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RESEARCH |
1 Centre for Human Development, Stem Cells and Regeneration
2 , Human Genetics Division
3 Developmental Origins of Health & Disease Division, School of Medicine, University of Southampton, Southampton General Hospital, Duthie Building (MP 808), Tremona Road, Southampton, SO16 6YD, UK
Correspondence should be addressed to F D Houghton; Email: f.d.houghton{at}soton.ac.uk
This is an Open Access article distributed under the terms of the Society for Reproduction and Fertility's Re-use Licence which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is roperly cited.
N A Hanley is now at Endocrine Sciences Research Group, Manchester Academic Health Science Centre, University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, UK 
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and MethodsDeclaration of interestReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestReferences |
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In terms of hES cells, atmospheric oxygen remains the culture environment of routine use. There is emerging evidence to suggest that reducing the oxygen concentration towards physiological levels is beneficial for the in vitro maintenance of hES cells in terms of decreasing the amount of spontaneous differentiation, supporting self-renewal (Ezashi et al. 2005, Ludwig et al. 2006, Westfall et al. 2008), and reducing spontaneous chromosomal aberrations (Forsyth et al. 2006). Thus, while hES cells can be maintained under atmospheric oxygen tensions, lowering the oxygen tension to 2–5% appears beneficial for the propagation of a highly proliferative, pluripotent population of cells. However, controversy still remains as a recent report suggests that there are no significant advantages of culturing hES cells under reduced oxygen tension (Chen et al. 2009).
Upon exposure to hypoxic conditions, cells mount a physiological response to ensure sufficient levels for oxygen-dependent processes. This response is regulated by hypoxia inducible factors (HIFs) which regulate the expression of over 200 genes including those involved in erythropoiesis, apoptosis and proliferation (Semenza 2000). HIFs are transcription factors consisting of three oxygen-dependent 
subunits: HIF1A, HIF2A (also known as EPAS1) and HIF3A, and a constitutively expressed β subunit, HIF1B (also known as ARNT). Under atmospheric oxygen tensions, HIF1A protein is rapidly degraded due to hydroxylation by prolyl hydroxylase proteins (PHDs). These hydroxylated proteins are then recognised by the Von Hippel–Lindau (VHL) protein which targets them for proteosomal degradation. Under hypoxic conditions, PHDs are unable to hydroxylate HIF1A which therefore cannot be targeted for degradation by the VHL protein (Semenza 2003). Stabilised HIF1A subunits translocate from the cytoplasm to the nucleus where they bind with HIF1B to activate target genes (Wenger 2002). HIF1A, the first HIF- subunit described, is thought to be the global regulator of the hypoxic response (Semenza & Wang 1992). However, the contribution of HIF2A and HIF3A, which are believed to be regulated in a similar manner (Ivan et al. 2001, Masson et al. 2001), remain to be fully characterised. All three subunits share many sequence similarities (reviewed by Lee et al. (2004)); they all possess the Per, Arnt/HIF1B and Sim domain essential for binding to HIF1B and the oxygen-dependent degradation domain, the target for degradation under normoxic conditions. HIF3A differs from HIF1A and HIF2A as it lacks the C-terminal activation domain required for co-activator binding. Thus, HIF3A is unable to recruit co-transcriptional regulators and basal transcriptional machinery to gene targets. HIF- subunits have been found to activate genes that contain the hypoxia response element sequence located in the promoter region of hypoxia responsive genes (Wenger 2002). HIF2A can target this sequence independently of HIF1A, suggesting that they have functionally diverse roles. For example, HIF1A, originally thought to be the main oxygen sensing subunit, predominantly regulates glycolytic genes (Hu et al. 2003), whereas HIF2A is the main regulator of hypoxia-induced erythropoietin in tissues which express both HIF1A and HIF2A (Warnecke et al. 2004, Rankin et al. 2007). HIF2A has also been shown to promote cell cycle progression in hypoxic renal clear cell carcinoma cells (Gordan et al. 2007). To date, little is known about which genes are targeted by HIF3A.
The functional significance of HIF genes has been highlighted using targeted gene inactivation. HIF1A–/– mice are non-viable, displaying developmental arrest by E9.0 with significant mesenchymal cell death and impaired vascular development (Kotch et al. 1999). HIF1B null mice are embryonic lethal by E10.5, displaying yolk sac and placental deficiencies and decreased numbers of haematopoietic progenitors (Nishi et al. 2004). HIF2A–/– mice develop severe vascular defects and show developmental arrest between E9.5 and E12.5 with variability depending on the genetic background (Compernolle et al. 2002, Nishi et al. 2004).
In mouse ES cells, both HIF1A and HIF2A are expressed but HIF1A appears to be central to regulating hypoxic responses, since it targets many oxygen-dependent genes that are not regulated by HIF2A (Hu et al. 2006). However, HIF2A has been found to be a direct upstream regulator of POU5F1 (OCT4) in mouse ES cells, suggesting that HIF2A is involved in the regulation of stem cell maintanence (Covello et al. 2006). In hES cells, the mechanism of hypoxic regulation appears to differ since HIF1A protein is only transiently expressed for 
48 h following exposure to low oxygen tension (Cameron et al. 2008).
While there has been controversy in the literature, this study aims to prove that hES cell culture is improved under physiological oxygen concentrations compared with ambient, atmospheric oxygen tension. We investigate the effect of oxygen tension on hES cell morphology, pluripotency and proliferation and determine the functional significance and potential hierarchy of HIFs in regulating these hypoxic responses.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestReferences |
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50% larger at 5% oxygen than those cultured under 20% oxygen on each day post-passage (P<0.01–P<0.001; Fig. 1b). In total, hES cells were maintained under both 5% and 20% oxygen for 10–12 months, or 36–40 passages and the same morphological differences were maintained throughout this period. To further quantify the increase in colony size, equal numbers of cells were seeded on Matrigel coated plates on day 0 under both oxygen tensions and total hES cell number determined on each subsequent day post-passage. A significant increase in cell number was apparent by 48 h post-passage under 5% oxygen compared with 20% oxygen and was maintained on days 3 and 4 (P<0.001; Fig. 1c). Using Ki67 labelling of hES cells on Matrigel on day 3 post-passage, there was a dramatic decrease in the number of proliferating cells at 20% oxygen compared with 5% oxygen with 50% of hES cells cultured under 20% oxygen being Ki67 positive compared with virtually all cells at 5% oxygen (Fig. 1d). Similar results were obtained for hES cells cultured on mouse embryonic fibroblasts (MEFs; data not shown).
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40% in hES cells cultured at 20% oxygen compared with 5% oxygen (P<0.01; Fig. 2c and d).
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subunits with respect to oxygen tension. Surprisingly, HIF1B was significantly upregulated under hypoxic conditions (Fig. 3a).
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Using immunocytochemistry, HIF1A was found to be only transiently expressed being present in the nucleus of hES cells cultured under 5% oxygen for 48 h but absent after long-term culture under hypoxic conditions (Fig. 3d). HIF1A was also not expressed in hES cells cultured under 20% oxygen. HIF2A and HIF3A both displayed a largely cytoplasmic expression following 48 h of culture at 5% oxygen. However, following long-term hypoxic culture they both displayed a predominantly nuclear localisation. Under 20% oxygen, HIF2A and HIF3A remained cytoplasmic. HIF1B, which is known to be constitutively expressed, was present in the nuclei of hES cells when cultured under both 20% and 5% oxygen tensions. It was therefore not necessary to investigate the localisation of HIF1B in hES cells cultured in 5% oxygen for 48 h.
HIF knockdown
Following siRNA real-time RT-PCR confirmed an 83%, 86% and 90% knockdown of HIF1A, HIF2A and HIF3A respectively compared with transfection control siRNA (Fig. 4a, c and e). Initial studies examined the effect of knocking down individual HIF- subunits for 48 h. When HIF2A was silenced HIF1A mRNA expression was not affected (Fig. 4a). However, the knockdown of HIF3A significantly upregulated HIF1A mRNA expression (P<0.05, Fig. 4a). As expected, HIF1A protein was not expressed in hES cells maintained at 5% oxygen and was not induced by silencing HIF2A. Interestingly, HIF1A protein was induced when HIF3A was silenced (Fig. 4b). When both HIF2A and HIF3A were silenced, HIF1A was expressed but at a significantly reduced level compared with when HIF3A alone was knocked down (P<0.001, Fig. 4b).
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HIF3A mRNA (Fig. 4e) and protein (Fig. 4f) expression were found to be significantly upregulated when HIF1A and HIF2A were knocked down independently.
Effect of HIFs on pluripotency marker expression
Using real-time RT-PCR there was a significant reduction in POU5F1 (Fig. 5a; P<0.001), SOX2 (Fig 5c; P<0.001, P<0.05) and NANOG (Fig. 5e; P<0.001) when HIF2A and HIF3A were silenced independently. As expected, silencing of HIF1A did not alter the mRNA expression of POU5F1, SOX2 and NANOG compared with transfection control siRNA (Fig. 5a, c and e). At the protein level, POU5F1, SOX2 and NANOG were significantly reduced when HIF2A (P<0.001) and HIF3A (P<0.05) were silenced (Fig. 5b, d and f).
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knockdown hES cells did not form colonies and did not survive in culture.
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30% when HIF2A expression was knocked down and displayed a further decrease (P<0.01) when HIF2A and HIF3A were silenced simultaneously. There was no additional increase in the diameter of HIF2A silenced cells 72 h post-transfection (data not shown). Colony size and cell number were unaffected by HIF1A or HIF3A knockdown. It was not possible to measure the colony size of double knockdown combinations of HIF1A and HIF2A or HIF1A and HIF3A or the triple knockdown due to the absence of colony formation occurring in these populations. Virtually all cells were positive for Ki67 when either HIF1A or HIF3A expression was silenced whereas Ki67 expression decreased to 85% when HIF2A was knocked down (P<0.001, Fig. 7c and d).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestReferences |
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Culture at 5% oxygen, whether on MEFs or feeder-free on Matrigel, increased the rate of cell proliferation which agrees with data from Ludwig et al. (2006), producing significantly larger hES cell colonies. This may be due to the observed increase in NANOG expression levels, since it has recently been shown that NANOG regulates S-phase entry in hES cells and that overexpression of NANOG significantly increases proliferation by binding to regulatory regions of CDK6 and CDC25A, two important cell cycle regulators (Neganova et al. 2009, Zhang et al. 2009). Similar increased rates of proliferation have also been observed in many other primitive populations including mesenchymal stem cells and neural progenitor cells when cultured under low oxygen tensions (Grayson et al. 2007, Zhao et al. 2008). Data from mesenchymal stem cells suggest the involvement of HIFs, specifically HIF2A (Grayson et al. 2007). This is supported in hypoxic carcinoma cells where HIF2A has been shown to promote cell cycle progression (Gordan et al. 2007).
In agreement with Forsyth et al. (2008) and Westfall et al. (2008), there was no significant increase in the mRNA expression of the HIF- subunits under hypoxia suggesting that altered protein levels are due to post-transcriptional regulation as found in other cell types (Huang et al. 1998, Lang et al. 2002). However, in contrast to Westfall et al. (2008), HIF1B mRNA expression was upregulated under hypoxia which may reflect differences in both the methodology and cell lines used. To date, most research has focused on the HIF1A subunit which is thought to be the master regulator of the hypoxic response (Mazure et al. 2004). However, our data suggests this is not the case, at least in hES cells since HIF1A was only transiently expressed in the nucleus for 48 h following exposure to hypoxia. These findings are in agreement with recent data from Cameron et al. (2008) using a 2% environmental oxygen tension, although contrary to those of Westfall et al. (2008), who found HIF1A protein to be expressed in hES cells after 10 days of culture under both 5 and 20% oxygen. The reason for this discrepancy is unknown although HIF1A would be expected to be rapidly degraded by PHDs under atmospheric conditions. Moreover, the HIF1A staining observed by the latter investigators was cytoplasmic and therefore would not be expected to regulate the expression of hypoxia regulated genes. Our study suggests that HIF1A may play a role in the initial adaptation of hES cells to hypoxia but some other factor(s) must be responsible for maintaining the hypoxic response. We propose that HIF2A or HIF3A, both of which are translocated from the cytoplasm to the nucleus and are upregulated following culture under hypoxic conditions, may take over the initial, transient role of HIF1A. This is the first time that HIF3A and HIF1B have been demonstrated in hES cells.
hES cell colonies cultured at 5% or 20% oxygen were both positive for pluripotency markers albeit at a significantly decreased level at 20% oxygen. These results were not apparent using immunocytochemistry and highlight the importance of performing quantitative techniques when assessing pluripotency. The current results suggest that although hES cells are capable of being maintained under atmospheric conditions pluripotency is reduced before the appearance of morphological differentiation. This shows that the manual removal of differentiated cells at the time of passage, a method often used during routine culture at 20% oxygen is not effective in ensuring the maintenance of a highly pluripotent population. However, culturing hES cells at 5% oxygen does not require the routine removal of differentiated areas and can therefore be considered a much more effective method of maintaining pluripotency during long term culture. Interestingly, our data shows that hES cells cultured at 5% oxygen have an increased expression of SOX2, NANOG and POU5F1 compared with cells cultured under 20% oxygen even on day 3 post-passage, before these cells appear to be morphologically differentiated. These data are in agreement with similar POU5F1 results obtained by Ludwig et al. (2006). However, they are contrary to those of Forsyth et al. (2008) and Westfall et al. (2008), who both observed no difference in the expression SOX2, POU5F1 and NANOG between hES cells cultured at 2 and 4% oxygen respectively compared with atmospheric oxygen tensions. However, the latter investigators did show that POU5F1-regulated genes such as left-right determination factor 2 and fibroblast growth factor receptor 2 (FGFR2) were down-regulated under atmospheric oxygen tensions, suggesting that although mRNA expression of pluripotency markers was not reduced under 20% oxygen, their downstream targets display decreased expression.
Taken together, our data suggest that either low oxygen tension is beneficial for the maintenance of pluripotency or that cultures at 5% oxygen contain a greater proportion of undifferentiated cells (or both). In favour of the former, an increase in POU5F1 mRNA and protein has also been observed in mouse ES cells when HIF2A expression was increased using a genetic knock-in strategy (Covello et al. 2006). It is tempting to speculate that HIF2A regulates the long-term response of hES cells to reduced oxygen but this should be interpreted with caution as the little studied HIF3A is also differentially regulated and may also be fundamental to the hypoxic responses.
Our concept is illustrated in Fig. 8, which summarises our data on HIF expression and localisation in hES cells. We suggest that HIF1A and HIF3A are able to regulate each other's expression because when HIF1A is knocked down HIF3A is upregulated and when HIF3A is silenced HIF1A expression increases. Thus, HIF1A may be key in the initial adaptation of hES to the hypoxic environment, but when this expression is lost after 48 h in hypoxic conditions, HIF3A is upregulated, translocates to the nucleus where it is transcriptionally active, and takes over from HIF1A in maintaining the long-term response to hypoxia. In addition, HIF3A appears to be an upstream regulator of HIF2A since when HIF3A expression is lost HIF2A expression significantly decreases. Thus, it appears that HIF3A regulates the expression of the other HIF- isoforms in hES cells. The precise role of HIF3A has previously been unknown but data from the human kidney suggests that it may be a negative regulator of HIF- gene expression (Hara et al. 2001) and therefore might have a role in the downregulation of HIF1A in hES cells exposed to long-term hypoxia. This is supported by data from mouse ES cells where hypoxic expression of HIF1A was shown to suppress LIF–STAT signalling leading to the inhibition of self renewal and the promotion of cell differentiation (Jeong et al. 2007).
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As well as maintaining pluripotency, HIF2A was found to regulate proliferation in hES cells with a significant decrease in both cell number and colony size occurring when HIF2A expression was silenced. This may be due to the reduced NANOG expression observed when HIF2A was silenced since a decrease in NANOG has been found to delay entry into S-phase of the cell cycle, thus decreasing proliferation (Zhang et al. 2009).
Colony morphology was severely affected when two HIF- subunits were silenced simultaneously, with a combination of HIF1A and either HIF2A or HIF3A being non-viable in culture. This may be due to HIF2A being instrumental in controlling pluripotency and HIF3A in regulating the expression of HIF2A. Not surprisingly, therefore, the simultaneous loss of HIF1A, HIF2A and HIF3A results in these cells being unable to survive hypoxia. hES cells in which both HIF2A and HIF3A were silenced formed viable colonies but contained large areas of differentiation. The survival of these cells may be due to compensation by HIF1A, which was switched back on at the protein level. This highlights the importance of HIF1A in maintaining the hypoxic response when the other subunits are not active as observed in the initial adaptation of hES cells to hypoxia.
In conclusion, these studies demonstrate that the culture of hES cells is preferential under a reduced oxygen environment. In contrast to other cell types, HIF1A is only responsible for the initial adaptation of cells to hypoxia, whereas HIF2A regulates the long-term hypoxic response by controlling hES cell pluripotency and proliferation. HIF3A acts to regulate the expression level of both HIF1A and HIF2A. These data provide a greater understanding of the mechanisms which regulate hES cell function and the pluripotent state.
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Materials and Methods |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestReferences |
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-irradiated MEFs in knockout (KO) DMEM (Invitrogen) supplemented with 10% KO serum replacement (Invitrogen), 1 mM L-glutamine, 50 mM β-mercaptoethanol, 1% non-essential amino acids, 10 ng/ml basic FGF factor (Peprotech Ltd, London, UK) and 100 µg/ml penicillin/streptomycin before being transferred on to plates pre-coated with Matrigel (BD Biosciences, San Diego, CA, USA), in medium that had been cultured over-night on -irradiated MEFs. hES cells were initially cultured under atmospheric (20%) oxygen before half were transferred into 5% O2, 5% CO2 and balanced nitrogen. Unless stated, cells were cultured at 5% oxygen for a minimum of three passages prior to use.
Quantitative real-time RT-PCR
mRNA was isolated from hES cells cultured on Matrigel on day 3 post-passage using TriReagent (Sigma) and RNA (2 µg) was reverse transcribed into cDNA using Moloney murine leukaemia virus reverse transcriptase (Promega). The standard curve method of relative quantification real-time PCR was performed using Applied Biosystems reagents in 20 µl reactions containing 4 µg of cDNA, 10 µl 2x Taqman Universal PCR Master Mix, 1 µl Probes and Primer Mix (HIF1A: Hs00936368_m1; HIF1B: Hs00231048_m1; HIF2A: Hs01026142_m1; HIF3A: Hs00541709_m1; POU5F1: Hs01895061_u1; NANOG: Hs02387400_g1; SOX2: Hs00602736_s1; ubiquitin C (UBC): Hs00824723_m1) using a 7500 Real-Time PCR System. The following conditions were used; 50 °C for 2 min, 95 °C for 10 min, 45 cycles at 95 °C for 15 s and 60 °C for 1 min. Placental cDNA (0–10 ng) was used to create a standard curve for each gene of interest as well as for UBC, used as an endogenous control. All target transcripts were analysed in duplicate and normalised to UBC.
Western blotting
hES cells cultured on Matrigel were rinsed with ice-cold PBS and treated with ice-cold radio immunoprecipitation assay lysis buffer (50 mM Tris–HCl (pH 7.4), 1% nonyl phenoxylpolyethoxylethanol, 0.25% Na-deoxycholate and complete protease inhibitors (Roche) supplemented with 1 mM phenylmethylsulphonyl fluoride, 1 mM Na3VO4 and 1 mM NaF) for 30 min with gentle agitation. Protein concentration was quantified using the Bradford assay (Bradford 1976) and lysates (75 µg) were resolved on an 8% SDS bisacrylamide gel. Positive controls were run for each protein of interest; hypoxic colorectal carcinoma cell protein for HIF1A, foetal heart (obtained following ethical approval from the Southampton & South West Hampshire Local Research Ethics Committee, under guidelines issued by the Polkinghorne Committee) for HIF1B and NT2 cells treated with 130 µM cobalt chloride for 4 h for HIF2A and HIF3A. The protein was electro-transferred to a nitrocellulose membrane and blocked in PBS containing 5% non-fat powdered milk and 0.1% Tween-20 for 1 h at room temperature. Primary antibodies were diluted in blocking buffer and incubated with the membrane overnight at 4 °C; HIF1A (BD Biosciences) 1:250, HIF1B (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) 1:250, HIF2A (Novus Biologicals, Cambridge, UK) 1:500, HIF3A (Santa Cruz) 1:1000, POU5F1 (Santa Cruz) 1:1000, SOX2 (Millipore, Billerica, MA, USA) 1:000 and NANOG (Abcam, Cambridge, UK) 1:1000. Membranes were washed and incubated with either peroxidase labelled anti-mouse antibody (Amersham) 1:50 000, or anti-goat antibody (Sigma) 1:200 000, in blocking buffer for 1 h at room temperature. Protein expression was quantified relative to β-actin expression which was detected with mouse anti-β-actin peroxidase-conjugated antibody (1:50 000; Sigma). Membranes were developed using ECL advanced western blotting detection kit (Amersham).
Immunocytochemistry
hES cells cultured on Matrigel were fixed in 4% paraformaldehyde for 20 min. Non-specific antibody binding was blocked with 3% donkey serum and where necessary cells were permeabilised with 0.1% tritonX-100 for 1 h before the addition of primary antibodies diluted in PBS and 3% donkey serum. Primary antibodies used were TRA-1-60 (Santa Cruz) 1:100, TRA-1-81 (gift from P Andrews) 1:50, SSEA1 (Santa Cruz) 1:100, POU5F1 1:100, SOX2 (Chemicon) 1:150, HIF1A 1:250, HIF1B 1:100, HIF2A 1:100, HIF3A 1:50, Ki67 (Novocastra Laboratories, Newcastle upon Tyne, UK) 1:100 and incubated overnight in a humidified chamber at 4 °C. Secondary antibody staining was performed with anti-mouse IgG conjugated-FITC 1:100, anti-mouse IgM conjugated-FITC 1:200 (Sigma) or anti-goat IgG Alexa Fluor 594 1:100 (Sigma) for 1 h in a humidified chamber. Nuclei were labelled with DAPI (Vecta Laboratories, Peterborough, UK).
siRNA
siRNA experiments were carried out on hES cells cultured on Matrigel under 5% oxygen for a minimum of three passages. hES cells cultured under 5% oxygen were passaged and incubated overnight. For each transfection 50 nM siRNA (HIF1A: Hs_HIF1A_5; HIF2A: Hs_EPAS1_5_HP; HIF3A: HsHIF3A_1_HP; Qiagen) along with 12 µl HiPerfect transfection reagent (Qiagen) were mixed into 200 µl of KO-DMEM and added in a dropwise manner to 1-well of a 6-well plate. At 48 h after transfection, cells were harvested and extracts were prepared for mRNA and protein. For HIF3A, siRNA targeting exon 5 was used which is present in all the major known HIF3A isoforms (Maynard et al. 2003). AllStars Negative Control (Transfection control, Qiagen) siRNA that has no homology to any known mammalian gene was used as a negative control for each transfection. For double and triple knockdowns 50 nM siRNA and 12 µl HiPerfect transfection reagent were added in 600 µl of KO-DMEM. Twice or three times the volume of Allstars Negative control was added to controls for double and triple knockdowns respectively. hES cells were analysed for knockdown using relative quantification real-time RT-PCR.
Statistical analysis
All data were analysed to determine whether they were normally distributed, using the Anderson–Darling normality test. Differences between oxygen tension and maximum colony diameter and cell number were analysed using a Student's t-test. Differences in relative gene and protein expression between cells cultured at 5 and 20% oxygen were analysed using a 1-sample t-test. Differences between negative control and knockdown results were analysed using a Student's t-test. All data represent at least three independent experiments. A value of P<0.05 was considered significant. Data are presented as mean±S.E.M.
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Declaration of interest |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestReferences |
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Funding |
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Received July 16, 2009
First decision August 18, 2009
Revised manuscript received September 8, 2009
Accepted September 14, 2009
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References |
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