Figures
Abstract
Numerous studies have focused on the transcriptional signatures that underlie the maintenance of embryonic stem cell (ESC) pluripotency. However, it remains unclear whether ESC retain transcriptional aberrations seen in in vitro cultured embryos. Here we report the first global transcriptional profile comparison between ESC generated from either in vitro cultured or in vivo derived primate embryos by microarray analysis. Genes involved in pluripotency, oxygen regulation and the cell cycle were downregulated in rhesus ESC generated from in vitro cultured embryos (in vitro ESC). Significantly, several gene differences are similarly downregulated in preimplantation embryos cultured in vitro, which have been associated with long term developmental consequences and disease predisposition. This data indicates that prior to derivation, embryo quality may influence the molecular signature of ESC lines, and may differentially impact the physiology of cells prior to or following differentiation.
Citation: Harvey AJ, Mao S, Lalancette C, Krawetz SA, Brenner CA (2012) Transcriptional Differences between Rhesus Embryonic Stem Cells Generated from In Vitro and In Vivo Derived Embryos. PLoS ONE 7(9): e43239. https://doi.org/10.1371/journal.pone.0043239
Editor: Jennifer Nichols, Wellcome Trust Centre for Stem Cell Research, United Kingdom
Received: April 12, 2012; Accepted: July 18, 2012; Published: September 18, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This study was supported by the National Institutes of Health grants HD045966, RR015395, RR021881 and HD046553. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Embryonic stem cells (ESC) derived from the inner cell mass (ICM) of preimplantation embryos have the potential to differentiate into any cell type of the three embryonic germ layers. ESC retain the ability to proliferate indefinitely, and maintain pluripotency through conserved regulatory networks; however require the provision of various extrinsic factors within the culture environment for continued growth and self-renewal capacity [1], [2]. Loss of pluripotency results in changes in gene expression that include down-regulation of key pluripotency and repressive markers and the up-regulation of regulators of differentiation [3]. Recent studies have documented the transcriptional profiles of various embryonic stem cell lines [4]–[7], establishing a common stem cell regulatory program underlying pluripotency. However, ESC exhibit significant heterogeneity between and within lines, displaying differences in gene expression and differentiation capacity, as well as changes with increasing passage number and culture environment [8]–[11], largely attributed to adaptation with long term culture [12], [13]. Significant differences have also been observed between human ESC lines attributed to differences in derivation techniques [14] and culture conditions [15]–[17]. Very little attention has been paid to other factors which may contribute to the overall normalcy of these cell lines, particularly the quality of the embryo from which a line is derived.
Preimplantation embryo development in vitro is associated with a number of perturbations in ultrastructure [18], [19], gene expression [20]–[25] and post-transfer development [26]–[30], when compared with embryos derived in vivo. These differences likely underlie the significant variation between ESC lines. There is also considerable evidence that the environment to which the preimplantation embryo is exposed, particularly the in vitro culture environment, predisposes the resulting fetus to increased risk of adult onset diseases and imprinting disorders [28], [31]–[36]. Recently, Horii et al [37] reported retention of epigenetic differences in mouse ESC dependent on the in vivo or in vitro origin of the embryo from which they were derived. While ESC transcriptional profiles are known to differ from that of the ICM [38], [39], these data raise the question as to whether ESC retain transcriptional memory of the embryos from which they were derived. Significantly, it is not clear whether current ESC models are similarly predisposed to developing disease characteristics post-transplantation, or whether they exhibit low levels of perturbation that are not easily distinguishable.
To explore the hypothesis that differences exist between ESC derived from in vitro and in vivo embryos, gene expression profiles of rhesus macaque ESC generated from either in vitro cultured (Ormes series [40]) or in vivo derived (R series [41]) embryos were compared.
Results
Expression Profiling of rhesus ESC generated from in vitro or in vivo derived embryos
The transcriptional profiles of undifferentiated ESC generated from either in vivo derived or in vitro produced rhesus embryos were compared using the Affymetrix GeneChip Rhesus Macaque Genome Array, enabling large scale gene expression profiling of 52,865 probe sets, representing over 20,000 genes. Initial data analysis using dChip software identified a total of 2537 transcripts as significantly different between in vitro and in vivo ESC, by a twofold or greater fold change (Table S2). Comparison between groups revealed 592 probe sets upregulated in rhesus ESC of in vitro origin. The reciprocal analysis identified 1945 probe sets upregulated in rhesus ESC of in vivo origin. Of the 2537, 1803 had known Entrez Gene IDs. As dChip is a model-based approach that only allows probe-level analysis, we undertook ChipInspector (Genomatix) analysis to assess differences at the level of each gene. ChipInspector identified a total of 3881 transcripts with differential expression of twofold or greater, of which 2706 were unique to the Genomatix analysis (Table S3), while 1175 transcripts overlapped with the dChip analysis. Of the 3881 transcripts, 560 genes were upregulated and 3321 were downregulated in in vitro ESC.
Further classification of the 3881 differentially expressed transcripts by biological function was undertaken using NetAffx (Affymetrix). Several significant (P<0.05) functional biological categories were represented including apoptosis, cell cycle, development and regulation of transcription (Figure 1A). Of the 3321 downregulated genes and 560 upregulated genes, 797 and 129 were specific to in vitro ESC respectively (Figure 1B). Hierarchical clustering demonstrated that gene expression profiles of in vivo ESC samples clustered together, separately from in vitro ESC samples (Figure 1C), indicating that gene expression differences observed between in vivo and in vitro ESC were greater than differences within the experimental groups.
A: Pie charts representing up- and down-regulated biological functions of 3881 differentially expression genes in ESC. Numbers represent percentages of 560 up- and 3321 down-regulated genes in ESC generated from in vitro cultured embryos, compared with ESC generated from in vivo derived embryos. B: Combination Venn diagram of shared and specific genes expressed in ESC originating from in vitro or in vivo derived embryos. The region of overlap between all areas represents the number of genes expressed in ESC from either origin. Regions not overlapping reflect genes expressed specifically in in vitro or in vivo ESC. There are 11521 genes categorized as present (dChip). Of the 3881 genes identified as significant genes from ChipInspector, 2955 genes are considered as present by dChip, the remaining 926 genes as absent. Of the 2955 genes, 2,524 are down-regulated and 431 are up-regulated; on the 926 absent genes, 797 are down-regulated, 129 are up-regulated. C: Dendrogram representing 3881 significantly different transcripts and hierarchical clustering of biological replicates. Colors indicate relative expression level of each gene in all analyzed samples, with red indicating higher expression and green indicating lower expression.
To identify functional relationships between transcripts, 3881 differentially expressed rhesus transcripts were uploaded into Bibliosphere (Genomatix) for literature based gene connection analysis. Bibliosphere identified 1388 transcripts significantly up- or downregulated in rhesus ESC. Further analysis of the 1388 genes, identified 202 transcription factors (Table 1), and 40 significantly enriched pathways (Table 2), involving a total of 544 genes.
Of the 202 transcription factors identified in Bibliosphere four known to be involved in the transcriptional control of pluripotency, POU5F1, Akt, SMAD2 and HIF1A, were further analyzed to establish literature based gene networks. The interactions of HIF1A and SMAD2 with other genes are presented in Figure 2. Regulatory mechanisms of the transcription factors HIF1A (Matrix family HIFF) and SMAD2 (Matrix family SMAD)'s were further studied as shown in Figure 2. The promoter regions of eleven genes were found to have HIFF binding sites. Likewise, the promoter regions of five genes contained SMAD binding sites.
sentences with expert curated information. Each rectangle depicts a single gene. Red indicates the gene is unregulated, blue downregulated. Arrows between two genes shows regulatory mechanisms: green indicates a transcription factor binding site match in the target promoter; open arrowhead indicates regulation; filled arrowhead indicates activation; blocked arrowhead indicates inhibition; blue dot on the edge indicates that the connection has been annotated by experts; A: Associations present between HIF1A and other genes at the expert level; B: Associations present between SMAD2 and other genes at the expert level. IN: gene is an input gene; TF: gene's product is a transcription factor; ST: gene product is part of signal transduction pathway.
Common framework, a pattern of transcription factor binding sites defined by a set of physical parameters such as order, distance, and strand orientation on the promoter region, is a promoter module that participates in transcription regulation in a certain context. The common frameworks were mined from the eleven genes' and five genes' promoter regions identified above. Frameworks CTCF-HIFF, ETSF-HIFF and SMAD-E2FF were identified in these two gene groups respectively and suggest that transcription factors CTCF and ETSF may work with HIFF, and that E2FF may work with SMAD, to regulate transcription (Table S4).
Expression of markers of pluripotency
Comparison of the 1388 significant differentially expressed genes with previous microarray data examining regulators of pluripotency [4]–[6], [16], [42]–[47] identified 225 significantly different genes documented by at least one publication, with 68 of these genes documented by at least two or more publications (Table 3). Among these genes FGF2 (basic FGF) and FGFR1 were significantly downregulated (2-fold) in in vitro ESC. Similarly, SOX2 expression was decreased more than 3-fold in in vitro ESC, while POU5F1 was reduced by 2-fold. Other genes, including those involved in transcriptional repression and TGFß signaling, were also identified. In particular TGFß1, FST, SMAD1, 4 and 5 and ID4 were downregulated in in vitro ES, while SMAD3 was upregulated (Table S3).
Differentially expressed genes correlate with differences observed in preimplantation embryos
Analysis was undertaken to determine whether ESC generated from in vitro cultured rhesus embryos displayed perturbations in gene expression reported in the literature as differentially expressed in in vitro and in vivo preimplantation embryos [19], [23], [26], [28], [31], [48]–[52], results of which are summarized in Table 4. These differences included significantly decreased expression of insulin-like growth factor receptor 1 and 2 (IGF-I, IGF-II), glucose transporters 3 and 5 (SLC2A3, SLCA2A5), activating transcription factor 1 (ATF1), cyclin D1, secreted phosphoprotein 1, and the antioxidant enzymes superoxide dismutase 1 (SOD1), peroxiredoxin 2 (PDX2) and glutathione peroxidase 4 (GPX4) was seen in in vitro ESC. Alterations in gene expression observed in mouse embryos as a result of the use of serum during embryo culture [52] were also detected, and included downregulation of platelet derived growth factor receptor (PDGFR), the metabolic genes pyruvate dehydrogenase isoenxyme 1, aldehyde dehydrogenase 2 (ADH2) and aldehyde dehydrogenase family 6 subfamily A1, and upregulation of solute carrier family 25 (mitochondrial carrier, citrate transporter) member 1.
Differential expression of oxygen-regulated and metabolic genes
Oxygen-regulated gene expression is known to be important for preimplantation embryo development [21]. The oxygen concentration in which the rhesus preimplantation embryo develops in vivo is reduced [53], [54] compared with in vitro culture. The HIF1A pathway was identified as over-represented in the significantly downregulated gene list by Bibliosphere, the 3881 significant gene list was further interrogated for HIF-regulated genes. Significantly, HIF1A transcript levels were 5.5 fold lower in in vitro ESC (q-value −2.462) than in in vivo ESC. In addition to the 18 genes identified in the HIF1A canonical pathway by Bibliosphere (Table 2), a further 17 genes known to be regulated by oxygen, including SLC2A3 (glucose transporter 3), ALDOA (aldehyde dehydrogenase A) and ENO1 (enolase 1), were identified in the 3881 differentially expressed gene list (Table 5). A comparison of the 3881 output with that of Rinaudo et al 2006 [55], examining the effect of oxygen on preimplantation mouse embryos, resulted in the identification of an additional 23 genes that appear to be regulated by oxygen during early development [55] (Table 6).
In addition to perturbed expression of metabolic genes previously reported in preimplantation embryos, including SLC2A1, SLC2A3, ALD2 and PDK1, regulatory genes controlling mitochondrial biogenesis were also identified as being downregulated in in vitro ESC, including mtSSB, POLG and TFAM, along with genes regulating mitochondrial dynamics (MFN1, KIF5C and OPA1; Table S3).
Confirmation of gene expression by RT-PCR
To confirm the fidelity of our results, we assessed the expression of 13 genes identified in the data analyses. Genes involved in metabolism and mitochondrial function (ATP5B, KIF5C, MFN1, PKM2, SLC2A3, UCP2), pluripotency (FGF2, POU5F1, SOX2, NANOG), transcriptional repression (PCGF2), aging (LMNA) and embryo development (FGF1R, IGF1R, IGFBP2) were examined in pooled ESC RNA from available cultures (Ormes 7 and R466) grown under the same conditions as the samples used for transcriptional profiling. Expression of these genes was confirmed by RT-PCR, with all transcripts detected in both in vitro and in vivo ESC (Figure S1).
Discussion
It is often overlooked that human ESC are generated from in vitro cultured, often surplus/‘discard’, embryos considered unsuitable for transfer in infertility clinics. While the classification of a good quality embryo is based largely on subjective criteria, it is well known that in vitro culture significantly perturbs embryo development, particularly in terms of gene expression, metabolism and subsequent development. With this in mind, we hypothesized that in vitro culture conditions would compromise gene expression in resulting ESC. To achieve this, we examined the transcriptional profiles of four different lines generated from in vivo derived embryos (R series) with that of four lines generated from in vitro derived embryos (Ormes series). Multiple passage numbers were analyzed to minimize passage related cell culture adaptation, with cells maintained under equivalent conditions known to support high quality ESC [56]. The data reported here represent selected passages between 8 and 37 for both in vitro and in vivo ESC. Transcriptional profiling of in vitro ESC and in vivo ESC identified a total of 3881 transcripts with twofold or greater differential expression, of which the majority were downregulated in in vitro ESC. Hierarchical clustering of ESC according to origin, irrespective of passage number, suggests that the differences in gene expression detected are stably maintained during long-term culture. It is important to consider that derivation of the R series (in vivo), and Ormes series (in vitro) carried out by different laboratories may contribute to some of the differences observed in the present study. However, as transcriptional profiles were compared over a range of early passage numbers, with all cell lines maintained under the same conditions by the same laboratory for each passage assessed, this contribution is likely to be minimal.
In vitro ESC and in vivo ESC differ in the expression of imprinted and cell cycle genes, a potential legacy of embryo culture
Aberrant imprinting has been reported in a number of species following preimplantation embryo culture in vitro [57], [58], including the rhesus macaque [59], with long-term consequences for fetal growth and adult health [29], [33]. Bertolini et al [26] and Yaseen et al [60] have reported significantly decreased expression of IGF1R and IGF2R following in vitro culture of bovine embryos, conditions also associated with altered fetal and placental development and large offspring syndrome [27]. The expression of these genes was significantly lower in in vitro ESC when compared with in vivo ESC, suggesting that the altered expression of these genes in cultured embryos is preserved during ESC isolation. In support of this, a number of other genes involved in epigenetic regulation, including histones, histone deactylases and lysine-specific demethylase 3A were identified as differentially expressed between in vitro ESC and in vivo ESC (Table S3). Studies have also reported aberrations in imprinted genes in mouse [61], monkey [62], [63] and human ESC [64]–[67], particularly that of IGF2 and IGF2R. Frost et al [68] reported genomic instability in human ESC, and suggested that derivation and ESC culture contributed to atypical methylation patterns, however it is possible that aberrant imprinting was inherent to the embryo from which the line was derived, in addition to any derivation and culture induced alterations. Significantly, epigenetic differences have been observed between mouse ESC generated from in vitro versus in vivo embryos [37], although these differences were lost by passage 5. Bioinformatic analysis of significantly different transcripts between in vitro and in vivo ESC also highlighted dysregulation of canonical pathways, particularly those regulating cyclins, cell cycle checkpoints and chromosomal stability (Table 2), including genes involved in the G1 to S phase known to be important in ESC [69], [70]. Mtango and Latham [71] have reported altered expression of cell cycle machinery in in vitro cultured rhesus embryos, suggesting that cell cycle control mechanisms may also be heritable from the embryo to resulting ESC. Misregulation of imprinted and cell cycle genes, previously documented following in vitro embryo culture, may therefore be preserved in resulting ESC, and may compromise the cells functionality during and/or following differentiation.
In vitro culture perturbs the expression of key pluripotency regulators
Among the genes identified as significantly altered between ESC of different origin were known pluripotency markers, including POU5F1 (OCT4), basic FGF and SOX2. Basic FGF (FGF2) is an important component of primate ESC culture media required for propagation and colony maintenance. FGFs play several roles in vivo during early development [72] and are known to mediate IGF expression [73], representing a positive feedback loop. Sato et al [6] reported that FGF2 and FGFR1 were important genes enriched in the undifferentiated state, regulated by OCT4, SOX2 and NANOG. Activation of SMAD2/3 signaling is required for human ESC pluripotency [74] as both SMAD2/3 and FGF2 regulate NANOG gene expression. While NANOG is not significantly different between in vivo and in vitro ESC, in vitro ESC displayed significantly increased SMAD2 expression. Upregulation of SMAD2 may support ongoing culture in reduced levels of other pluripotency regulators. A reduction in the expression of OCT4 and SOX2, in addition to a reduction in FGF2 and FGF receptor expression, suggests that in vitro ESC may be more prone to spontaneous differentiation. Indeed, Byrne et al [55] reported significant variability in OCT4 expression across the same Ormes lines examined in the present study. Less than a two-fold difference in the level of OCT4 expression has been shown to have significant effects on ESC maintenance [75]. In support of this, Mtango et al [76] documented changes in pluripotency and differentiation marker expression during the early stages of rhesus macaque blastocyst outgrowth, and in Ormes 6 ESC, when compared with gene expression profiles of rhesus inner cell mass cells. Data therefore suggests that ESC derived from in vitro cultured embryos display alterations in pluripotency markers, however cells have potentially compensated by modulating other pathways to maintain self-renewal.
The effects of oxygen on in vitro cultured embryos are sustained in ESCs
A significant difference between in vivo derived embryos and in vitro cultured embryos is the oxygen environment in which they develop. In vivo the oxygen concentration approximates 2–7% [52], [53], with an oxygen concentration of 2% reported in rhesus macaque uteri, considerably lower than the atmospheric conditions commonly used for in vitro embryo culture, and lower than the 5% oxygen concentration used to generate the embryos from which the in vitro ESC were derived. The oxygen environment is known to alter blastocyst gene expression and embryo development [21], [77]. Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors that mediate cellular adaptation to reduced oxygen conditions. HIF1 protein levels increase exponentially at oxygen concentrations lower than 6% [78]. The response to hypoxia leads to the activation of signaling pathways involved in the regulation of mitochondrial function, glycolytic metabolism and cell survival. In the present study, HIF1 alpha was significantly reduced in in vitro ESC (Table 1). Further analysis demonstrated enrichment (P = 0.0004) of HIF1 alpha regulated genes (Table 5). Physiological oxygen concentrations also regulate human ESC pluripotency, proliferation, karyotypic stability and differentiation [15], [79]–[82], mediated by HIFs [83]. Consistent with our findings, significant differences in OCT4 levels [83], [84] and SOX2 mRNA expression [83] have been reported in human ESC lines derived under 5% and 20% oxygen, or following transfer to reduced oxygen culture conditions. Significantly reduced expression of FGFR1 and FGFR2 [80] and SLC2A3, PKM2, ALDOC, and LGALS1 [17] have also been reported in human ESC in response to atmospheric oxygen conditions, and differences in SLC2A1, SLC2A3 and PGK1 have been reported between in vivo derived and in vitro produced rhesus macaque blastocysts [85]. These results suggest that underlying alterations in metabolism may exist. This is further supported by downregulation of regulatory genes controlling mitochondrial biogenesis and dynamics in in vitro ESC, including mtSSB, POLG and TFAM, as well as MFN1, KIF5C and OPA1 (Table S3). Differences in the expression of genes regulating mitochondrial biogenesis has also been reported between in vivo and in vitro rhesus blastocysts [86]. Significantly, Wale and Gardner [87] demonstrated that developmental perturbations observed following culture of preimplantation mouse embryo under atmospheric conditions were not restored by transferring cultures to a low oxygen environment, suggesting that adaptation of ESC will likewise not resolve underlying differences in ESC physiology. ESC properties may therefore be dependent on reduced oxygen conditions not only during derivation and subsequent expansion, but also during embryo culture prior to derivation.
Conclusions
Results of the present study document significant differences at the transcriptional level between embryonic stem cells derived from in vitro cultured embryos, and those derived from in vivo derived embryos. Data suggests that embryonic stem cells may retain a transcriptional memory representative of the environment of the preimplantation embryo from which the cells were derived. In vitro ESC exhibit transcriptional perturbations seen in in vitro cultured embryos, including alterations in markers of pluripotency and differences impacted by oxygen concentration. These differences may impact cell physiology, although it is unclear whether these differences will contribute to long-term functionality following ESC differentiation and transplantation. Further investigation into the differences between in vitro and in vivo ESCs, particularly in terms of imprinting, metabolism and functionality following differentiation, is warranted to ensure their therapeutic potential. Attention needs to be directed towards physiological measures of functionality, coupled with transcriptional, epigenetic and proteomic characterizations of pluripotency, to assess the impact the culture environment has throughout stem cell isolation, maintenance and differentiation. As methods become more refined and more efficient, and xeno-free isolation becomes routine, the examination of not only embryonic stem cells, but also induced pluripotent stem cells will be pivotal in establishing fundamental properties necessary to supply normal, safe and efficient cells for therapeutic translation.
Materials and Methods
Embryonic Stem Cell culture
Four rhesus (Macaca mulatta) ESC lines generated from in vitro cultured embryos cultured up to day 9 (Ormes 6, 7, 10 and 13, [40]; referred to as ‘in vitro ESC’) and four lines generated from in vivo derived embryos flushed from uteri 6 days post ovulation (R-series 278, 366, 394 and 511, [41]; referred to as ‘in vivo ESC’) were cultured as previously described [56] and were generously provided by Dr Shoukhrat Mitalipov. Briefly, ESC were grown on mitotically inactivated mouse embryonic fibroblast feeder cells (MEF; cell line isolation was approved by the Oregon Health and Sciences University's Institutional Animal Care and Use Committee issued to S. Mitalipov) in Dulbecco's Modified Eagle Medium (DMEM/F12) (Invitrogen, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS) (Hyclone, Logan, UT), 0.1 mM ß-mercaptoethanol, 1% nonessential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), and 4 ng/ml FGF2 (Sigma), at 37°C under a 5% CO2-balance air atmosphere, and were passaged by manual scraping. To account for variability between derivation conditions, cultures were sampled from varying passage numbers (range 8–37) and cultures characterized to ensure that pluripotent ESC morphology, marker expression and karyotype were maintained.
RNA extraction, microarray probe preparation and hybridisation
ESC colonies were collected following manual removal of MEFs and careful dissection to ensure no feeder cell transfer prior to lysis. Total RNA was isolated from cultures for each respective ESC line using TRIZOL reagent (Invitrogen), followed by further purification with a RNeasy MinElute Cleanup Kit (Qiagen). The RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the quality of the RNA was assessed using Lab-on-a-Chip RNA Pico Chips and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Samples with electropherograms showing a size distribution pattern predictive of acceptable microarray assay performance were considered to be of good quality. Twenty nanograms of total RNA from each line was amplified and labeled using a two-cycle cDNA synthesis and an in vitro transcription cRNA-RNA labeling system (GeneChip One-Cycle Target Labeling and Control Reagents; Affymetrix, Inc., Santa Clara, CA). Following successful cRNA amplification, 10 µg of labeled target cRNA was hybridized to Rhesus Macaque Genome Arrays (Affymetrix, Santa Clara, CA) using standard protocols, as described in the Affymetrix GeneChip Expression Analysis manual. Arrays were scanned using the GeneChip laser scanner (Affymetrix).
Bioinformatic analysis
All microarray data complies with MIAME guidelines, and all microarray information and individual cell intensity (CEL) files are available online at the Gene Expression Omnibus (GEO; GSE25198). Analysis of Affymetrix output files was performed with DNA-Chip Analyzer (dChip; Harvard School of Public Health, Boston, MA) and Genomatix (www.genomatix.de) software. In vivo ESC samples were used as the baseline for comparison. For dChip analysis, data normalization and model expression was undertaken using default dChip settings, with analysis of the False Discovery Rate (FDR) also performed. A gene was defined as significantly up- or down-regulated if the signal fold-change between the target samples was greater than 2, at a significance level of alpha = 0.05. For Genomatix data analysis, statistical significance of differential gene expression was assessed by computing a q-value (logarithm) for each gene. Genes were considered to be up- or down-regulated when the logarithm of the gene expression ratio was more than 1 or less than -1, that is, a 2-fold or greater difference in expression, where alpha<0.05. Bibliosphere Pathway Edition (Genomatix), which combines literature analysis with genome annotation and promoter analysis, was used to create a directed regulatory network from transcripts identified by ChipInspector. To establish pathway and common framework information for significantly different transcripts, data was uploaded into GePS (www.genomatix.de). To further classify differentially expressed genes, Entrez gene IDs from the Genomatix analyses were used to search for over-represented biological processes against the rhesus and human genomes. Gene Ontology was performed using NetAffx (www.affymetrix.com).
RT- PCR validation
To validate the microarray results, RT-PCR was carried out on representative rhesus ESC samples (Ormes 7 in vitro and R475 in vivo) for 13 genes identified as significantly altered by the microarray analyses. RNA was extracted using an Absolutely RNA Nanoprep Kit (Stratagene, La Jolla, CA, USA), from which 1 µg was reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen) and random primers (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Resulting cDNA was amplified with 1U Taq polymerase (Qiagen, Valencia, CA) in a final volume of 50 µl containing 1× buffer, 1.5 mM MgCl2, 10 pmol of each sequence-specific primer and 10 mM of each dNTP. The mixture was amplified for 40 cycles in a BioRad DNA Engine thermal cycler (BioRad, Hercules, CA), where each cycle included denaturation at 94°C for 1 min, reannealing for 30 sec at 60°C, and primer extension at 72°C for 30 sec, followed by a final extension at 72°C for 7 min. PCR products were analyzed by electrophoresis through 2% agarose gels containing 0.5 mg/ml ethidium bromide and were photographed using a Kodak GL100 Imaging System equipped with Kodak Molecular Imaging software (Eastman Kodak Co., Rochester, NY). Primers were designed using Primer Express software (Applied Biosystems, Foster City, CA) and are listed in Table S1.
Supporting Information
Figure S1.
RT-PCR analysis of undifferentiated rhesus ESC generated from in vitro (A) or in vivo (B) derived embryos.
https://doi.org/10.1371/journal.pone.0043239.s001
(TIF)
Table S1.
PCR primer sequences used for validation of microarray results.
https://doi.org/10.1371/journal.pone.0043239.s002
(DOCX)
Table S2.
dChip output generated from CEL files (GEO: GSE25198; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25198).
https://doi.org/10.1371/journal.pone.0043239.s003
(XLSX)
Table S3.
Genomatix output generated from CEL files (GEO: GSE25198; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25198).
https://doi.org/10.1371/journal.pone.0043239.s004
(XLSX)
Table S4.
Transcripts identified within common frameworks CTCF-HIFF, ETSF-HIFF and SMAD-E2FF.
https://doi.org/10.1371/journal.pone.0043239.s005
(XLSX)
Acknowledgments
We gratefully acknowledge Dr Shoukhrat Mitalipov and Dr James A. Byrne for the provision and preparation of samples used in this study. The authors also wish to thank Dr Joy Rathjen for valuable discussion regarding the manuscript.
Author Contributions
Conceived and designed the experiments: AJH CAB. Performed the experiments: AJH SM CL. Analyzed the data: AJH SM CL SAK CAB. Contributed reagents/materials/analysis tools: SM CL SAK. Wrote the paper: AJH SM CL SAK CAB.
References
- 1. Beattie GM, Lopez AD, Bucay N, Hinton A, Firpo MT, et al. (2005) Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23: 489–495.
- 2. Xu RH, Peck RM, Li DS, Feng X, Ludwig T, et al. (2005) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2: 185–190.
- 3. Bhattacharya B, Puri S, Puri RK (2009) A review of gene expression profiling of human embryonic stem cell lines and their differentiated progeny. Curr Stem Cell Res Ther 4: 98–106.
- 4. Bhattacharya B, Miura T, Brandenberger R, Mejido J, Luo Y, et al. (2004) Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 103: 2956–2964.
- 5. Player A, Wang Y, Bhattacharya B, Rao M, Puri RK, et al. (2006) Comparisons between transcriptional regulation and RNA expression in human embryonic stem cell lines. Stem Cells Dev 15: 315–323.
- 6. Sato N, Sanjuan IM, Heke M, Uchida M, Naef F, et al. (2003) Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 260: 404–413.
- 7. Skottman H, Mikkola M, Lundin K, Olsson C, Stromberg AM, et al. (2005) Gene expression signatures of seven individual human embryonic stem cell lines. Stem Cells 23: 1343–1356.
- 8. International Stem Cell Initiative Consoritum (2010) Akopian V, Andrews PW, Beil S, Benvenisty N, et al. (2010) Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell Dev Biol Anim 46: 247–258.
- 9. Allegrucci C, Young LE (2007) Differences between human embryonic stem cell lines. Hum Reprod Update 13: 103–120.
- 10. Skottman H, Hovatta O (2006) Culture conditions for human embryonic stem cells. Reproduction 132: 691–698.
- 11. Tavakoli T, Xu X, Derby E, Serebryakova Y, Reid Y, et al. (2009) Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V. BMC Cell Biol 10: 44.
- 12. Enver T, Soneji S, Joshi C, Brown J, Iborra F, et al. (2005) Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells. Hum Mol Genet 14: 3129–3140.
- 13. Grandela C, Wolvetang E (2007) hESC adaptation, selection and stability. Stem Cell Rev 3: 183–191.
- 14. Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, et al. (2007) Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum Reprod 22: 567–577.
- 15. Forsyth NR, Musio A, Vezzoni P, Simpson AH, Noble BS, et al. (2006) Physiologic oxygen enhances human embryonic stem cell clonal recovery and reduces chromosomal abnormalities. Cloning Stem Cells 8: 16–23.
- 16. Skottman H, Stromberg AM, Matilainen E, Inzunza J, Hovatta O, et al. (2006) Unique gene expression signature by human embryonic stem cells cultured under serum free conditions correlates with their enhanced and prolonged growth in an undifferentiated stage. Stem Cells
- 17. Westfall SD, Sachdev S, Das P, Hearne LB, Hannink M, et al. (2008) Identification of oxygen-sensitive transcriptional programs in human embryonic stem cells. Stem Cells Dev 17: 869–881.
- 18. Abe H, Otoi T, Tachikawa S, Yamashita S, Satoh T, et al. (1999) Fine structure of bovine morulae and blastocysts in vivo and in vitro. Anatomy and Embryology (Berlin) 199: 519–527.
- 19. Rizos D, Fair T, Papadopoulos S, Boland MP, Lonergan P (2002) Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro. Mol Reprod Dev 62: 320–327.
- 20. Corcoran D, Fair T, Park S, Rizos D, Patel OV, et al. (2006) Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos. Reproduction 131: 651–660.
- 21. Harvey AJ, Kind KL, Pantaleon M, Armstrong DT, Thompson JG (2004) Oxygen-regulated gene expression in bovine blastocysts. Biol Reprod 71: 1108–1119.
- 22. Nowak-Imialek M, Wrenzycki C, Herrmann D, Lucas-Hahn A, Lagutina I, et al. (2008) Messenger RNA expression patterns of histone-associated genes in bovine preimplantation embryos derived from different origins. Mol Reprod Dev 75: 731–743.
- 23. Wrenzycki C, Herrmann D, Keskintepe L, Martins Jnr A, Sirisathien S, et al. (2001) Effects of culture system and protein supplementation on mRNA expression in pre–implantation bovine embryos. Human Reproduction 16: 893–901.
- 24. Wrenzycki C, Herrmann D, Lucas-Hahn A, Korsawe K, Lemme E, et al. (2005) Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development. Reprod Fertil Dev 17: 23–35.
- 25. Wrenzycki C, Herrmann D, Lucas-Hahn A, Lemme E, Korsawe K, et al. (2004) Gene expression patterns in in vitro-produced and somatic nuclear transfer-derived preimplantation bovine embryos: relationship to the large offspring syndrome? Anim Reprod Sci 82–83: 593–603.
- 26. Bertolini M, Beam SW, Shim H, Bertolini LR, Moyer AL, et al. (2002) Growth, development, and gene expression by in vivo- and in vitro-produced day 7 and 16 bovine embryos. Molecular Reproduction and Development 63: 318–328.
- 27. Bertolini M, Mason JB, Beam SW, Carneiro GF, Sween ML, et al. (2002) Morphology and morphometry of in vivo- and in vitro-produced bovine concepti from early pregnancy to term and association with high birth weights. Theriogenology 58: 973–994.
- 28. Lazzari G, Wrenzycki C, Herrmann D, Duchi R, Kruip T, et al. (2002) Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome. Biology of Reproduction 67: 767–775.
- 29. Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, et al. (2001) Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nature Genetics 27: 153–154.
- 30. Sinclair KD, Young LE, Wilmut I, McEvoy TG (2000) In-utero overgrowth in ruminants following embryo culture: lessons from mice and a warning to men. Hum Reprod 15 Suppl 5: 68–86.
- 31. Fernandez-Gonzalez R, Moreira P, Bilbao A, Jimenez A, Perez-Crespo M, et al. (2004) Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. Proc Natl Acad Sci U S A 101: 5880–5885.
- 32. Lawrence LT, Moley KH (2008) Epigenetics and assisted reproductive technologies: human imprinting syndromes. Semin Reprod Med 26: 143–152.
- 33. Sinclair KD, Singh R (2007) Modelling the developmental origins of health and disease in the early embryo. Theriogenology 67: 43–53.
- 34. Turan N, Katari S, Gerson LF, Chalian R, Foster MW, et al. (2010) Inter- and intra-individual variation in allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology. PLoS Genet 6: e1001033.
- 35. Watkins AJ, Papenbrock T, Fleming TP (2008) The preimplantation embryo: handle with care. Semin Reprod Med 26: 175–185.
- 36. Watkins AJ, Platt D, Papenbrock T, Wilkins A, Eckert JJ, et al. (2007) Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure. Proc Natl Acad Sci U S A 104: 5449–5454.
- 37. Horii T, Yanagisawa E, Kimura M, Morita S, Hatada I (2010) Epigenetic differences between embryonic stem cells generated from blastocysts developed in vitro and in vivo. Cell Reprogram 12: 551–563.
- 38. Brink TC, Sudheer S, Janke D, Jagodzinska J, Jung M, et al. (2008) The origins of human embryonic stem cells: a biological conundrum. Cells Tissues Organs 188: 9–22.
- 39. Reijo Pera RA, DeJonge C, Bossert N, Yao M, Hwa Yang JY, et al. (2009) Gene expression profiles of human inner cell mass cells and embryonic stem cells. Differentiation 78: 18–23.
- 40. Mitalipov S, Kuo HC, Byrne J, Clepper L, Meisner L, et al. (2006) Isolation And Characterization Of Novel Rhesus Monkey Embryonic Stem Cell Lines. Stem Cells 24: 2177–2186.
- 41. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, et al. (1995) Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 92: 7844–7848.
- 42. Cai J, Chen J, Liu Y, Miura T, Luo Y, et al. (2006) Assessing self-renewal and differentiation in hESC lines. Stem Cells 24: 516–530.
- 43. Kim CG, Lee JJ, Jung DY, Jeon J, Heo HS, et al. (2006) Profiling of differentially expressed genes in human stem cells by cDNA microarray. Mol Cells 21: 343–355.
- 44. Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA (2002) “Stemness”: transcriptional profiling of embryonic and adult stem cells. Science 298: 597–600.
- 45. Rao RR, Calhoun JD, Qin X, Rekaya R, Clark JK, et al. (2004) Comparative transcriptional profiling of two human embryonic stem cell lines. Biotechnol Bioeng 88: 273–286.
- 46. Wei CL, Miura T, Robson P, Lim SK, Xu XQ, et al. (2005) Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23: 166–185.
- 47. Zeng X, Miura T, Luo Y, Bhattacharya B, Condie B, et al. (2004) Properties of pluripotent human embryonic stem cells BG01 and BG02. Stem Cells 22: 292–312.
- 48. Gutierrez-Adan A, Rizos D, Fair T, Moreira PN, Pintado B, et al. (2004) Effect of speed of development on mRNA expression pattern in early bovine embryos cultured in vivo or in vitro. Mol Reprod Dev 68: 441–448.
- 49. Li X, Kato Y, Tsunoda Y (2006) Comparative studies on the mRNA expression of development-related genes in an individual mouse blastocyst with different developmental potential. Cloning Stem Cells 8: 214–224.
- 50. Lonergan P, Rizos D, Gutierrez-Adan A, Moreira PM, Pintado B, et al. (2003) Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biology of Reproduction 69: 1424–1431.
- 51. Ho Y, Wigglesworth K, Eppig JJ, Schultz RM (1995) Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol Reprod Dev 41: 232–238.
- 52. Fernandez-Gonzalez R, de Dios Hourcade J, Lopez-Vidriero I, Benguria A, De Fonseca FR, et al. (2009) Analysis of gene transcription alterations at the blastocyst stage related to the long-term consequences of in vitro culture in mice. Reproduction 137: 271–283.
- 53. Fischer B, Kunzel W, Kleinstein J, Gips H (1993) Oxygen tension in follicular fluid falls with follicle maturation. European Journal of Obstetrics & Gynecology and Reproductive Biology 43: 39–43.
- 54. Mastroianni L, Jones R (1965) Oxygen tension within the rabbit fallopian tube. J Reprod Fertil 9: 99–102.
- 55. Rinaudo PF, Giritharan G, Talbi S, Dobson AT, Schultz RM (2006) Effects of oxygen tension on gene expression in preimplantation mouse embryos. Fertil Steril 86: 1252–1265, 1265 e1251–1236.
- 56. Byrne JA, Mitalipov SM, Clepper L, Wolf DP (2006) Transcriptional profiling of rhesus monkey embryonic stem cells. Biol Reprod 75: 908–915.
- 57. Huntriss J, Picton HM (2008) Epigenetic consequences of assisted reproduction and infertility on the human preimplantation embryo. Hum Fertil (Camb) 11: 85–94.
- 58. Pannetier M, Feil R (2007) Epigenetic stability of embryonic stem cells and developmental potential. Trends Biotechnol 25: 556–562.
- 59. Vassena R, Dee Schramm R, Latham KE (2005) Species-dependent expression patterns of DNA methyltransferase genes in mammalian oocytes and preimplantation embryos. Mol Reprod Dev 72: 430–436.
- 60. Yaseen MA, Wrenzycki C, Herrmann D, Carnwath JW, Niemann H (2001) Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF- IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems. Reproduction 122: 601–610.
- 61. Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM 3rd, et al. (2001) Epigenetic instability in ES cells and cloned mice. Science 293: 95–97.
- 62. Mitalipov S, Clepper L, Sritanaudomchai H, Fujimoto A, Wolf D (2007) Methylation status of imprinting centers for H19/IGF2 and SNURF/SNRPN in primate embryonic stem cells. Stem Cells 25: 581–588.
- 63. Fujimoto A, Mitalipov SM, Kuo HC, Wolf DP (2006) Aberrant genomic imprinting in rhesus monkey embryonic stem cells. Stem Cells 24: 595–603.
- 64. Allegrucci C, Wu YZ, Thurston A, Denning CN, Priddle H, et al. (2007) Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome. Hum Mol Genet 16: 1253–1268.
- 65. Kim KP, Thurston A, Mummery C, Ward-van Oostwaard D, Priddle H, et al. (2007) Gene-specific vulnerability to imprinting variability in human embryonic stem cell lines. Genome Res 17: 1731–1742.
- 66. Rugg-Gunn PJ, Ferguson-Smith AC, Pedersen RA (2005) Epigenetic status of human embryonic stem cells. Nat Genet 37: 585–587.
- 67. Rugg-Gunn PJ, Ferguson-Smith AC, Pedersen RA (2007) Status of genomic imprinting in human embryonic stem cells as revealed by a large cohort of independently derived and maintained lines. Hum Mol Genet 16 Spec No. 2: R243–251.
- 68. Frost JM, Monk D, Moschidou D, Guillot PV, Stanier P, et al. (2010) The effects of culture on genomic imprinting profiles in human embryonic and fetal mesenchymal stem cells. Epigenetics 6
- 69. Becker KA, Ghule PN, Therrien JA, Lian JB, Stein JL, et al. (2006) Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol 209: 883–893.
- 70. Fluckiger AC, Marcy G, Marchand M, Negre D, Cosset FL, et al. (2006) Cell cycle features of primate embryonic stem cells. Stem Cells 24: 547–556.
- 71. Mtango NR, Latham KE (2008) Differential expression of cell cycle genes in rhesus monkey oocytes and embryos of different developmental potentials. Biol Reprod 78: 254–266.
- 72. Zhong W, Wang QT, Sun T, Wang F, Liu J, et al. (2006) FGF ligand family mRNA expression profile for mouse preimplantation embryos, early gestation human placenta, and mouse trophoblast stem cells. Mol Reprod Dev 73: 540–550.
- 73. Park SB, Yu KR, Jung JW, Lee SR, Roh KH, et al. (2009) bFGF enhances the IGFs-mediated pluripotent and differentiation potentials in multipotent stem cells. Growth Factors 27: 425–437.
- 74. James D, Levine AJ, Besser D, Hemmati-Brivanlou A (2005) TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132: 1273–1282.
- 75. Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24: 372–376.
- 76. Mtango NR, VandeVoort CA, Latham KE (2011) Ontological aspects of pluripotency and stemness gene expression pattern in the rhesus monkey. Gene Expr Patterns 11: 285–298.
- 77. Kind KL, Collett RA, Harvey AJ, Thompson JG (2005) Oxygen-regulated expression of GLUT-1, GLUT-3, and VEGF in the mouse blastocyst. Mol Reprod Dev 70: 37–44.
- 78. Jiang B-H, Semenza GL, Bauer C, Marti HH (1996) Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. American Journal of Physiology: Cell Physiology 271: C1172–1180.
- 79. Abaci HE, Truitt R, Luong E, Drazer G, Gerecht S (2010) Adaptation to oxygen deprivation in cultures of human pluripotent stem cells, endothelial progenitor cells, and umbilical vein endothelial cells. Am J Physiol Cell Physiol 298: C1527–1537.
- 80. Chen HF, Kuo HC, Lin SP, Chien CL, Chiang MS, et al. (2010) Hypoxic culture maintains self-renewal and enhances embryoid body formation of human embryonic stem cells. Tissue Eng Part A 16: 2901–2913.
- 81. Ezashi T, Das P, Roberts RM (2005) Low O2 tensions and the prevention of differentiation of hES cells. Proc Natl Acad Sci U S A 102: 4783–4788.
- 82. Forsyth NR, Kay A, Hampson K, Downing A, Talbot R, et al. (2008) Transcriptome alterations due to physiological normoxic (2% O2) culture of human embryonic stem cells. Regen Med 3: 817–833.
- 83. Forristal CE, Wright KL, Hanley NA, Oreffo RO, Houghton FD (2010) Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions. Reproduction 139: 85–97.
- 84. Lengner CJ, Gimelbrant AA, Erwin JA, Cheng AW, Guenther MG, et al. (2010) Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 141: 872–883.
- 85. Zheng P, Vassena R, Latham KE (2007) Effects of in vitro oocyte maturation and embryo culture on the expression of glucose transporters, glucose metabolism and insulin signaling genes in rhesus monkey oocytes and preimplantation embryos. Mol Hum Reprod 13: 361–371.
- 86. Mtango NR, Harvey AJ, Latham KE, Brenner CA (2008) Molecular control of mitochondrial function in developing rhesus monkey oocytes and preimplantation-stage embryos. Reprod Fertil Dev 20: 846–859.
- 87. Wale PL, Gardner DK (2010) Time-lapse analysis of mouse embryo development in oxygen gradients. Reprod Biomed Online 21: 402–410.