MPSS profiling of human embryonic stem cells

  • Ralph Brandenberger1,

    Affiliated with

    • Irina Khrebtukova3,

      Affiliated with

      • R Scott Thies2,

        Affiliated with

        • Takumi Miura1,

          Affiliated with

          • Cai Jingli,

            Affiliated with

            • Raj Puri4,

              Affiliated with

              • Tom Vasicek3,

                Affiliated with

                • Jane Lebkowski2 and

                  Affiliated with

                  • Mahendra Rao1, 5Email author

                    Affiliated with

                    Contributed equally
                    BMC Developmental Biology20044:10

                    DOI: 10.1186/1471-213X-4-10

                    Received: 30 March 2004

                    Accepted: 10 August 2004

                    Published: 10 August 2004

                    Abstract

                    Background

                    Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells.

                    Results

                    Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS) of approximately three million signature tags (signatures) identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS.

                    Conclusions

                    Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture conditions

                    Background

                    Multiple large-scale analytical techniques to assess gene expression in defined cell populations have been developed. These include microarray analysis, EST enumeration, SAGE and MPSS. Each of these techniques offers unique advantages and disadvantages. Technique selection largely depends on the expertise of the investigator, the cost, the availability of the techniques, the amount of RNA/DNA that is available, and the existence of the genome databases. The human genome dataset is the best annotated one available [1, 2]- making large scale gene expression analysis of human tissues and cells uniquely fruitful for investigators due to the increased ability to identify full length transcripts with predicted gene function instead of EST's.

                    Human ES cells have been isolated relatively recently and ES cell genes are underrepresented in current databases. More importantly, recent evidence has suggested that mouse ES and human ES cells differ significantly in their fundamental biology [3, 4] and one cannot readily extrapolate from one species to another. However, comparing results between species may provide unique insights. Given the wealth of SAGE and microarray data available from rodent ES cells examining human ES cells with similar techniques as has been done recently by several investigators [311] should be very useful in furthering our understanding of this special stem cell population. Until recently however, it has been difficult to obtain RNA from a homogenous population of undifferentiated hESC for such an analysis as cells could not be grown without feeders and few unambiguous ES cell markers had been described. However, we and others have now described markers that will clearly assess the state of ES cells using a combination of immunocytochemistry and RT-PCR [3, 12, 13] In addition, techniques of harvesting ES cells away from feeder layers have been developed and verified (our unpublished results) and methods of growing ES cells without feeders have been described [14]. These techniques, have allowed us (and others) to obtain large amounts of validated RNA/cDNA samples for comparison by microarray [311], SAGE [8] or EST enumeration [9].

                    We selected MPSS for this analysis as it offers some unique advantages over other methods including SAGE [15, 16]. MPSS offers sufficient depth of coverage when over one million transcripts are sequenced [16] and is efficient, as the numbers of sequences obtained are an order of magnitude larger than with shotgun sequencing or SAGE. It is relatively rapid with a turnaround of a six to ten weeks, and if done with human tissues, more than 80% of transcripts can be mapped to the human genome with current tools. Further, independent analysis has suggested that expression at greater than 3 tpm (transcripts per million) is predictive of detectable, reliable expression, equivalent to roughly one transcript per cell – a sensitivity that is unparalleled when compared to other large-scale analysis techniques [16]. Finally, MPSS libraries can be translated into SAGE libraries and compared to existing SAGE library sets using freely available tools such as digital differential display, allowing ready comparisons to existing SAGE/MPSS libraries of mouse ES cells. It is important to note that we found 14 base pair SAGE tags are generally not as specific as 17 base MPSS signatures and that SAGE sampling depth is usually insufficient. Newer technologies such as extended sequencing to 20 base pairs in MPSS, 24 base pairs in SAGE or cheaper bead alternatives such as those described by Illumina may offer additional depth of coverage and a cheaper price but these at present remain limited in availability.

                    We have utilized MPSS using a pooled sample of three human ES cell lines grown in feeder-free culture conditions over multiple passages [17, 18] to assess the overall state of undifferentiated ES cells. Our rationale for using pooled sample rather than individual samples was based on the fact that no standardized medium and culture conditions have been established for growing and propagating ES cell lines. Variation observed by sampling single lines may be due to culture conditions rather than intrinsic differences. We reasoned therefore that a need existed to establish a reference baseline using pooled samples to enhance the similarities and provide evidence for candidate genes that should be examined for differences such as expression of HLA genes, Y chromosome and X chromosome genes, imprinted genes and genes regulating the methylation state. Our results show that MPSS provides a greater depth of coverage than EST scan or microarray and provides a comprehensive expression profile for this stem cell type. The data set generated allows us and others to identify multiple genes that were not previously known to be expressed in this population, including novel gene as well as obtain a global overview of pathways that are active during the process of self-renewal.

                    Results

                    MPSSS analysis of pooled samples

                    A pooled sample of undifferentiated human ES cell lines H1, H7, and H9 grown in feeder-cell free conditions [19] was used for the preparation of mRNA as previously described [20]. Growth without feeders avoids complication from feeder contamination, which even with good harvesting techniques [14, 21] ranges between 1–3% (unpublished data) and is sufficient to be detected by MPSS (Dr. B. Lim-Harvard University personal communication). Under these conditions, 80–95% of the cells express SSEA-4, 91–94% express TRA-1-60, and 88–93% express TRA-1-81, previously described markers for undifferentiated hESC [19]. Microarray analysis of 2802 genes suggests that these cells are remarkably similar in their gene expression profiles, with only 5 genes being more than 2-fold different between the three cell lines [17, 18] (and data not shown). The undifferentiated state of the cells was also assessed by RT-PCR of known markers of undifferentiated hESC on mRNA of the pooled hESC sample (Figure 1). In addition absence of early markers of differentiation was assessed. No expression of GATA, Sox-1, nestin, Pdx-1 or markers of trophoectoerm were detected in samples used (Supplementary table 3a, see also 3)
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-4-10/MediaObjects/12861_2004_Article_45_Fig1_HTML.jpg
                    Figure 1

                    RT-PCR analysis (a), cumulative tpm (b) and tpm of known ES cell markers (c) is shown. Note that MPSS identifies most known markers of huES cells and expression is at high tpm levels. * – signature maps to >100 location in the genome (class 0); ** – artifactual (class 5) signature

                    Pooled mRNA of the three hESC lines was subjected to MPSS analysis at Lynx Therapeutics (Hayward, CA), generating 22,136 distinct and significant signature sequences from a total of 2,786,765 sequences (see Methods and additional file 1). Each signature was ranked, as outlined in Methods (Table 1), based on its position and orientation within the transcript, and the presence of a polyadenylation signal and polyA in the transcript sequence. 16,675 signatures (75%) mapped to UniGene transcripts; 40 signatures (0.2%) mapped to mitochondrial transcripts; 3,818 signatures (17%) matched genomic sequences but did not map to a UniGene cluster; 927 (4%) signatures matched sequences present at more than 100 genome locations (class 0, representing transcripts containing repetitive elements in their 3' UTR). 676 (3%) signatures did not match to genome or UniGene sequences. Some UniGene clusters contain multiple signatures. These signatures likely represent either transcripts of alternative termination sites, or artefacts of MPSS library construction. Signature classification helps to distinguish artifactual signatures from signatures representing expressed transcripts. For example, signatures of class 1 to 3 are 3'most signatures in mRNA or EST sequences with poly (A) signal and/or polyA tail and most likely represent transcripts with multiple polyadenylation sites. Artifactual signatures constituted 1–3% of the tpm count of the "real" signature, although occasionally close counts were observed (data not shown; see supplementary data tables, additional files 2, 3). To simplify the MPSS data analysis and pair-wise comparison of ES cell data from this study to other datasets, multiple signatures mapping to the same Unigene ID (Hs build 169) were combined into one tpm count as the sum of tpm for signatures of class 1, 2, 3, 22, 23 if any found. These are 3'most signatures close to polyA signal and/or polyA tail, most probably representing true transcripts with alternative termination. If no signatures of above classes were found, then sum of class 4 (3'most, no polyA features) was used. If none the above, the sum of class 5 signatures was used for the tpm calculation per unigene cluster. Resulting table containing data for 8679 unigene clusters, 11 mitochondrial genes, and including 1991 signatures that did not map to unigene but uniquely matched genomic sequences (potential novel transcripts), is presented in supplementary table (additional file 4) and available for download from Lynx [27].
                    Table 1

                    Classification of the MPSS cDNA signatures. The signature classification used for annotation is shown * The Class 0 signatures are the signatures that hit genome more than 100 times, which is treated as a "repeat sequence". ** The polyA tail is defined as a stretch of A's (at least 13 out of 15 bases) that is no more than 50 bases away from the end of the source sequence. The polyA signal is either AATAAA or ATTAAA that has at least one base within the last 50 base before the end of the source sequence or the polyA tail. *** All the virtual signatures extracted from the genomic sequences are classified as class 1000 signatures.

                    Virtual Signature Class

                    MRNA Orientation

                    Poly-Adenelation Features **

                    Position

                    0*

                    Either – Repeat Warning

                    Not applicable

                    Not applicable

                    1

                    Forward Strand

                    Poly-A Signal, Poly-A Tail

                    3' most

                    2

                     

                    Poly-A Signal

                    3' most

                    3

                     

                    Poly-A Tail

                    3' most

                    4

                     

                    None

                    3' most

                    5

                     

                    None

                    Not 3' most

                    6

                     

                    Internal Poly-A

                    Not 3' most

                    11

                    Reverse Strand

                    Poly-A Signal, Poly-A Tail

                    5' most

                    12

                     

                    Poly-A Signal

                    5' most

                    13

                     

                    Poly-A Tail

                    5' most

                    14

                     

                    None

                    5' most

                    15

                     

                    None

                    Not 5' most

                    16

                     

                    Internal Poly-A

                    Not 5' most

                    22

                    Unknown

                    Poly-A Signal

                    Last before signal

                    23

                     

                    Poly-A Tail

                    Last before tail

                    24

                     

                    None

                    Last in sequence

                    25

                     

                    None

                    Not last

                    26

                     

                    Internal Poly-A

                    Not 3' most

                    1000***

                    Unknown – Derived from Genomic Sequence

                    Not applicable

                    Not applicable

                    The frequency distribution of the signatures shows that the 200 most abundant signatures represent 99% of the total number of signature counts obtained from the hESC (Figure 1). Most of top 200 genes (unigene clusters, additional file 5) represent ribosomal genes and genes involved in protein and nucleic acid synthesis and are consistent with results obtained by EST scan and other analyses (data not shown, and [5, 8, 9]). We note that several ribosomal genes were identified as being overexpressed by microarray, SAGE and EST scan as well (see additional files 16, 17, 18). Comparison of the pattern of gene expression with other cell types showed a very similar expression profile with housekeeping genes being the predominant population of sequences in all cell types examined (data not shown). Only three known ES cell specific genes were present in the top 200 genes (additional file 5 and Figure 1). These included SOX-2, DNMT3β, and Oct-4. As in other cells cell type specific genes, transcription factors and cytokines were present at much lower abundance (<50 tpm on average). These low tpm level genes were often not detected by other methods (discussed below). The expression level of cell surface receptors for fibronectin are high (ITGB1 – 578 tpm) and their presence was confirmed by immunocytochemistry and RT-PCR, suggesting that feeder-free clones may grow well on this substrate (data not shown, see also Figure 2 and [14, 21]). The major signaling pathways represented in the top 200 most abundant genes are the FGF signaling pathway, with FGFR1 being most abundant (673 tpm, Figure 2), and the ras activated pathway, with two members of the ras family (NRAS-related and ran) being present in the top 200. This is consistent with data that E-Ras is critical for rodent ES cell self-renewal [22]. No transcripts for HRASP (Homologue of ERAS pseudogene) were detected however (Figure 2), suggesting that these other ras family members may subserve this critical role of self-renewal [9]. The absence of E-Ras was confirmed by RT-PCR (data not shown), as was the presence of FGFR1 (Figure 2, [22], and data not shown).
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-4-10/MediaObjects/12861_2004_Article_45_Fig3_HTML.jpg
                    Figure 3

                    RT-PCR for E-ras/RASP, FGFR1 and novel genes identified as enriched in undifferentiated ES cells is shown in Panel A and B. Localization of E-cadherin and β-catenin in undifferentiated ES cell is shown in Panel C. All of the genes identified by MPSS and tested were present in undifferentiated ES cells and most were significantly downregulated as cells differentiated. Note the high expression at the cell surface and low or undetectable levels of β-catenin in the nucleus.

                    Major pathways present at detectable levels by MPSS

                    To gain a broad overview of the properties of hESC, we mapped the genes found in the hESC cells to the human genome to get an overview of the chromosomal distribution of genes expressed in hESC (Figure 3 and additional files 6, 7, 8, 9, 10, 11). Overall, MPSS detected gene expression in most of the previously identified zones of transcriptional activity within chromosomes. Two chromosomal regions contained more genes expressed in hESC – than expected, and several regions where fewer genes were expressed, compared to the total number of genes located within a particular chromosomal region. No bias to chromosome 17, 12 or X was seen either in overall gene expression or in a particular cytoband. The failure to detect a bias was confirmed by mapping EST scan data [8] as well. The overall distribution patterns were similar and did not show any bias at this level of resolution. Interestingly, gene expression from both X and Y chromosomes was observed. Unlike rodent ES lines both male and female ES lines have been obtained with roughly equal frequency [20] suggesting that when individual cell lines are examined differences between levels of expression between male and female will be present and detectable.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-213X-4-10/MediaObjects/12861_2004_Article_45_Fig2_HTML.jpg
                    Figure 2

                    Cytoband mapping of ES cell expressed genes and regions of relatively high and low transcription relative to the refseq database is shown. More detailed mapping information is presented in supplementary tables.

                    Likewise, MPSS detected expression of several MHC Class I and II genes, suggesting that MPSS can identify differences between ES cell samples when HLA gene expression is used to type cells [17, 18]. We also note that both H19 and Igf2 were expressed at detectable levels. H19 and Igf2 are located adjacent to each other on chromosome 11p15.5 and are reciprocally regulated by imprinting, H19 being paternally imprinted, and IGF2 being maternally imprinted [23, 24]. It is therefore likely that their ratio of expression is likely to differ between cell populations and may represent a simple assessment of the imprinting status of cells.

                    We classified genes expressed into ECM related, homeobox containing, zinc finger proteins, novel genes as well as genes which could assigned to major signaling pathways such as wnt, BMP/TGFβ, LIF, receptors, etc. This data is provided in excel files in the supplementary information provided (additional files 12, 13). Overall certain general themes emerged when genes were classified into such a fashion. We find that: A) hESC express markers characteristic of ES cells in general and few markers characteristic of differentiated cells confirming the initial purity of ES cells used for this analysis and the fidelity of the analysis B) Ribosomal protein transcripts, and mitochondrial genes are highly expressed in ES cells (relative to other transcripts) and constitute more than 50% of the total transcripts analyzed (Figure 1, additional files 5, 16, 17, 18). And this is similar to other samples analyzed [311], (Lynx Inc. data not shown) C) Positive regulators of the cell cycle, TERT and antisenescence related genes and DNA repair pathway regulators are expressed at high levels while proapototic genes, Rb and p53 pathways regulators are expressed at low levels (see table 2 for an example of TERT related gene expression, see supplementary tables (additional files 12, 13) for cell cycle, apoptosis and other pathways) D) The number of novel genes or genes of unknown function is high (2600/11,000) and constitutes approximately 25% of the unique signatures (see additional file 13 for a listing of genes of unknown function, their chromosomal mapping, and UniGene identity). Comparison with other samples suggest that the number of novel genes or genes of unknown function seen are higher in ES cells (25% versus 20%). E) Components of most major signaling pathways are present but so are negative regulators (including zinc finger proteins), suggesting that inhibition plays an important role in maintaining cells in an undifferentiated state (see additional file 13).
                    Table 2

                    Senesence and Aging related genes A subset of genes related to senescence and aging that may regulate the lack of senescense in ES cells is shown. Note that the telomerase, morf's, nortalins and sirtuins are all expressed in ES cells. *The TERT gene has a signature uniquely mapping to an intron (cryptic exon?), which was present in all runs of the ES cell analysis and was not found in other human samples (not shown).

                    HuES_TPM

                    Gene Bank

                    Hs169

                    Gene

                    chr

                    802

                    BC029378

                    Hs.442707

                    TERF1

                    8q13

                    56

                    AF289599

                    Hs.274428

                    TERF2IP

                    16q23.1

                    38

                    AI742882

                    Hs.409194

                    TNKS

                    8p23.1

                    15

                    AF002999

                    Hs.63335

                    TERF2

                    16q22.1

                    10

                    AW271065

                    Hs.9645

                    TNKS1BP1

                    11q12.1

                    9

                    BC005030

                    Hs.7797

                    TINF2

                    14q11.2

                    7

                    AF264912

                    Hs.280776

                    TNKS2

                    10q23.3

                    10*

                    NM_003219

                    Hs.439911

                    TERT

                    5p15.33

                    321

                    NM_004134

                    Hs.184233

                    HSPA9B

                    5q31.1

                    94

                    AF070664

                    Hs.374503

                    MORF4L1

                    15q24

                    80

                    BC017305

                    Hs.528641

                    SIRT7

                    17q25

                    42

                    AF100620

                    Hs.411358

                    MORF4L2

                    Xq22

                    27

                    NM_012238

                    Hs.31176

                    SIRT1

                    10q22.1

                    10

                    BM803485

                    Hs.511950

                    SIRT3

                    11p15.5

                    16

                    AL579291

                    Hs.282331

                    SIRT5

                    6p23

                    Examination of signaling pathways suggest that wnt, TGFβ and FGF signaling pathways are likely important in regulating the ES cell state while LIF/gp130 signaling is not as important. These conclusions are based on examining the expression of the positive and negative regulators of a particular pathway by MPSS, and EST scan. When critical components are low or absent we have tentatively assumed that the pathway is unlikely to be active. An example of the Igf/PTEN pathway is shown to illustrate the logic (Table 3) and other pathways along with verification with EST scan are summarized in the supplementary tables (additional files 12, 13). Note the high levels of soluble frizzled receptors and the expression of E-cadherin (negatively regulating β-catenin translocation). The expression of cadherin and β-catenin was confirmed by immunocytochemistry (Figure 2). The relatively fidelity of the conclusion was confirmed by examining the expression of E-cadherin by immunocytochemistry and localizing β-catenin expression.
                    Table 3

                    IGF-/PTEN/Akt and Ras/Raf/MAP pathway A subset of genes related to Igf/PTEN pathway that are expressed in undifferentiated ES cells is shown. Note that the overall pattern of expression suggest that this pathway is active in undifferentiated ES cells.

                    Tpm ES

                    Tpm EB

                    Unigene ID

                    Locus ID

                    Description

                    14

                    32

                    Hs.239176

                    3480

                    IGF-1 receptor

                    N.D.

                    N.D.

                    Hs.390242

                    3667

                    IRS-1

                    0

                    0

                    Hs.253309

                    5728

                    PTEN

                    N.D.

                    N.D.

                    Hs.32942

                    5294

                    PI3K

                    11

                    8

                    Hs.433611

                    5163

                    PDK1

                    0

                    15

                    Hs.92261

                    5164

                    PDK2

                    N.D.

                    N.D.

                    Hs.6196

                    3611

                    ILK

                    75

                    157

                    Hs.368861

                    207

                    AKT1

                    15

                    82

                    Hs.170133

                    2308

                    FKHR (FoxO1A)

                    78

                    54

                    Hs.14845

                    2309

                    FKHRL1 (FoxO3A)

                    15

                    88

                    Hs.282359

                    2932

                    GSK3beta

                    39

                    14

                    Hs.238990

                    1027

                    p27

                    280

                    240

                    Hs.371468

                    595

                    Cyclin D1

                    0

                    594

                    Hs.370771

                    1026

                    p21

                    1

                    10

                    Hs.329502

                    842

                    Caspase 9

                    0

                    39

                    Hs.76366

                    572

                    Bad

                    98

                    193

                    Hs.260523

                    4893

                    N-Ras

                    2

                    0

                    Hs.37003

                    3265

                    H-Ras

                    35

                    128

                    Hs.257266

                    5894

                    Raf1

                    N.D.

                    N.D.

                    Hs.132311

                    5604

                    MEK1

                    128

                    218

                    Hs.366546

                    5606

                    MEK2

                    37

                    75

                    Hs.324473

                    5594

                    ERK (p42 MAPK)

                    We compared the signature sequences detected in the hESC to an MPSS database of 36 human tissues and cell lines to look for genes that are unique to, or highly overexpressed in hESC. A list of several hundred was generated when a cutoff of 30 tpm or higher (ten fold above detection level) that were elevated in ES cells when compared to neural stem cells examined in a similar manner was used. This list is provided in supplementary materials (additional file 14). A list of 13 highly enriched genes of unknown function is shown in Table 4, and the tpm values for the corresponding signatures in each of 36 tissues or cell lines is provided in the supporting information (additional file 15). The expression in ES cells, of these 13 genes was confirmed by designing PCR primers to different regions and examining gene expression (Figure 2). Several of these genes are highly expressed in hESC and absent in most other tissues tested (Table 4, additional file 15, and data not shown), are downregulated as ES cells differentiate (Figure 2), and are good novel, candidate markers for undifferentiated hESC.
                    Table 4

                    Novel genes enriched in hESC as assessed by MPSS A short list of genes of unknown function that are highly enriched in three ES cell lines comparing to 36 different tissues and cells are shown. A complete list of unknown genes expressed in pooled hESC cells is presented in supplementary tables. * NS-neural stem cells, TH-thymus, HY-hypothalamus, PG-pituitary gland, TE-testis ** this gene (Hs.507833 in the unigene Hs.169) is transcribed in antisense to HDCMA18P (Hs.278635)

                    SIGNATURE

                    HuES,TPM

                    Chr

                    GB:description

                    Other 36, TPM*

                    GATCTCCAGTAGACTTA

                    1646

                    4

                    CD250365:Homo sapiens transcribed sequence **

                    NS-10

                    GATCTGTTAACAAAGGA

                    967

                    16

                    BC008934:claudin 6

                    ND

                    GATCTAGAAGTTGCAAC

                    489

                    1

                    NM_019079:hypothetical protein FLJ10884

                    ND

                    GATCTTTTTTTTTGCCC

                    455

                    3

                    NM_018189:hypothetical protein FLJ10713

                    TH-47, HY-3, PG-3

                    GATCCCCATCCAAAAGA

                    366

                    7

                    AI636928:Homo sapiens transcribed sequences

                    MCF7-2

                    GATCCACCTAGGACCTC

                    244

                    X

                    CD174249:Homo sapiens transcribed sequence

                    ND

                    GATCCGCCTCCTTGGCC

                    240

                    4

                    AK092578:Sapiens cDNA FLJ35259 fis

                    ND

                    GATCCTAGCCAAGCCCC

                    169

                    3

                    BF223023:Homo sapiens transcribed sequences

                    ND

                    GATCTGGCCCGCCACCA

                    150

                    16

                    NM_032805:hypothetical protein FLJ14549 (ZNF206)

                    ND

                    GATCGTTGTGGTGGACT

                    146

                    3

                    XM_067369:similar to Heterochronic gene LIN-41

                    ND

                    GATCCACCACATGGCGA

                    92

                    11

                    CD176172:Homo sapiens transcribed sequence

                    ND

                    GATCCAACAATTCTACT

                    78

                    U

                    CD173198:Homo sapiens transcribed sequences

                    TE-33

                    GATCTTCTAAACCCATC

                    75

                    12

                    BU608353:Homo sapiens transcribed sequence

                    ND

                    Comparing with other data sets

                    Recently we and others have begun examining hESC with EST scan [10] and microarray analysis to develop a characteristic profile of this unique population [310]. We used this data to compare the sensitivity of MPSS with EST scan and microarray analysis. We have previously reported a set of 90 genes reported common to 6 different hESC lines [10]. Of these, eighty-five were detected by MPSS showing a high degree of concordance (>90%). Of the five genes missing from the MPSS hESC data set, four of the genes had valid MPSS signatures (Table 5) and were readily detected in other human samples (data not shown). One gene (SNRPF) lacked a DpnII (GATC) site making it non-detectable by MPSS. GDF3 was detected at non-significant level in the hESC, though was detected by MPSS at higher level (10–30 tpm) in other ES cells tested (Dr. B. Lim-Harvard University personal communication, and additional file 17). Sperger et al., also used microarray to examine gene expression in undifferentiated cell lines [11]. They compared expression in undifferentiated cells with expression in EC carcinoma lines and with microarray data from several other cell lines. They have identified 895 genes (GenBank accession numbers) which reduce to 718 number of unigene identities when mapped to the unigene build Hs161. We have compared this data with the MPSS data and see that MPSS identified the large majority of these genes as well (additional file 16). Similar results were obtained when data was compared with that reported by Sato et al., and Abetya et al., [6, 7] and a similar concordance in gene expression was observed (data not shown). Thus, MPSS provides an independent verification of the microarray results and in addition identifies other genes that may not be present on the arrays or detectable by current microarray techniques.
                    Table 5

                    MPSS tpm of genes reported as enriched by microarray in hESC Table 5 Tpm of genes identified as overexpressed microarray analysis of six pooled human ES cell lines. Note that most of them have high tpm values and are detected by MPSS. * – PSIP2 and PSIP1 have 3' alternate termination and distinguished by MPSS (but not by microarray); ** – PODXL: TPM for signature of class 5; 3' most signature has double palindrome and underrepresented. *** – higher expression of GDF3 was detected in other ES cells (suppl.table for BG02 and not shown). **** – expression detected in other human samples (not shown).

                    GB_accession

                    Gene Symbol

                    HuES_TPM

                    X85372

                    SNRPF

                    No GATC

                    NM_002295

                    LAMR1

                    6135

                    D23660

                    RPL4

                    5269

                    NM_001002

                    RPLP0

                    4656

                    NM_002520

                    NPM1

                    3207

                    X69391

                    RPL6

                    3745

                    M31520

                    RPS24

                    3183

                    AF070600

                    OK/SW-cl.56

                    2702

                    X57958

                    RPL7

                    1923

                    NM_024674

                    LIN-28/

                    1692

                    NM_145899

                    HMGIY

                    1618

                    NM_018407

                    LAPTM4B

                    1326

                    M94314

                    RPL24

                    1279

                    X62534

                    HMGB2

                    989

                    D13748

                    EIF4A1

                    1070

                    NM_006086

                    TUBB4

                    809

                    J04164

                    IFITM1

                    788

                    X69804

                    SSB

                    874

                    M93651

                    SET

                    1323

                    D00760

                    PSMA2

                    673

                    AL162079

                    SLC16A1

                    991

                    AF225425

                    SEMA6A

                    742

                    U28386

                    KPNA2

                    542

                    X74929

                    KRT8

                    543

                    NM_002300

                    LDHB

                    527

                    M97856

                    NASP

                    536

                    AF311912

                    SFRP2

                    457

                    AF020038

                    IDH1

                    450

                    D83174

                    SERPINH1

                    477

                    S74445

                    CRABP1

                    437

                    NM_000165

                    GJA1

                    392

                    AB040903

                    TD-60

                    524

                    AF063020

                    PSIP2*

                    389

                    U76713

                    HNRPAB

                    166

                    NM_000224

                    KRT18

                    302

                    NM_021144

                    PSIP1*

                    389

                    M94856

                    FABP5

                    257

                    NM_016304

                    Ribo 60S L30

                    247

                    AK094423

                    HNPRA1 like

                    214

                    AF055270

                    HSSG1 (SFRS7)

                    201

                    M77140

                    GAL

                    199

                    AF257659

                    CALU

                    100

                    AF098158

                    C20orf1

                    338

                    U41387

                    DDX21

                    179

                    AD001528

                    SMS

                    175

                    NM_006548

                    IMP-2

                    177

                    AJ223953

                    PTTG1

                    154

                    X54326

                    EPRS

                    210

                    D13627

                    CCT8

                    167

                    NM_012247

                    SEPHS1

                    306

                    D00762

                    PSMA3

                    123

                    AF005418

                    CYP26A1

                    121

                    M25753

                    CCNB1

                    168

                    NM_000884

                    IMPDH2

                    174

                    X16396

                    MTHFD2

                    113

                    NM_005159

                    ACTC

                    98

                    U31814

                    HDAC2

                    112

                    J04031

                    MTHFD1

                    104

                    NM_006341

                    MAD2L2

                    95

                    J03746

                    MGST1

                    88

                    NM_020997

                    LEFTB

                    62

                    M74091

                    CCNC

                    86

                    AK001962

                    BRIX

                    66

                    M36981

                    NME2

                    93

                    AL133611

                    Novel

                    63

                    X05360

                    CDC2

                    62

                    AB040930

                    LRRN1

                    46

                    AF071592

                    KIF4A

                    71

                    AF015254

                    STK12

                    41

                    X14253

                    TDGF1

                    37

                    AB023420

                    HSPA4

                    42

                    M19309

                    TNNT1

                    54

                    BC004200

                    PPAT

                    34

                    NM_024090

                    ELOVL6

                    23

                    NM_014366

                    NS

                    30

                    U97519

                    PODXL

                    26**

                    AF048722

                    PITX2

                    25

                    NM_024498

                    ZNF117

                    32

                    NM_001878

                    CRABP2

                    24

                    X59244

                    ZNF43

                    13

                    BC001068

                    C20orf129

                    17

                    NM_024865

                    Nanog

                    15

                    NM_024900

                    Jade-1

                    11

                    AB046793

                    KIAA1573

                    11

                    Z26317

                    DSG2

                    18

                    NM_020634

                    GDF3

                    1***

                    AF070651

                    ZNF257

                    0****

                    NM_016448

                    RAMP

                    0****

                    U88573

                    NBR2

                    0****

                    AB044157

                    GSH1

                    0****

                    Comparison with an EST scan analysis of 37,081 EST sequenced from a similar pooled sample of hESC [9, 10] also showed a high degree of concordance. The EST scan analysis detected 8,801 distinct UniGene clusters in hESC versus 9,996 distinct UniGene clusters expressed at 4 tpm or higher in the MPSS dataset. Of the 8,801 UniGene clusters identified by the EST scan, 1,139 are singletons, i.e. identified by only one EST out of the 37,081 total EST's. 5,286 UniGene clusters have 5 or more ESTs as evidence, and only 118 UniGene clusters have more than 100 EST's as evidence. In contrast, all 9,996 UniGene clusters identified by MPSS were detected at 4 or more tpm and identified in multiple sequencing runs. More than 8,000 have at least 10 tpm, and over 1,000 have more than 100 tpm. Thus, although the EST's are longer in length and thus easier to assign to a particular gene, MPSS appears more sensitive than EST scan. MPSS for example identified almost twice as many genes as EST scan consistent with the difference in the depth of analysis (No of sequences MPSS/EST).

                    Richards et al [8] have used SAGE analysis to two ES cell lines. Their analysis revealed expression of approximately four thousand genes which was significantly fewer than that identified by MPSS consistent with the fewer number of gene tags sequenced. Comparison of the data sets however showed good concordance particularly for genes expressed at higher tpm levels. The entire comparison is presented in supplementary table (additional file 18) and is available for download from Lynx [27]. Overall MPSS could identify genes that other methods identified with an average concordance rate of 70%. The depth of analysis with MPSS at 2.4 million signatures however was significantly greater. MPSS in general identified many more genes than microarray or EST scan or SAGE (see above). The most direct comparison is with EST scan or SAGE, which do not rely on comparative gene expression to establish significance of gene expression. Overall our comparison suggests that MPSS results provide a complementary global overview of the transcriptome of the ES cell. The data supplement and extend the microarray, SAGE and EST scan data sets and provide an independent verification of the same. MPSS in addition identifies additional genes expressed particularly at lower tpm, that are either not present on microarrays or not detected with a lower resolution analysis.

                    Discussion

                    Our results provide a global overview of the gene expression pattern of undifferentiated human ES cells and allow comparisons with other data sets. These results suggest the hESC are an actively dividing population of cells that exhibit high metabolic activity. Our analysis detected expression of approximately 10,600 unique transcripts, a figure that about a third of the total number of mapped genes. Unlike other cell types, however, a much larger fraction of unknown or novel genes was present. This high ratio likely represents the paucity of information available in existing libraries on this relatively newly characterized cell population rather than the possibility that ES cells use radically different pathways for self-renewal, survival, proliferation and differentiation.

                    Our results confirm the reported differences between rodent and human ES cells. We confirm the absence of expression of ERAS, Ehox and the orthologs PEPP1 and 2. The apparent lack of LIF requirement of hESC is reflected by the absence or low tpm levels for genes of the LIF pathway and high tpm for suppressors of LIF mediated signalling (see supporting information). The high level of expression of genes in the FGF pathway likely reflects the requirement of hESC for bFGF. The high level of FGFR1 expression suggests that FGFR1 is an important signal transducer and that FGF's other than FGF4 are important in hESC self-renewal. The high tpm of the fibronectin receptor also suggest that fibronectin or vitronectin are likely useful substitutes for matrigel and that activation of ras mediated signalling is likely critical, as has been described in the rodent ES cell analysis [20].

                    Comparing data from the MPSS analysis with microarray, SAGE and EST scan analyses suggest that MPSS is a powerful alternative to these techniques. MPSS identified virtually all of the genes highlighted as genes common between six different human ES cell lines surveyed by microarray. We noted that most genes detected by microarray were expressed at high tpm indicating that MPSS is more sensitive than microarray analysis. MPSS however appeared to be able to identify genes detected by microarray. Analysing an additional 400 markers detected by MPSS using focused microarray or RT-PCR confirmed their expression [3], (data not shown). Likewise, MPSS analysis showed good concordance with the EST scan data at a fraction of the price. In contrast to the EST scan, tpm levels determined by MPSS are highly correlated to the mRNA levels present in the cells, even at low tpm values [25], and (Lynx unpublished results). Due to the low sampling number of most EST scans, this is not true for relatively low number of EST's found for a particular gene, and can be used only as a rough estimate of gene expression. Unlike other in depth analyses, the absence of markers in MPSS runs is also a powerful control provided that the marker possesses a GATC site. The chromosomal distribution of the genes expressed in hESC did not reveal any bias for a particular chromosome or chromosomal region. While a couple of "hotspots" and several "cold spots" were identified, in no case was any region comprised of all transcribed or all silent genes.

                    Another important conclusion from our analysis is that selection of input RNA is critical. In our case we tested samples repeatedly to assess their purity and made considerable efforts to establish subclones that did not require feeder cells that could be potentially contribute transcripts to the analysis. Given the range of tpm of biologically relevant molecules (5 to 32,000 in this experiment) we predict that even a 5% contamination can confound results or detailed comparisons across different laboratories.

                    We note also that gene transcription from both the X and Y chromosome is observed indicating that at least subtle differences will exist between male and female lines even in the undifferentiated state. Sex-based gene expression, along with MHC gene expression and ratio of expression of imprinted genes could serve to distinguish between different ES cell populations. The present results further suggest that analysing embryoid bodies that differentiate stochastically or analysing tissue samples (with variable proportions of cells) by MPSS will prove more difficult and that results will be variable. We suggest that variability can be reduced by pooling samples, normalizing by careful testing for known markers of differentiation, by semi quantitative PCR, or by focused microarray analysis.

                    While MPSS is cost-effective and sensitive, it is by no means perfect. MPSS is limited by the requirement that DpnII sites (GATC) be present in a gene and be present in a unique locus such that the signature obtained is unique. For example, SNRF expression could not be assessed directly, as no GATC site is present. The signatures for ZFP42 are ambiguous and map to multiple transcripts. Although MPSS can distinguish between alternate transcript termination sites, MPSS cannot distinguish between alternative splicing events and possible incomplete digestion during the sample preparation process. Signature lengths are relatively short and it is possible to have to select between multiple genome hits (reviewed in [16]. Sequencing is performed four bases at a time and transcripts that contain palindromic sequences (in particular double palindromes) are often undetected because of self-hybridization of single DNA strands on the bead. A survey of the genome suggests that this is a rare event (approximately 3% of all virtual signatures in human MGC database have double palindromes). The NODAL gene is an example for such an event, where the class 1 signature was lost and NODAL expression is detected only by a signature resulting from incomplete digestion during library construction (see results). The success of MPSS analyses also depends to a large extent on the quality of genomic information available and, in our opinion, currently is best utilized to analyse human cells. Furthermore, MPSS itself may not be the best method for routine, lower throughput analyses, given price per sample, sample processing time and the large amount of data generated, which requires considerable analysis. However, the database, once developed, is extremely valuable provided it is freely available to make comparisons and to select subsets of genes for further analysis. MPSS information can be effectively utilized by establishing a common database of markers expressed at a defined stage in the differentiation of cells. Additional data sets from sampling of cells at well-controlled stages of differentiation that can be readily accessed and compared to existing datasets will provide the most information while still being cost effective. The genome database is an example of such sharing that has proven to be an invaluable resource for our experiments. Such a strategy requires cooperative pooling of information and free sharing such that individual results can be readily compared against validated datasets. Our future experiments will be directed and developing additional data sets of ES cell differentiation, which can be shared in a manner similar to the present set.

                    Conclusions

                    Our results provide a comprehensive data set that can be effectively utilized to analyse expression patterns of known and unknown genes. Comparison with other data sets provides independent confirmation of results and shows a high level of concordance. The caveats to all such large-scale comparisons are discussed and the importance of pooling data and comparing across multiple data sets is demonstrated.

                    Methods

                    Cell culture

                    The human ES cell lines H1, H7, and H9 were maintained under feeder-free conditions in MEF-conditioned medium supplemented with bFGF as described previously [19, 26].

                    MPSS

                    MPSS was performed using RNA from three pooled ES cell lines (H1, H7, and H9) that had been maintained in feeder free culture conditions and evaluated for the presence of ES cell markers and absence of markers of differentiation. The mRNA was converted to cDNA and digested with DpnII. The last DpnII site and the downstream 16 bases were cloned into Megaclone vectors and their sequences determined according to the MPSS protocol [15, 16, 25]. A total of 2.786.765 sequences were read from four different runs and 48,388 unique signatures were identified. The abundance for each signature was converted to transcripts per million (tpm) for the purpose of comparison between samples. Signatures at an abundance of less than 4 tpm or those that were not detected in at least two runs were removed and a total of 22,136 sequences were analyzed further. All data is available for download from Lynx [27]

                    MPSS signature classification and annotation

                    To generate a complete, annotated human signature database, we extracted all the possible signatures ("virtual signatures") from the human genome sequence, the human Unigene sequences, and human mitochondrion. Each virtual signature was ranked, as outlined in the table 1a, based on its position and orientation in the original sequence. Unigene, genomic, and mitochondrial hits were combined and grouped by signature. The annotation was then assigned to the signature in following order of preference: repeat warnings (signature hits more than 100 genome locations); mitochondrial hits; Unigene hits; genome hits (if no transcript match found). If a signature matched only one Unigene cluster, the MPSS signature class is the lowest class of the member sequences of the cluster. If a signature hits multiple Unigene clusters, the best cluster hit is selected based on the lowest MPSS signature class or the largest number of member sequences. The resulting signature database was used to annotate the data from the experiments Initially the signatures were annotated using genome version hg15 (April 2003, Golden Path, UCSC,) and Unigene build #161 (additional file 2). Recently we re-annotated all signatures using genome version hg16 (July 2003, Golden Path, UCSC) and Unigene build #169 (additional file 3). Both annotations are available for download in supplemental tables [27].

                    Microarray

                    Analysis was performed as described in Bhattacharya et al., [9] using six different samples. These included two lines from Bresagen (01 and 02), the pooled sample from Geron comprising feeder free subclones of (H1, H7, H9), H1, grown in our laboratory on feeders and H9 and I6 from Dr. Itskovitz-Eldor grown following their published protocols.

                    EST-enumeration

                    EST frequency counts of genes expressed in human ES cells were done as described ([8]). Statistical significance was determined using the Fisher Exact Test [28].

                    Chromosomal mapping of MPSS signatures and UniGene clusters to the human genome

                    MPSS signatures with a hit to a UniGene cluster were mapped to the Giemsa staining cytobands of the hg15 release of the human genome (April 10, 2003 freeze, [29]). By this method, 7731 MPSS signatures were mapped to the cytobands of the human genome. Similar mapping was done for all UniGene clusters for which the chromosomal mapping is known. In order to achieve a gene-based rather than a transcript (i.e. splice variant) based distribution of genes splice variants the UniGene clusters were filtered using LocusLink data [30], since LocusLink captures all characterized splice variants of a particular gene. 23,828 UniGene clusters were identified by this method and mapped to the cytobands of the human genome. To discover differences in the number of genes mapped to each cytoband, the number of genes mapped to each cytoband was compared to the total number of genes analyzed, for both the MPSS signatures as well as for the UniGene clusters. The Fisher test [28] was used to determine the statistical significance, using a p-value = 0.05 as cutoff.

                    Gene detection by RT-PCR

                    Total RNA was isolated from cell pellets using RNAeasy Qiagen mini protocol and kit. cDNA was synthesized using 100 ng of total RNA in a 20-μl reaction. Superscript II (Gibco-BRL), a modified Maloney murine leukemia virus RT, and Oligo (dT)12–18 primers were used according to the manufacturer's instructions (Gibco-BRL). The list of primers used for RT-PCR and annealing conditions are described previously [3]).

                    Declarations

                    Acknowledgements

                    This work was supported by the NIA and an ALS center grant. JC, TM, MR were supported by the NIA. RP was supported by the FDA. ST, RB, JL are employees of Geron Inc. IK and TV are employees of Lynx Inc. We thank Drs. Ginis and Limke for careful manuscript reading and all members of our laboratories for constant stimulating discussions. MSR acknowledges the contributions of Dr. S. Rao that made undertaking this project possible.

                    Authors’ Affiliations

                    (1)
                    National Institute on Aging; GRC, Laboratory of Neuroscience
                    (2)
                    Geron Corporation
                    (3)
                    Lynx Therapeutics, Inc.
                    (4)
                    Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration
                    (5)
                    Department of Neuroscience, School of Medicine, Johns Hopkins University

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                    Copyright

                    © Brandenberger et al; licensee BioMed Central Ltd. 2004

                    This article is published under license to BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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