.007628 0.006461 0.000922 0.025144 0.001277 0.002846 0.000043 0.00579 0.00522 0.046674 0.000018 0.003212 0.026541 0.033153 0.002125

0.57 0.99 5.36 43.88 1.95 14.93 17.62 162.85 250.92 56.7

13.98 19.04 40.48 383.2 12.53 88.51 88.95 871.13 1316.87 293.75

24.39 19.17 7.56 7.47 6.43 5.93 5.37 5.35 5.25 5.18

0.046685 0.002526 0.000154 0.000007 0.000049 0.000004 0.000436 0.000707 0.000017 0.019006

(A) H1 Homo sapiens, clone IMAGE:5198544, mRNA C9orf117 AQP4 ZNF248 SLC6A2 DLL1 SNF1LK SERHL2/SERHL DCN DPPA5 Homo sapiens, clone IMAGE:4513059, mRNA GYPA PRICKLE1 IRX2 INDO HGPD APOBEC3G ABAT CLIP4 gb:AW513652 (Hs.437414, clone IMAGE:5268367, mRNA) CDKN1A TMEM64 (B) H9 OSBPL7 MYOCD ADIPOQ Nasopharyngeal carcinoma-associated antigen (NPC-A)-5 HES2 TNFRSF10C ARRB1 GDF15 CDKN1A Homo sapiens, clone IMAGE:5198544, mRNA

upregulated at 2% O2 in H1, H9 and RH1, respectively (FIGURE 3A; SUPPLEMENTARY DOCUMENTS 1 & 2) . Within the genes associated with ontology terms across all hESC lines, there was a solitary gene held in common, EGR1 (FIGURE 3B) . This finding agrees strongly with previous reports detailing substantial heterogeneity between hESC lines [9] . However, despite this low similarity there were six GO terms held in common across the hESC lines for genes significantly upregulated in response to 2% O2 ; anatomical structure development, multicellular organismal process, organ development, response to wounding, extracellular region part and extracellular space (FIGURE 3A; SUPPLEMENTARY DOCUMENT 1) . No common ontology groups were noted for genes upregulated in 21% O2 (as compared with 2% O2 ). future science group

Ontology ana lysis of grouped 21% O2 revealed a solitary gene SERPINE1 (upregulated in late passage), which associated significantly with seven GO terms (SUPPLEMENTARY DOCUMENT 1) . There were 302 genes upregulated in grouped 2% O2 and of these 113 genes displayed significant over-representation across 82 GO terms (TABLE 6A; S UPPLEMENTARY DOCUMENTS 1 & 2) . Within the GO-organizing principles, these GO terms clustered predominantly in ‘Biological processes’ (72% [59/82]) and ‘Molecular functions’ (25.6% [21/82]) with slight representation from ‘Cellular components’ (2.4% [2/82]) (TABLE 6C) . Further grouping of the GO terms according to broad functionality groups revealed that the main focus of over-represented GO terms were concerned with ‘Proliferation, signaling and www.futuremedicine.com

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Table 5. Total numbers of genes upregulated and associated gene ontology terms in direct base versus experimental comparison of grouped analysis. Base

Experimental

Value

(A) The number of significantly over-represented GO terms is indicated in parentheses after the gene number H1 21% p0p10 H1 2% p10 H9 21% p0p10 H9 2% p10 RH1 21% p0p10 RH1 2% p10 All 21% p0p10 All 2% p10

H1 2% p10 H1 21% p0p10 H9 2% p10 H9 21% p0p10 RH1 2% p10 RH1 21% p0p10 All 2% p10 All 21% p0p10

262 (53) 98 (0) 239 (46) 84 (0) 119 (14) 36 (0) 302 (82) 56 (0)

(B) The number of significantly under-represented GO terms is indicated in parentheses after the gene number All 21% p0p10 All 2% p10

All 2% p10 All 21% p0p10

302 (9) 56 (0)

Gene upregulation is determined through > twofold upregulation, p < 0.05 and > 10REU. GO: Gene ontology.

behavior’ (43.9%) and ‘Differentiation and development’ (28%) (BOX 1) . Upregulation of signaling, behavioral and proliferation pathways reinforces previous observations on clonality and pluripotentiality of hESC in 2% O2 [11–13] . The relative abundance of terms concerned with differentiation and development implies an increase in potency as cells in 2% O2 maintain an undifferentiated phenotype in vitro [12,13,15] . No ontological over-representation was noted within the 56 genes upregulated in 21% O2 grouped (vs 2% O2 grouped).

Ontological under-representation Over-representation of ontologies within defined gene lists can be used to identify pathways undergoing upregulation. In this instance, overrepresentation was observed for subsets of genes in response to 2% O2 culture. Conversely, the under-representation of ontology groups demonstrates that for a given subset of genes there is less representation that would be expected from a random set. We observed 114 genes that were under-represented across nine GO terms in the upregulated gene set from grouped 2% O2 (TABLE 6B; SUPPLEMENTARY DOCUMENT 1) . Unlike the 2% O2 over-represented GOs, these clustered strongly within ‘Cellular component’ (77.8% [7/9]) and less so in ‘Biological process’ (1/9 [11.1%]) and ‘Molecular function’ (1/9 [11.1%]) categories. We have previously described hESC as being smaller and less complex in 2% O2 versus 21% O2 [11] . In this instance, the underrepresented cellular component ontology groups 826

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are predominantly concerned with intracellular characteristics and organelles providing a potential molecular rational to our previous description. From this analysis we conclude that in response to 2% O2 hESC upregulated a large genetic cohort. At the individual cell line level these differences were mostly unique with little homology. Grouped ana lysis increased statistical strength, revealing a common transcriptional upregulation; a physiological O2-response biomarker comprising 358 (302 upregulated and 56 downregulated) genes. Of the upregulated genes approximately two out of five were significantly over-represented for ontological groupings, some of which can be predicted as hypoxic response (angiogenesis, glycolysis and inflammatory response), and others which are unexpected (receptor binding, extracellular space and hormone activity). Conversely, in 21% O2 we saw no ontological over-representation for upregulated genes. Therefore, genes upregulated in 2% O2 fall into distinct predicted and novel pathways, whereas those downregulated in 2% O2 (upregulated in 21% O2) are potentially representative of transcriptional noise through transcriptional deregulation as a consequence of inappropriate culture conditions. Discussion This study was designed to determine and define the extent of the transcriptional consequence of culturing hESCs in 2% O2 (approximately physiological) when compared with 21% O2 (air and supraphysiological). We identified 302 genes that passed our stringent inclusion parameters and were upregulated in 2% O2. These genes were of both known and unknown function and included many predicted to be functionally involved in response to 2% O2. Examination of ontological associations of these 302 genes revealed significant clustering within 82 GO terms, which included known hypoxic response mechanisms (TABLE 6; BOX 1; SUPPLEMENTARY TABLE 9; SUPPLEMENTARY DOCUMENTS 1 & 2) . Conversely, we identified 56 genes that were downregulated in response to 2% O2. These genes did not form any significant ontological associations. These findings demonstrate that the previously described consequences of culturing hESCs in 2% O2 (reduced spontaneous differentiation, enhanced chromosomal stability and increased clonality) are accompanied by widespread transcriptional changes [11–13,15,16] . In performing this study, we sought to address two important parameters: transcriptional change as a consequence of adaptation through future science group


extended culture and environmental parameters. In the first instance, we introduced experimental design, which would reveal changes as

RESEARCH ARTICLE

a consequence of culture. We analyzed each hESC line in standard 21% O2 culture at p0, bifurcated each line into 21% O2 and 2% O2,

A Genes with ontological associations in 2% O2 cultured human embryonic stem cell

H1

H9

adcy8; ahnak; angpt1; barx1; bhlhb3; calca; cd300a; centa1; chrna9; creb5; dact1; ddr2; dgkg; dtna; ednrb; eomes; epha3; fos; fosl1; fzd10; gas8; gata6; gdf15; gjb6; gpr177; grem1; id4; igfbp5; il32; inhbe; isg20; kifc3; kiss1r; lsamp; mxi1; nckipsd; ndrg1; nefl; nppb; nrn1; p2ry12; page1; paqr5; pcdh10; pcdh11x; pde4c; pdk4; pgam2; pgk1; plau; ppargc1a; ppp1r3c; ptprb; rho; scarf1; scg2; sepp1; serpine2; sfn; sgcg; slc22a3; slc8a3; sox10; sox17; syde1; tac1; tnc; tnfrsf12a; trib3; trpm1; upk1a; vegfa; zbtb17

adamts20; adamts5; adm; apold1; bnip3; ccl26; ch25h; crybb2; cxcl5; ddit4; fgf1; gabbr2; gbe1; hk2; igfbp2; insig2; ldhc; nefm; pdk1; pfkfb3; pfkfb4; phlda1; ppfia4; ptx3; rab20; rhob; serpine1; sox14; tbc1d2; trim54

adamtsl2; alcam; aldoa; aldoc; alox12; ccl4; cd59; cd8b; col1a1; col7a1; crip2; dazl; dclk1; dusp9; egr2; egr3; extl1; gabra2; gys1; hoxb4; htr2c; itgad; jmjd1a; ldha; leprel1; mpi; msn; mtp18; nab2; nnt; nol3; per3; pes1; pf4; pthlh; rasef; s100a1; serpinb1; serpinb3; slc1a1; slc8a1; sntg2; srpinb3; tbx1; thrb; wsb1

egr1

ankrd1; anxa1; cer1; cxcl14; id3

edn1; egln3; fjx1; igfbp6; ptger2; sox15; sox3

acta1; actg2; arhgdib; cav1; cdh13; cnn1; col11a1; ctgf; ebi3; emp3; fam110c; fzd7; hspb2; il17ra; il24; il6; insm1; itgbl1; jub; jun; krtap9-3; lamc2; lgals1; marco; myl2; myocd; nexn; nox4; npr3; pdlim7; rasgrf2; runx2; serpinb4; tfpi; timp3; tmsl8; tshz3; vil1

RH1

B Common ontological associations in 2% O2 cultured human embryonic stem cell

H1

H9 13

22

27

6 3

1 Anatomical structure development 0

2 Multicellular organismal process 3 Organ development

5

4 Response to wounding 5 Extracellular region part

RH1

6 Extracellular space

Figure 3. Shared genes and gene ontology terms identified through ontology. (A) A comparison of genes upregulated in 2% O2 which were associated with significant Gene ontology (GO) terms for H1, H9 and RH1 line specific analysis. No common genes were identified. (B) A comparison of statistically over-represented GO terms for genes upregulated in 2% O2 in H1, H9 and RH1 line specific analysis. The five common GO terms are associated with differentiation capacity (anatomical structure development and anatomical structure morphogenesis) and signaling (extracellular region part, extracellular space and response to wounding).



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Table 6. Over-represented and under-represented gene ontology terms in 2% O2 upregulated genes. GO term

Genes in cluster

p-value

% genes

26 43 20 35 26 23 26 47 39 21 52 6 16 13 11 16 6 16 8 12 49 10 6 10 7 7 7 7 8 4 4 8 8 8 6 4 10 8 44 16 16 8 12 4 9 29 29 6 4

3.51e-11 3.9e-11 4.22e-11 4.53e-10 2.4e-08 5.32e-08 1.17e-07 2.92e-07 3.48e-07 1.04e-06 1.04e-0 1.24e-05 3.18e-05 4.44e-05 6.43e-05 7.92e-05 0.000118 0.000177 0.000188 0.000228 0.000275 0.000296 0.0003 0.0003 0.000374 0.000374 0.000374 0.000374 0.000379 0.000416 0.000416 0.000416 0.000416 0.000416 0.000482 0.000591 0.000677 0.000841 0.000841 0.000841 0.000841 0.000872 0.000907 0.00102 0.00102 0.00115 0.00115 0.00127 0.00133

8.6 14.2 6.6 11.6 8.6 7.6 8.6 15.6 12.9 6.9 17.2 2.0 5.3 4.3 3.6 5.3 1.9 5.3 2.6 4.0 16.2 3.3 2.0 3.3 2.3 2.3 2.3 2.3 2.6 1.3 1.3 2.6 2.6 2.6 2.0 1.3 3.3 2.6 14.6 5.3 5.3 2.6 4.0 1.3 3.0 9.6 9.6 2.0 1.3

(A) Over-represented GO terms for 2% O2 upregulated genes 0044421: extracellular region part 0048856: anatomical structure development 0005615: extracellular space 0048731: system development 0009653: anatomical structure morphogenesis 0008283: cell proliferation 0048513: organ development 0032501: multicellular organismal process 0007275: multicellular organismal development 0005102: receptor binding 0032502: developmental process 0001664: G-protein-coupled receptor binding 0009605: response to external stimulus 0009611: response to wounding 0007610: behavior 0007267: cell–cell signaling 0005179: hormone activity 0042127: regulation of cell proliferation 0001525: angiogenesis 0006952: defense response 0007154: cell communication 0019318: hexose metabolic process 0004867: serine-type endopeptidase inhibitor activity 0005996: monosaccharide metabolic process 0006935: chemotaxis 0030414: protease inhibitor activity 0042330: taxis 0004866: endopeptidase inhibitor activity 0048514: blood vessel morphogenesis 0042379: chemokine receptor binding 0008009: chemokine activity 0005125: cytokine activity 0048646: anatomical structure formation 0006006: glucose metabolic process 0006096: glycolysis 0006000: fructose metabolic process 0004857: enzyme inhibitor activity 0001568: blood vessel development 0007165: signal transduction 0022610: biological adhesion 0007155: cell adhesion 0001944: vasculature development 0006066: alcohol metabolic process 0046058: cAMP metabolic process 0009888: tissue development 0048869: cellular developmental process 0030154: cell differentiation 0006007: glucose catabolic process 0005520: IGF binding

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Table 6. Over-represented and under-represented gene ontology terms in 2% O2 upregulated genes. 0009887: organ morphogenesis 0007626: locomotory behavior 0048519: negative regulation of biological process 0019798: procollagen–proline dioxygenase activity 0006954: inflammatory response 0019320: hexose catabolic process 0046365: monosaccharide catabolic process 0046164: alcohol catabolic process 0009187: cyclic nucleotide metabolic process 0048523: negative regulation of cellular process 0031543: peptidyl-proline dioxygenase activity 0008285: negative regulation of cell proliferation 0031418: L-ascorbic acid binding 0007399: nervous system development 0016701: oxidoreductase activity* 0051213: dioxygenase activity 0016702: oxidoreductase activity‡ 0008083: growth factor activity 0003714: transcription corepressor activity 0003014: renal system process 0003091: renal water homeostasis 0030146: diuresis 0045110: intermediate filament bundle assembly 0051482: elevation of cytosolic calcium§ 0004332: fructose-bisphosphate aldolase activity 0030147: natriuresis 0006931: substrate-bound cell migration, cell attachment to substrate 0044275: cellular carbohydrate catabolic process 0016052: carbohydrate catabolic process 0050770: regulation of axonogenesis 0006171: cAMP biosynthetic process 0016706: oxidoreductase activity ¶ 0044262: cellular carbohydrate metabolic process

11 7 24 3 8 6 6 6 4 23 3 9 4 14 5 5 5 6 7 2 2 2 2 2 2 2 2 6 6 3 3 4 11

0.00156 0.00156 0.00159 0.00212 0.00219 0.00219 0.00245 0.00271 0.00271 0.00286 0.00324 0.00326 0.00326 0.00395 0.00423 0.00423 0.00423 0.00456 0.00456 0.00456 0.00456 0.00456 0.00456 0.00456 0.00456 0.00456 0.00456 0.00674 0.00674 0.00829 0.00829 0.0088 0.00996

3.6 2.3 7.9 1.0 2.6 2.0 2.0 2.0 1.3 7.6 1.0 3.3 1.3 4.6 1.7 1.7 1.7 2.0 2.3 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 2.0 2.0 1.0 1.0 1.3 3.6

0005622: intracellular

73

-3.04e-05

24.2

0044424: intracellular part 0043227: membrane-bound organelle 0043226: organelle 0043229: intracellular organelle 0043283: biopolymer metabolic process 0003676: nucleic acid binding 0044464: cell part

71 47 56 26 27 13 105

-0.000288 -0.000458 -0.00082 -0.00083 -0.00244 -0.00317 -0.00317

23.5 15.6 18.5 18.5 8.9 4.3 34.8

21% p0p10 vs 2% p10

Biological process

Cellular component

Molecular function

Over-represented

72% (59/82)

2.4% (2/82)

25.6% (21/82)

Under-represented

11.1% (1/9)

77.8% (7/9)

11.1% (1/9)

(B) Under-represented GO terms for 2% O2 upregulated genes

The GO term (numerical identifier and description), number of genes (up- or downregulated in this study) associated with the term, p-value and number of genes expressed as a percentage of the total upregulated (A) or downregulated (B) population are described. * Acting on single donors with incorporation of molecular oxygen; ‡Acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen; §Elevation of cytosolic calcium ion concentration during G-protein signaling, coupled to IP3 second messenger; ¶ Acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors. GO: Gene ontology.



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Box 1. Broad assignation of gene ontology terms into macro groupings. Proliferation, signaling and behavior Extracellular region part Extracellular space Cell proliferation Receptor binding G-protein-coupled receptor binding Response to external stimulus Response to wounding Behavior Cell–cell signaling Hormone activity Regulation of cell proliferation Defense response Cell communication Serine-type endopeptidase inhibitor activity Chemotaxis Protease inhibitor activity Taxis Endopeptidase inhibitor activity Chemokine receptor binding Chemokine activity Cytokine activity Enzyme inhibitor activity Biological adhesion Cell adhesion Signal transduction Insulin-like growth factor binding Locomotory behavior Negative regulation of biological process Inflammatory response Negative regulation of cellular process Negative regulation of cell proliferation L-ascorbic acid binding Growth factor activity Transcription corepressor activity Elevation of cytosolic calcium* Substrate-bound cell migration, cell attachment to substrate 43.9% (36/82) Differentiation and development Anatomical structure development System development Anatomical structure morphogenesis Organ development Multicellular organismal process Multicellular organismal development Developmental process Angiogenesis Blood vessel morphogenesis Anatomical structure formation Blood vessel development Vasculature development

and then reanalyzed at p10 in both air and 2% O2. Through this process, we were able to demonstrate that in late-passage (p10) air cells there 830

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Box 1. Broad assignation of gene ontology terms into macro groupings (cont.). Tissue development Cellular developmental process Cell differentiation Organ morphogenesis Nervous system development Renal system process Renal water homeostasis Diuresis Intermediate filament bundle assembly Natriuresis Regulation of axonogenesis 28% (23/82) Metabolism Hexose metabolic process Monosaccharide metabolic process Glucose metabolic process Glycolysis Fructose metabolic process Alcohol metabolic process cAMP metabolic process Glucose catabolic process Hexose catabolic process Monosaccharide catabolic process Alcohol catabolic process Cyclic nucleotide metabolic process Fructose-bisphosphate aldolase activity Cellular carbohydrate catabolic process Carbohydrate catabolic process cAMP biosynthetic process Cellular carbohydrate metabolic process 21% (17/82) Oxygen response Procollagen-proline dioxygenase activity Peptidyl-proline dioxygenase activity Oxidoreductase activity‡ Dioxygenase activity Oxidoreductase activity§ Oxidoreductase activity¶ 7.1% (6/82) The 82 gene ontology (GO) terms associated with 113 genes upregulated in 2% O2 group into four broad classifications. Within each classification the terms are sorted numerically-alphabetically. The shaded row describes the percentage of GO terms within each broad category. * Acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors; ‡ Elevation of cytosolic calcium ion concentration during G-protein signaling, coupled to IP3 second messenger; § Acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen; ¶Acting on single donors with incorporation of molecular oxygen.

were a total of six genes significantly upregulated and a solitary gene downregulated when compared with p0 air (SUPPLEMENTARY TABLE 5) . This future science group


argues strongly against any significant contribution from extended culture to the transcriptional upregulation evidenced in 2% O2. In the second instance, it was imperative that we achieved as robust a control of O2 concentration as possible. We selected a modular incubator system, which allowed inspection of cells without disruption of environment. This provided greater control compared with other available systems and allowed preplanning on passaging and media changes. All cell manipulations were performed as rapidly as possible and cells replaced into the original hermetic environment as swiftly as possible. To our knowledge, this is the fi rst reported description of the transcriptional consequences of 2% O2 on hESC culture. A previous study has reported that the global gene-expression patterns of 2% O2 in vitro-developed murine blastocysts more closely resemble their in vivo counterparts than 21% O2 in vitro-developed murine blastocysts [21,22] . Unfortunately, a complementary study has not yet been performed with murine ESCs in 2% O2 versus 21% O2, preventing comparison with this study. The purpose of this study was to identify consensus transcriptional changes across three hESC lines in response to 2% O2. Notwithstanding this, we described earlier the detailing of a consensus pluripotency ‘fi ngerprint’ of five genes from earlier studies (CD24, NASP, POU5F1, SEMA6A and SLC16A1) [4–6] . This footprint was generated by drawing comparisons between undifferentiated hESCs and differentiated progeny or tissue samples. None of these genes underwent significant alterations in response to 2% O2 . However, we did note substantial upregulation of SLC16A3 (17.8-fold), which, along with SLC16A1, belongs to the monocarboxylate transporter family responsible for pyruvate uptake in pre-implantation blastocysts (TABLE 3D; S UPPLEMENTARY TABLE 9) [23] . In addition, both share common substrates (lactate, pyruvate and ketone bodies), suggesting potential redundancy across the two family members. Hierarchical clustering of fi ltered gene lists for all samples demonstrated that hESC cultured in 2% O2 demonstrated a strong transcriptional resemblance (FIGURE 2) . This resemblance was sufficient to distinguish them from 21% O2 cultured cells, implying a reduction of line-specific heterogeneity [9] . Heterogeneity within cultures of hESCs results in suboptimal differentiation frequencies and difficulties in generating standardized profi les, for instance, the pluripotency ‘fingerprint’ defined earlier. The precise reasons for the reduction future science group

RESEARCH ARTICLE

in transcriptional heterogeneity are not clear. There are known hypoxia transcriptional response pathways that are both hypoxiainducible factor (HIF) dependant and independent. The HIF transcription factor family contains three known α-subunits (HIF-1α, -2α and-3α) and one common β-subunit (HIF-β/ aryl hydrocarbon receptor nuclear translocator [ARNT]) [24] . The dimerization of each independent α-subunit with ARNT creates a unique nonredundant transcription factor. The HIF transcription factors activate target genes in response to hypoxia in a prolyl hydroxylasedependent manner [24] . Transcriptional upregulation of HIF components was not observed in this study. However, we did observe transcriptional upregulation of known HIF-dependant target genes including transcription factors (BHLHB2 and BHLHB3) and HIF control factors (EGLN1 and EGLN3). We also observed upregulation of HIF-independent hypoxiainducible genes including the transcription factor EGR1 [24] . The cascade of transcriptional changes occurring as a consequence of physiological normoxia (not hypoxia in this instance) and resultant translational changes could underpin the reduction in transcriptional heterogeneity. Further studies, including global proteome ana lysis, will clarify the mechanisms involved. Coupled to the observed transcriptional changes there may also be alterations occurring at the epigenetic level as a consequence of physiological normoxia. Previous studies have shown that HIF-1 must associate with the coactivator p300 to activate target genes [25,26] . The p300 coactivator has histone acetyl transferase activity. In addition to p300, HIF-1α interacts with the histone deacetylase HDAC7 [27] . Other reports indicate a global decrease in methylation after exposure to chronic hypoxia [28] . We observed transcriptional upregulation of three jumonji domaincontaining histone modifiers within this study: JMJD1A, JMJD2B and JMJD3. At least one of these genes, JMJD1A, is known to be stimulated by hypoxia [29] . No studies exist to demonstrate a global epigenetic consequence as a result of hESC culture in physiological normoxia. Taken together, the findings from this study illustrate transcriptional upregulation of HIF-dependent and -independent genes and histone demethylases. The reduction in heterogeneity is potentially a consequence of both transcriptional and epigenetic alterations. Controlled experimentation using histone deacetylase or methylation inhibitors is necessary to extend this body of www.futuremedicine.com

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knowledge. The implication of this is that culture of hESCs in 2% O2, combined with standardized conditions, may be sufficient to generate a hESC transcriptional ‘fingerprint’ suitable for use in high-throughput screening of potentially pluripotent cell lines. Interestingly, we did not detect any significant alteration of the previously described key regulators of pluripotency: POU5F1, NANOG or SOX2 [30] . Functional annotation using GO terms provides a mechanism to discover common pathways within groups of upregulated genes. Analysis of significant GO-term associations with genes upregulated in 2% O2 in individual cell lines identified six common terms: anatomical structure development, multicellular organismal process, organ development, response to wounding, extracellular region part and extracellular space. The common theme across these individual lines is that in response to 2% O2 genes associated with development and differentiation are undergoing significant upregulation above an expected background level. In addition, there is a generic over-representation of terms concerned with modifying the extracellular environment. Despite there being six GO terms held in common across the three hESC lines examined, there was a solitary gene held in common within those associated with GO terms: EGR1. EGR1 is a zinc-finger transcription factor that is upregulated in ‘hypoxic’ conditions [31] . In addition to an involvement in transcriptional activity of genes associated with differentiation and mitogenesis, an association with HIF-1α activity has also been recently demonstrated [32] . We identified 82 GO terms associated with 113/302 upregulated genes with p < 0.01 (TABLE 6A; SUPPLEMENTARY DOCUMENTS 1 & 2) . Within these GO terms were a number that served to reinforce earlier observations, including enhanced clonogenicity, for example, cell proliferation, regulation of cell proliferation, biological

adhesion, cell adhesion, cell–cell signaling and negative regulation of cell proliferation [11,12,16] . A large number of GO terms concerned with signaling pathways were also over-represented. These included receptor binding, G-proteincoupled receptor binding, cell–cell signaling, hormone activity, cell communication, chemokine receptor binding, chemokine activity and cytokine activity. The particular hormones and cytokines upregulated in this instance do not fit into classical self-renewal pathways; NPPB, CALCA, PTHLH, EDN1, ADM, INHBE, CXCL5, IL32, CXCL14, CCL26, VEGFA, CXCL1, GREM1 and CER1. The nine GO terms (114/302 genes) under-represented in our upregulated gene set were predominantly concerned with intracellular organelles. This serves to provide a molecular rationale to our earlier observations describing reduction of complexity in 2% O2 cultured cells. In summary, this report identifies a core physiological O2 response ‘fingerprint’ in hESCs. This ‘fingerprint’ consists of 302 upregulated and 56 downregulated genes, where a large number of the upregulated genes were significantly over-represented across expected and surprising pathways described by GO terms. The functional testing of the pathway components and proteomic translation will have implications for routine hESC culture, cell therapy and the development of further understanding in stem cell biology. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Physiological normoxia (2% O2) induces widespread transcriptional alterations in human embryonic stem cells (hESCs). Combined analysis reveals a physiological normoxia transcriptional ‘fingerprint’. Transcriptional fingerprint comprises of 302 genes upregulated and 56 genes downregulated. Ontology analysis of upregulated component of transcriptional fingerprint shows ontological over-representation of genes involved in signaling, developmental and metabolic pathways, as well as ontological under-representation of genes involved in intracellular organelles. Ontology analysis of downregulated component of transcriptional fingerprint shows no ontological association. Hierarchical clustering of differentially regulated probes in all hESC samples illustrates a reduction in transcriptional heterogeneity across all cell lines in 2% O2.

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RESEARCH ARTICLE


Website 101 GOStat: Find statistically overrepresented

GO terms within a group of genes. http://gostat.wehi.edu.au/

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