The Transcriptome Profile of Human Embryonic Stem Cells as Defined by SAGE
http://www.100md.com
《干细胞学杂志》
a Department of Obstetrics and Gynecology, National University of Singapore, National University Hospital, Singapore;
b Department of Biological Sciences, National University of Singapore, Singapore
Key Words. SAGE ? Human embryonic stem cells ? Transcriptome ? POU5F1 ? REX1 ? SOX2 ? NANOG
Woon-Khiong Chan, Ph.D., 14 Science Drive 4, S117543, Singapore. Fax: 65-6779-2486; e-mail: dbscwk@nus.edu.sg; Ariff Bongso, Ph.D., D.Sc., National University Hospital, S119074, Singapore. Fax: 65-6779-4752; e-mail: obongso@nus.edu.sg
ABSTRACT
Immortal human embryonic stem (ES) cells and their derivatives promise to revolutionize the future of reparative medicine through the development of stem cell-based therapies . ES cells form teratomas when injected into severe combined immunodeficient (SCID) mice and can differentiate into a variety of cell types from all three primitive germ layers in vitro and in vivo ; this distinguishes ES cells from other stem cells. Several lines of evidence suggest that human and mouse ES cells do not represent equivalent embryonic cell types . In vitro differentiation of human ES cells leads to the expression of AFP and HCG, which are typically produced by trophoblast cells in the developing human embryo, while mouse ES cells are generally believed not to differentiate along this lineage. In addition, human ES cells express stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor rejection antigen (TRA)-1-60, and TRA-1-81 surface antigens prior to differentiation but only SSEA-1 upon differentiation, while mouse ES cells only express SSEA-1 prior to differentiation . Human ES cell lines have heterogeneous genetic backgrounds and appear to behave differently in culture. For example, not all human ES cell lines are amenable to bulk and feeder-free culture protocols, doubling times differ considerably between different lines, and the degree of spontaneous differentiation in vitro also appears to show much variation .
Several groups have used comparative data from microarray studies to propose a blueprint for the molecular basis of "stemness" in human and mouse stem cells . They have also demonstrated that a large proportion of the transcripts expressed in stem cells are expressed sequence tags (ESTs) with indeterminate functions. Recent evidence has suggested that a small, unique network of transcription factors, including Nanog, Oct-4, and Sox-2 may be sufficient to establish self-renewal and/or suppress lineage differentiation in mouse ES cells . Nevertheless, despite the proposed stemness molecular blueprint, many of the genes and molecular mechanisms involved in self-renewal, pluripotency, and differentiation in human ES cells are poorly understood. Moreover, considering the uniqueness of the human ES cell phenotype and the difficulty in obtaining embryonic tissues and preimplantation embryos for research due to ethical reasons, it is probable that many novel genes important for the stemness phenotype in human ES cells remain to be discovered.
We have shown previously that undifferentiated, pluripotent human ES cell lines can be derived from the inner cell masses (ICMs) of 5-day-old human embryos . Since human ES cells lines are capable of differentiating into all three germ layers despite the reported differences in their behavior in vitro, we hypothesized that a quantitative comparison of the transcriptome profiles of selected human ES cells lines might allow the determination of key regulators involved in the maintenance of the stemness property, as previously defined , as well as help identify a basis for line-specific cellular and behavioral differences.
Serial analysis of gene expression (SAGE) allows quantitative characterization and has the added value over microarray expression profiling in its ability to identify novel splice variants, exons, and genes . Since SAGE libraries comprise discrete data, they can be subjected to pairwise comparison to statistically analyze the differential expression of genes and to generate a comparative digital gene expression profile .
We have used SAGE to obtain the transcriptome profiles of two human ES cell lines, HES3 and HES4, which have different gender and ethnic backgrounds. SAGE should identify genes that comprise a distinct molecular signature of human ES cells. To delineate genes that were differentially regulated in human ES cells, the human ES SAGE libraries were subjected to pairwise comparisons with 21 normal and cancer SAGE libraries. Finally, comparison with the mouse ES SAGE library was conducted to determine differences between the SAGE molecular signatures of ES cells between these two mammalian species.
MATERIALS AND METHODS
HES3 and HES4 SAGE Libraries
Transcriptional profiling of mRNA isolated from undifferentiated human ES cells was performed using SAGE. Undifferentiated human ES colonies were carefully selected and individual SAGE libraries were constructed. A combined total of 145,015 SAGE tags were sequenced from HES3 (67,807) and HES4 (77,208) SAGE libraries. This translated into 31,852 distinct transcripts. Approximately 64.2% (20,447) of these distinct transcripts were found only once in the combined human ES SAGE library (HES3: 9,977; HES4: 10,470). This is probably indicative of the abundance of rare transcripts in human ES cells, although it is possible that some singletons might have resulted from sequencing errors or leaky transcription as a result of epigenetic dysregulation . A vast majority of singletons that could be reliably assigned to UniGene clusters matched ESTs or hypothetical genes (46.2%), although we have also noted the existence of distinct transcripts that matched to genes like FOXD3 and GBX2, which have been previously identified to be important to mouse ES cells or are expressed in the ICM of mouse blastocysts. We omitted these singletons from our analysis to provide a more accurate estimation of distinct transcripts.
Few early markers of differentiation were detected in our human ES SAGE libraries. Tags for early ectodermal markers of differentiation like SOX1, NESTIN, and ?III–TUBULIN; early endodermal markers like PDX1, MIXER, and SOX17b; and mesodermal markers like cardiac ACTIN and ?-GLOBIN were not detected in both SAGE libraries, indicating that contamination of our starting material with differentiated cells was indeed very low.
The Overall Transcriptome Profiles of HES3 and HES4 Are Similar
The exclusion of singletons from the combined human ES SAGE dataset left us with 11,404 distinct transcripts. Among these transcripts, 1.0% had more than 135 copies, 11.2% had between 14–135 copies, 14.1% had between 7–13 copies, and 73.7% had fewer than 6 copies. Altogether, 1,511 distinct transcripts (13.2%) could not be reliably assigned (orphan transcripts) to UniGene clusters. The remaining 9,893 distinct transcripts were matched to 12,721 UniGene clusters. Of these, 4,475 (37.6%) matched only ESTs or hypothetical genes, while 313 (2.5%) have unknown functions (Fig. 1A). A putative functional breakdown of the genes expressed in HES3 and HES4 revealed that a preponderance of the genes are involved in DNA repair, stress responses, apoptosis, cell cycle regulation, and development (Fig. 1A). Based on the presence of numerous distinct transcripts that could not be reliably assigned to UniGene clusters and the prevalence of hypothetical proteins and ESTs, we conclude that a large proportion of the mRNA species in human ES cells is likely to be novel and expressed only in ES cells or cells derived from the ICM.
Figure 1. SAGE analysis of undifferentiated human ES cells. A) A pie chart depicting the percentage of distinct genes encoding proteins in various functional categories in the combined human ES SAGE libraries. B) The distribution of distinct transcripts (SAGE tags) and genes expressed in human ES cells. Numbers in parentheses represent the number of genes that can be assigned to the distinct transcripts. Figures above the values in parentheses represent the total number of distinct transcripts while those below represent transcripts with no reliable UniGene assignment. C) A scatter plot showing the comparative distribution of distinct transcripts of the two human ES SAGE libraries. Pairwise comparison was performed using the compare function of the SAGE2000 software. Tag frequencies were plotted on a logarithmic scale and p values calculated using the Z-test.
Of the 9,917 and 9,828 distinct transcripts that were identified in the HES3 and HES4 SAGE libraries, respectively, 8,341 were common to both (Fig. 1B). Moreover, most of the 3,063 transcripts that were not detected in both human ES cell lines are rare transcripts (<3 copies). With a more detailed SAGE profiling, it is likely that the majority of these transcripts would be detected in both human ES cell lines. More importantly, the vast majority of the highly expressed transcripts were expressed in both HES3 and HES4. A pairwise comparison of all the distinct transcripts found in HES3 and HES4 was also performed and the results are presented as a scatterplot (Fig. 1C). Statistical analysis using the Z-test indicated that the expression levels of the majority of the distinct transcripts in HES3 and HES4 were highly correlated.
Highly Expressed Genes in Human ES Cells
The most abundant transcripts in the combined human ES SAGE libraries include many housekeeping genes important for key metabolic processes such as glycolysis, the ETS pathway, and protein synthesis, or genes that encode for cytoskeletal related proteins, transporters, and RNA processing. Furthermore, with few exceptions, human ES cells expressed these genes to a much higher extent than any other cell types. Notably, POU5F1, a POU transcription factor, and SOX2, which is important for the pluripotency of ES cells , were the two most highly expressed transcription factors. Additional transcription factors that were highly expressed include HMGA1, ERH, and BTF3. As a result of a single nucleotide polymorphism (SNP) within the SAGE tag sequence, BTF3 has two matched tags, CTGAGACGAA and CTGAGACAAA. Table 1 lists the 30 most abundantly expressed genes in human ES cells. Emphasizing the view that the transcriptome of human ES cells is not well characterized, 6 of the top 200 most abundant transcripts had no UniGene match. Another unusual aspect of human ES cells is the high abundance of two tight junction proteins, CLDN6 and GJA1. Several cytoskeletal and actin-binding proteins like profilin, cofilin, thymosin, and a vasa-type RNA helicase, DDX5, were also very highly expressed.
Table 1. The 30 most abundant transcripts expressed in human ES cells
Overall, one of the most striking observations is the high expression level of genes that are involved in protein synthesis and mRNA processing. In particular, genes that encode for the 73 ribosomal proteins were, on average, about 3.70–8.41 times more abundant than in normal tissues like brain cortex, cerebellum, colon, kidney, stomach, and liver. Only the pancreas has a higher proportion of SAGE tags that were derived from ribosomal genes. This is indicative that the human ES cells must devote a large proportion of their cellular resources to the synthesis of proteins, which is certainly not unexpected given the rapid cellular proliferation rate of human ES cells.
Genes Differentially Expressed in HES3 and HES4
Although the general transcriptome profiles of the two human ES cell lines we profiled were similar, a number of genes were found to be differentially represented. A pairwise comparison of the HES3 and HES4 SAGE libraries (Fig. 1C) using the Z-test statistical analysis (p 0.01) and fold differences revealed 175 differentially expressed transcripts. Monte Carlo simulation gave identical results (data not shown). A list of 25 differentially expressed HES3 and HES4 genes with the greatest fold difference is presented in Table 2. Most conspicuously, the transcript for REX1 was absent in the HES4 line. SNPs and splice variants/isoforms account for some of the differences in the HES3 and HES4 SAGE transcriptomes. For example, six differentially expressed genes were found to have two assigned SAGE tags. RPS27A, NDUFB1, and BTF3 were represented by two different SAGE tags containing an SNP within each tag sequence, while the second alternative tag for TPI1, FSCN1, and SLC2A3 resulted from the expression of a second isoform in HES3. Several transcription factors, REX1, BTF3, ZFX and XBP1, were upregulated in HES3, but only CTBP1 was upregulated in HES4.
Table 2. The top 25 differentially expressed transcripts in HES3 or HES4 cells showing the greatest fold difference
In contrast to HES3, the upregulated genes in HES4 included mainly ribosomal proteins, cytoskeletal proteins, and enzymes involved in metabolic pathways, which probably reflect the higher metabolic and proliferation rates of HES4. Three genes, LECT1, TGF, and IFRD1, which are associated with differentiation, were upregulated in HES3, perhaps indicative of a small subpopulation of differentiating cells. Some of the cell line-specific differential gene expression could be attributed to different gender backgrounds. For instance, the Y-linked RPS4 was found only in HES4, while all five X-linked genes were more highly expressed in HES3. About 8.7% of the differentially expressed transcripts were ESTs or hypothetical proteins and 9.1% were orphan SAGE tags.
Genes Differentially Upregulated in Human ES Cells
To determine genes that were upregulated in ES cells, we compared the combined human ES SAGE dataset with 21 publicly available SAGE libraries from normal adult and fetal peripheral tissues and cancer tissues. Upregulated transcripts were identified based on p values (p < 0.01) and fold differences (fold difference > 4) in 21 pairwise comparisons. The 192 upregulated transcripts included known ES-specific transcription factors like POU5F1, SOX2, REX1, and NANOG as well as other less well-characterized transcription factors, hypothetical proteins, and several DNA/RNA-modifying proteins like LIN28 and DNMT3B, an embryonic DNA methyltransferase . A large number of orphan SAGE tags, hypothetical genes, and ESTs were found to be abundantly expressed and highly restricted in their expression to human ES cells. A selected list of differentially upregulated transcripts is presented in Table 3.
Table 3. Differentially upregulated genes in human ES cells
Pairwise statistical comparisons also revealed that the medulloblastoma (886), embryonic kidney (941), and ovarian carcinoma (1,720) have the least number of differentially expressed transcripts and thus most closely resemble human ES cells. The scatterplots depicting the distribution of distinct transcripts in these three tissues and adult kidney with respect to human ES cells are shown in Figure 2. Many of the upregulated genes in the combined human ES data set were also highly represented in cancer SAGE libraries, therefore, although human ES cells do not closely resemble cancer cells in their generalized transcriptome profiles, they do appear to share certain characteristics.
Figure 2. Scatter plots showing the comparative distribution of distinct transcripts in four selected tissues. The combined human ES SAGE library was compared with (A) embryonic kidney, (B) adult kidney, (C) medulloblastoma, and (D) ovarian carcinoma. Tag frequencies were plotted on a logarithmic scale and p values calculated using the Z-test.
Independent Confirmation of SAGE Expression Data by qRT-PCR
To confirm the SAGE tag frequency results, we performed qRT-PCR on total RNA derived from undifferentiated (7D) and high-density (20D) differentiated human ES cells. Genes studied were POU5F1, SOX2, REX1 HESX1, DNMT3B, ERH, STAT3, LIF, LIFR, IL6ST, AFP, BMP4, NEUROD1, and FGF4 (Table 4). While ES cell markers like HESX1, POU5F1, REX1, SOX2, and STAT3 showed a decline, there was a strong increase in the expression of AFP and NEUROD1, but not BMP4, in the differentiated human ES cells. HESX1 expression showed the greatest decline during ES cell differentiation. Interestingly, there was also a significant decrease in DNMT3B expression during human ES cell differentiation. FGF4 could not be detected in undifferentiated or differentiated human ES cells with qRT-PCR or SAGE. For LIF and LIFR, although SAGE tags were not detected, qRT-PCR indicated that both were expressed at low levels, with LIFR expression showing an increase during HES3 and HES4 differentiation. Expression data for HES3 and HES4 matched very well; overall correlation between qRT-PCR and SAGE analyses was 0.67, which is similar to that reported for mouse ES cells .
Table 4. Real-time RT-PCR gene expression between undifferentiated and differentiated human ES cells
Expression of Candidate Human ES Cell-Specific Genes
We examined the expression profiles of 18 known and candidate ES-specific genes identified by our SAGE analysis by semiquantitative RT-PCR. The expression of these genes was determined in undifferentiated and differentiated human ES cells: six adult peripheral tissues and two fetal tissues (Fig. 3). Of the known ES transcription factors, POU5F1, SOX2, and REX1 were expressed only in human ES cells, while low levels of NANOG expression were detected in fetal brain and adult testis. Several new candidate human ES-specific genes such as DNMT3B, an embryonic DNA methyltransferase; LIN28, an RNA-binding protein; NPM1, a nucleolar protein; OC90, a PLA2-like protein; and FLJ14549, a germ cell Zn-finger transcription factor, were expressed only in human ES cells and showed decreased expression during ES cell differentiation.
Figure 3. Gene expression of candidate human ES-specific marker genes. Transcriptional analysis of the 19 genes and ACTB, which is included as loading control, were carried out by RT-PCR with total RNA prepared from fetal brain, fetal liver, adult brain, placenta, adult testis, adult kidney, adult lung, adult heart, undifferentiated (7D) HES3 and HES4 cells, and differentiated (20D) HES3 and HES4. Input RNA amounts were controlled for all first-strand RT reactions. Ten percent of the PCR product was loaded into each lane and analyzed on a 1.5% agarose gel.
The expression of DNMT3B was further evaluated with qRT-PCR to confirm a decline during ES cell differentiation (Table 4). De novo methylation of genomic DNA is a developmentally regulated process that is believed to play a pivotal role in development, genome imprinting, and gene silencing in mammals . LIN28, an RNA-binding and heterochronic gene, was downregulated during ES differentiation. LIN28 is a negative regulator controlling the embryonic development of a variety of somatic cell types in many organisms . Downregulation of LIN28 expression has also been associated with a progress to differentiation in embryonal carcinoma cells. Other genes, such as CLDN6, GJA1, CKS1B, ERH, and HMGA1, were expressed in some peripheral tissues, but the expression levels appeared to be much higher in human ES cells. However, no marked decline in the expression of these genes was detected during the onset of ES differentiation. Of the five transcription factors assayed by qRT-PCR (Table 4), HESX1 gene expression showed the most dramatic decline during ES differentiation. However, HESX1 was also expressed in several peripheral adult and fetal tissues.
DISCUSSION
This study was supported by a grant from Embryonic Stem Cell International (ESI) Pte. Ltd.
FOOTNOTES
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Received July 27, 2003; accepted for publication September 15, 2003.(Mark Richardsa, Siew-Peng)
b Department of Biological Sciences, National University of Singapore, Singapore
Key Words. SAGE ? Human embryonic stem cells ? Transcriptome ? POU5F1 ? REX1 ? SOX2 ? NANOG
Woon-Khiong Chan, Ph.D., 14 Science Drive 4, S117543, Singapore. Fax: 65-6779-2486; e-mail: dbscwk@nus.edu.sg; Ariff Bongso, Ph.D., D.Sc., National University Hospital, S119074, Singapore. Fax: 65-6779-4752; e-mail: obongso@nus.edu.sg
ABSTRACT
Immortal human embryonic stem (ES) cells and their derivatives promise to revolutionize the future of reparative medicine through the development of stem cell-based therapies . ES cells form teratomas when injected into severe combined immunodeficient (SCID) mice and can differentiate into a variety of cell types from all three primitive germ layers in vitro and in vivo ; this distinguishes ES cells from other stem cells. Several lines of evidence suggest that human and mouse ES cells do not represent equivalent embryonic cell types . In vitro differentiation of human ES cells leads to the expression of AFP and HCG, which are typically produced by trophoblast cells in the developing human embryo, while mouse ES cells are generally believed not to differentiate along this lineage. In addition, human ES cells express stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor rejection antigen (TRA)-1-60, and TRA-1-81 surface antigens prior to differentiation but only SSEA-1 upon differentiation, while mouse ES cells only express SSEA-1 prior to differentiation . Human ES cell lines have heterogeneous genetic backgrounds and appear to behave differently in culture. For example, not all human ES cell lines are amenable to bulk and feeder-free culture protocols, doubling times differ considerably between different lines, and the degree of spontaneous differentiation in vitro also appears to show much variation .
Several groups have used comparative data from microarray studies to propose a blueprint for the molecular basis of "stemness" in human and mouse stem cells . They have also demonstrated that a large proportion of the transcripts expressed in stem cells are expressed sequence tags (ESTs) with indeterminate functions. Recent evidence has suggested that a small, unique network of transcription factors, including Nanog, Oct-4, and Sox-2 may be sufficient to establish self-renewal and/or suppress lineage differentiation in mouse ES cells . Nevertheless, despite the proposed stemness molecular blueprint, many of the genes and molecular mechanisms involved in self-renewal, pluripotency, and differentiation in human ES cells are poorly understood. Moreover, considering the uniqueness of the human ES cell phenotype and the difficulty in obtaining embryonic tissues and preimplantation embryos for research due to ethical reasons, it is probable that many novel genes important for the stemness phenotype in human ES cells remain to be discovered.
We have shown previously that undifferentiated, pluripotent human ES cell lines can be derived from the inner cell masses (ICMs) of 5-day-old human embryos . Since human ES cells lines are capable of differentiating into all three germ layers despite the reported differences in their behavior in vitro, we hypothesized that a quantitative comparison of the transcriptome profiles of selected human ES cells lines might allow the determination of key regulators involved in the maintenance of the stemness property, as previously defined , as well as help identify a basis for line-specific cellular and behavioral differences.
Serial analysis of gene expression (SAGE) allows quantitative characterization and has the added value over microarray expression profiling in its ability to identify novel splice variants, exons, and genes . Since SAGE libraries comprise discrete data, they can be subjected to pairwise comparison to statistically analyze the differential expression of genes and to generate a comparative digital gene expression profile .
We have used SAGE to obtain the transcriptome profiles of two human ES cell lines, HES3 and HES4, which have different gender and ethnic backgrounds. SAGE should identify genes that comprise a distinct molecular signature of human ES cells. To delineate genes that were differentially regulated in human ES cells, the human ES SAGE libraries were subjected to pairwise comparisons with 21 normal and cancer SAGE libraries. Finally, comparison with the mouse ES SAGE library was conducted to determine differences between the SAGE molecular signatures of ES cells between these two mammalian species.
MATERIALS AND METHODS
HES3 and HES4 SAGE Libraries
Transcriptional profiling of mRNA isolated from undifferentiated human ES cells was performed using SAGE. Undifferentiated human ES colonies were carefully selected and individual SAGE libraries were constructed. A combined total of 145,015 SAGE tags were sequenced from HES3 (67,807) and HES4 (77,208) SAGE libraries. This translated into 31,852 distinct transcripts. Approximately 64.2% (20,447) of these distinct transcripts were found only once in the combined human ES SAGE library (HES3: 9,977; HES4: 10,470). This is probably indicative of the abundance of rare transcripts in human ES cells, although it is possible that some singletons might have resulted from sequencing errors or leaky transcription as a result of epigenetic dysregulation . A vast majority of singletons that could be reliably assigned to UniGene clusters matched ESTs or hypothetical genes (46.2%), although we have also noted the existence of distinct transcripts that matched to genes like FOXD3 and GBX2, which have been previously identified to be important to mouse ES cells or are expressed in the ICM of mouse blastocysts. We omitted these singletons from our analysis to provide a more accurate estimation of distinct transcripts.
Few early markers of differentiation were detected in our human ES SAGE libraries. Tags for early ectodermal markers of differentiation like SOX1, NESTIN, and ?III–TUBULIN; early endodermal markers like PDX1, MIXER, and SOX17b; and mesodermal markers like cardiac ACTIN and ?-GLOBIN were not detected in both SAGE libraries, indicating that contamination of our starting material with differentiated cells was indeed very low.
The Overall Transcriptome Profiles of HES3 and HES4 Are Similar
The exclusion of singletons from the combined human ES SAGE dataset left us with 11,404 distinct transcripts. Among these transcripts, 1.0% had more than 135 copies, 11.2% had between 14–135 copies, 14.1% had between 7–13 copies, and 73.7% had fewer than 6 copies. Altogether, 1,511 distinct transcripts (13.2%) could not be reliably assigned (orphan transcripts) to UniGene clusters. The remaining 9,893 distinct transcripts were matched to 12,721 UniGene clusters. Of these, 4,475 (37.6%) matched only ESTs or hypothetical genes, while 313 (2.5%) have unknown functions (Fig. 1A). A putative functional breakdown of the genes expressed in HES3 and HES4 revealed that a preponderance of the genes are involved in DNA repair, stress responses, apoptosis, cell cycle regulation, and development (Fig. 1A). Based on the presence of numerous distinct transcripts that could not be reliably assigned to UniGene clusters and the prevalence of hypothetical proteins and ESTs, we conclude that a large proportion of the mRNA species in human ES cells is likely to be novel and expressed only in ES cells or cells derived from the ICM.
Figure 1. SAGE analysis of undifferentiated human ES cells. A) A pie chart depicting the percentage of distinct genes encoding proteins in various functional categories in the combined human ES SAGE libraries. B) The distribution of distinct transcripts (SAGE tags) and genes expressed in human ES cells. Numbers in parentheses represent the number of genes that can be assigned to the distinct transcripts. Figures above the values in parentheses represent the total number of distinct transcripts while those below represent transcripts with no reliable UniGene assignment. C) A scatter plot showing the comparative distribution of distinct transcripts of the two human ES SAGE libraries. Pairwise comparison was performed using the compare function of the SAGE2000 software. Tag frequencies were plotted on a logarithmic scale and p values calculated using the Z-test.
Of the 9,917 and 9,828 distinct transcripts that were identified in the HES3 and HES4 SAGE libraries, respectively, 8,341 were common to both (Fig. 1B). Moreover, most of the 3,063 transcripts that were not detected in both human ES cell lines are rare transcripts (<3 copies). With a more detailed SAGE profiling, it is likely that the majority of these transcripts would be detected in both human ES cell lines. More importantly, the vast majority of the highly expressed transcripts were expressed in both HES3 and HES4. A pairwise comparison of all the distinct transcripts found in HES3 and HES4 was also performed and the results are presented as a scatterplot (Fig. 1C). Statistical analysis using the Z-test indicated that the expression levels of the majority of the distinct transcripts in HES3 and HES4 were highly correlated.
Highly Expressed Genes in Human ES Cells
The most abundant transcripts in the combined human ES SAGE libraries include many housekeeping genes important for key metabolic processes such as glycolysis, the ETS pathway, and protein synthesis, or genes that encode for cytoskeletal related proteins, transporters, and RNA processing. Furthermore, with few exceptions, human ES cells expressed these genes to a much higher extent than any other cell types. Notably, POU5F1, a POU transcription factor, and SOX2, which is important for the pluripotency of ES cells , were the two most highly expressed transcription factors. Additional transcription factors that were highly expressed include HMGA1, ERH, and BTF3. As a result of a single nucleotide polymorphism (SNP) within the SAGE tag sequence, BTF3 has two matched tags, CTGAGACGAA and CTGAGACAAA. Table 1 lists the 30 most abundantly expressed genes in human ES cells. Emphasizing the view that the transcriptome of human ES cells is not well characterized, 6 of the top 200 most abundant transcripts had no UniGene match. Another unusual aspect of human ES cells is the high abundance of two tight junction proteins, CLDN6 and GJA1. Several cytoskeletal and actin-binding proteins like profilin, cofilin, thymosin, and a vasa-type RNA helicase, DDX5, were also very highly expressed.
Table 1. The 30 most abundant transcripts expressed in human ES cells
Overall, one of the most striking observations is the high expression level of genes that are involved in protein synthesis and mRNA processing. In particular, genes that encode for the 73 ribosomal proteins were, on average, about 3.70–8.41 times more abundant than in normal tissues like brain cortex, cerebellum, colon, kidney, stomach, and liver. Only the pancreas has a higher proportion of SAGE tags that were derived from ribosomal genes. This is indicative that the human ES cells must devote a large proportion of their cellular resources to the synthesis of proteins, which is certainly not unexpected given the rapid cellular proliferation rate of human ES cells.
Genes Differentially Expressed in HES3 and HES4
Although the general transcriptome profiles of the two human ES cell lines we profiled were similar, a number of genes were found to be differentially represented. A pairwise comparison of the HES3 and HES4 SAGE libraries (Fig. 1C) using the Z-test statistical analysis (p 0.01) and fold differences revealed 175 differentially expressed transcripts. Monte Carlo simulation gave identical results (data not shown). A list of 25 differentially expressed HES3 and HES4 genes with the greatest fold difference is presented in Table 2. Most conspicuously, the transcript for REX1 was absent in the HES4 line. SNPs and splice variants/isoforms account for some of the differences in the HES3 and HES4 SAGE transcriptomes. For example, six differentially expressed genes were found to have two assigned SAGE tags. RPS27A, NDUFB1, and BTF3 were represented by two different SAGE tags containing an SNP within each tag sequence, while the second alternative tag for TPI1, FSCN1, and SLC2A3 resulted from the expression of a second isoform in HES3. Several transcription factors, REX1, BTF3, ZFX and XBP1, were upregulated in HES3, but only CTBP1 was upregulated in HES4.
Table 2. The top 25 differentially expressed transcripts in HES3 or HES4 cells showing the greatest fold difference
In contrast to HES3, the upregulated genes in HES4 included mainly ribosomal proteins, cytoskeletal proteins, and enzymes involved in metabolic pathways, which probably reflect the higher metabolic and proliferation rates of HES4. Three genes, LECT1, TGF, and IFRD1, which are associated with differentiation, were upregulated in HES3, perhaps indicative of a small subpopulation of differentiating cells. Some of the cell line-specific differential gene expression could be attributed to different gender backgrounds. For instance, the Y-linked RPS4 was found only in HES4, while all five X-linked genes were more highly expressed in HES3. About 8.7% of the differentially expressed transcripts were ESTs or hypothetical proteins and 9.1% were orphan SAGE tags.
Genes Differentially Upregulated in Human ES Cells
To determine genes that were upregulated in ES cells, we compared the combined human ES SAGE dataset with 21 publicly available SAGE libraries from normal adult and fetal peripheral tissues and cancer tissues. Upregulated transcripts were identified based on p values (p < 0.01) and fold differences (fold difference > 4) in 21 pairwise comparisons. The 192 upregulated transcripts included known ES-specific transcription factors like POU5F1, SOX2, REX1, and NANOG as well as other less well-characterized transcription factors, hypothetical proteins, and several DNA/RNA-modifying proteins like LIN28 and DNMT3B, an embryonic DNA methyltransferase . A large number of orphan SAGE tags, hypothetical genes, and ESTs were found to be abundantly expressed and highly restricted in their expression to human ES cells. A selected list of differentially upregulated transcripts is presented in Table 3.
Table 3. Differentially upregulated genes in human ES cells
Pairwise statistical comparisons also revealed that the medulloblastoma (886), embryonic kidney (941), and ovarian carcinoma (1,720) have the least number of differentially expressed transcripts and thus most closely resemble human ES cells. The scatterplots depicting the distribution of distinct transcripts in these three tissues and adult kidney with respect to human ES cells are shown in Figure 2. Many of the upregulated genes in the combined human ES data set were also highly represented in cancer SAGE libraries, therefore, although human ES cells do not closely resemble cancer cells in their generalized transcriptome profiles, they do appear to share certain characteristics.
Figure 2. Scatter plots showing the comparative distribution of distinct transcripts in four selected tissues. The combined human ES SAGE library was compared with (A) embryonic kidney, (B) adult kidney, (C) medulloblastoma, and (D) ovarian carcinoma. Tag frequencies were plotted on a logarithmic scale and p values calculated using the Z-test.
Independent Confirmation of SAGE Expression Data by qRT-PCR
To confirm the SAGE tag frequency results, we performed qRT-PCR on total RNA derived from undifferentiated (7D) and high-density (20D) differentiated human ES cells. Genes studied were POU5F1, SOX2, REX1 HESX1, DNMT3B, ERH, STAT3, LIF, LIFR, IL6ST, AFP, BMP4, NEUROD1, and FGF4 (Table 4). While ES cell markers like HESX1, POU5F1, REX1, SOX2, and STAT3 showed a decline, there was a strong increase in the expression of AFP and NEUROD1, but not BMP4, in the differentiated human ES cells. HESX1 expression showed the greatest decline during ES cell differentiation. Interestingly, there was also a significant decrease in DNMT3B expression during human ES cell differentiation. FGF4 could not be detected in undifferentiated or differentiated human ES cells with qRT-PCR or SAGE. For LIF and LIFR, although SAGE tags were not detected, qRT-PCR indicated that both were expressed at low levels, with LIFR expression showing an increase during HES3 and HES4 differentiation. Expression data for HES3 and HES4 matched very well; overall correlation between qRT-PCR and SAGE analyses was 0.67, which is similar to that reported for mouse ES cells .
Table 4. Real-time RT-PCR gene expression between undifferentiated and differentiated human ES cells
Expression of Candidate Human ES Cell-Specific Genes
We examined the expression profiles of 18 known and candidate ES-specific genes identified by our SAGE analysis by semiquantitative RT-PCR. The expression of these genes was determined in undifferentiated and differentiated human ES cells: six adult peripheral tissues and two fetal tissues (Fig. 3). Of the known ES transcription factors, POU5F1, SOX2, and REX1 were expressed only in human ES cells, while low levels of NANOG expression were detected in fetal brain and adult testis. Several new candidate human ES-specific genes such as DNMT3B, an embryonic DNA methyltransferase; LIN28, an RNA-binding protein; NPM1, a nucleolar protein; OC90, a PLA2-like protein; and FLJ14549, a germ cell Zn-finger transcription factor, were expressed only in human ES cells and showed decreased expression during ES cell differentiation.
Figure 3. Gene expression of candidate human ES-specific marker genes. Transcriptional analysis of the 19 genes and ACTB, which is included as loading control, were carried out by RT-PCR with total RNA prepared from fetal brain, fetal liver, adult brain, placenta, adult testis, adult kidney, adult lung, adult heart, undifferentiated (7D) HES3 and HES4 cells, and differentiated (20D) HES3 and HES4. Input RNA amounts were controlled for all first-strand RT reactions. Ten percent of the PCR product was loaded into each lane and analyzed on a 1.5% agarose gel.
The expression of DNMT3B was further evaluated with qRT-PCR to confirm a decline during ES cell differentiation (Table 4). De novo methylation of genomic DNA is a developmentally regulated process that is believed to play a pivotal role in development, genome imprinting, and gene silencing in mammals . LIN28, an RNA-binding and heterochronic gene, was downregulated during ES differentiation. LIN28 is a negative regulator controlling the embryonic development of a variety of somatic cell types in many organisms . Downregulation of LIN28 expression has also been associated with a progress to differentiation in embryonal carcinoma cells. Other genes, such as CLDN6, GJA1, CKS1B, ERH, and HMGA1, were expressed in some peripheral tissues, but the expression levels appeared to be much higher in human ES cells. However, no marked decline in the expression of these genes was detected during the onset of ES differentiation. Of the five transcription factors assayed by qRT-PCR (Table 4), HESX1 gene expression showed the most dramatic decline during ES differentiation. However, HESX1 was also expressed in several peripheral adult and fetal tissues.
DISCUSSION
This study was supported by a grant from Embryonic Stem Cell International (ESI) Pte. Ltd.
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Received July 27, 2003; accepted for publication September 15, 2003.(Mark Richardsa, Siew-Peng)