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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
Correspondence:
Woon-Khiong Chan, Ph.D., 14 Science Drive 4, S117543, Singapore. Fax: 65-6779-2486; e-mail: dbscwk{at}nus.edu.sg; Ariff Bongso, Ph.D., D.Sc., National University Hospital, S119074, Singapore. Fax: 65-6779-4752; e-mail: obongso{at}nus.edu.sg
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ABSTRACT |
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INTRODUCTION |
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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 [14–16]. 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 [17–21]. 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 [5, 13]. 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 [15, 16], 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 [22–24]. Since SAGE libraries comprise discrete data, they can be subjected to pairwise comparison to statistically analyze the differential expression of genes [25] and to generate a comparative digital gene expression profile [24].
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 [26] was conducted to determine differences between the SAGE molecular signatures of ES cells between these two mammalian species.
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MATERIALS AND METHODS |
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SAGE Library Construction, Clone Preparation, and Sequencing
Total RNA was extracted from ~800,000 undifferentiated human ES cells using TRIzol (Invitrogen; Carlsbad, CA; http://www.invitrogen.com) and Poly [A+] RNA subsequently prepared with Oligo(dT)-conjugated magnetic beads (Dynal Biotech; Oslo, Norway; http://www.dynal.no). SAGE library construction was performed with the I-SAGE kit (Invitrogen) according to the manufacturer’s protocol. The anchoring enzyme was NlaIII and the tagging enzyme used was BsmFI. Concatemerized ditags were cloned into pZERO-1TM (Ecogen; Barcelona, Spain; http://www.ecogen.com). The ligated products were transformed into One Shot® Top 10 electrocompetent Escherichia Coli (Invitrogen), and transformants were selected on low-salt LB/zeocin agar plates. Blue-white selection was used to enhance the efficiency of selecting clones with longer concatemerized inserts [29]. Plasmids were prepared with the Wizard SV 96 plasmid purification kit (Promega; Madison, WI; http://www.promega.com). DNA sequencing was performed using ABI Big Dye V3.0 or V3.1 sequencing kit (Applied BioSystems; Foster City, CA; http://www.appliedbiosystems.com) and analyzed on the ABI 3100 capillary DNA sequencer (Applied BioSystems). The Gene Expression Omnibus (GEO) accession numbers for the human HES3 and HES4 SAGE libraries were GSM9220 and GSM9221, respectively.
qRT-PCR
Predesigned Assays on Demand and Assay by Design TaqManTM probes and primer pairs were obtained from Applied BioSystems. Total RNA was extracted from undifferentiated and differentiated human ES cells and reverse transcribed using SuperScript II (Invitrogen). Differentiated human ES cells were obtained by subjecting them to high-density culture conditions for an extended period of 20 days. qRT-PCR analysis was conducted using the ABI PRISM 7000 Sequence Detection System (Applied BioSystems). After an initial denaturation for 10 minutes at 95°C, qRT-PCR was carried out using 40 cycles of PCR (95°C for 15 seconds, 60°C for 1 minute). Changes in gene expression levels were calculated using the 
Ct method after the data (in triplicates) were normalized to the 18S rRNA levels. qRT-PCR experiments were repeated at least once with reproducible results.
RT-PCR
Gene expression was also determined by semiquantitative RT-PCR. Initial denaturation was carried out at 94°C for 2 minutes, followed by 35 cycles of PCR (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute). Primers used were: ACTB: product 400 bp, 5'-TGGCACCACACC TTTCTACAATGAGC-3', 5'-GCACAGCTTCTCCTTAA TGTCACGC-3; BTF3: product 281 bp, 5'-GAACTGCTC GCAGAAAGAAG-3', 5'-ACTAGTCAGACTATCCGC AC-3'; CKS1B: product 409 bp, 5'-ACATGTCATGCTGC CCAAGG-3', 5'-ACACTCAGCTTAGGCTGTGG-3'; CLDN6: product 373 bp, 5'-AGATGCAGTGCAAGGTG TAC-3', 5'-CAAGTGCAGCACAGCAACC-3'; DNMT3B: product 433 bp, 5'-CTCTTACCTTACCATCGACC-3', 5'-CTCCAGAGCATGGTACATGG-3'; ERH: product 495 bp, 5'-GAATGAATCCCAACAGTCCC-3', 5'-TGGAACCAA CATTAAGTGACG-3'; FLJ10713: product 285 bp, 5'-CA GAGAAGTCGAGGGAAGAG-3', 5'-GCTCAGCTTCA ATTGTTGGC-3'; FLJ21837: product 449 bp, 5'-GCAG CTTCTGAACATTTGGAC-3', 5'-GCAGTAGTCTAGAA CACACC-3'; GJA1: product 492 bp, 5'-GGAGTTCAAT CACTTGGCGTG-3, 5'-CTTACCATGCTCTTCAATAC CG-3'; HESX1: product 309 bp, 5'-GGATTTCATTCCCT AGCGTGG-3', 5'-GTGATTCTCTATGGGACCTTTTC-3'; HMGA1: product 469 bp, 5'-GAAGTGCCAACACCTAA GAG-3', 5'-AGTGGGATGTTAGCCTTGTC-3'; LIN-28: product 420 bp, 5'-AGTAAGCTGCACATGGAAGG-3', 5'-ATTGTGGCTCAATTCTGTGC-3'; NANOG: product 493 bp, 5'-GGCAAACAACCCACTTCTGC-3', 5'-TGTT CCAGGCCTGATTGTTC-3'; NPM1: product 343 bp, 5'-TGGTGCAAAGGATGAGTTGC-3', 5'-GTCATCATCTT CATCAGCAGC-3'; POU5F1: product 247 bp, 5'-CGRG AAGCTGGAGAAGGAGAAGCTG-3', 5'-CAAGGGCC GCAGCTTACACATGTTC-3'; REX1: product 418 bp, 5'-TCTAGTAGTGCTCACAGTCC-3', 5'-TCTTTAGGTAT TCCAAGGACT-3'; SOX2: product 370 bp, 5'-CCGCATG TACAACATGATGG-3', 5'-CTTCTTCATGAGCGTCT TGG-3'; and TNFRSF6: product 396 bp, 5'-AGAGTGACA CACAGGTGTTC-3', 5'-TGGCAGAATTGGCCATCATG-3'.
SAGE Data Analysis
Tag extraction and pairwise comparison were performed with the SAGE2000 software v.B (Invitrogen) and database construction and management with Microsoft Access and Excel. Tags with ambiguous bases, duplicate ditags, and ditags with abnormal length (< 22 or > 24 bp) were removed by SAGE2000. The SAGE tag to gene database based on UniGene Build #157 was used. Approximately 60% of all SAGE tags match more than one clustered UniGene entry [22, 30]. To partially overcome the problem of multiple ambiguous tag-to-gene assignments associated with the SAGE technique, we used two publicly available SAGE resources, the CGAP SAGEgenie (http://cgap.nci.nih.gov/SAGE/AnatomicViewer) [24] and the NCBI SAGEmap (http://www.ncbi.nih.gov/SAGE/) [31, 32] to assist in identifying the best SAGE tag for a particular gene. The assignment of molecular function of the proteins was based on the LocusLink database (http://www.ncbi.nih.gov/LocusLink/).
Statistical Treatment
The Z-test [33], based on the normal approximation of the binomial distribution, was used to determine p values for all pairwise library comparisons:
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Since no a priori knowledge about the direction of the effects is available in SAGE experiments, all decision rules were formulated for a 2-sided test of the null [25]. The GEO accession numbers for the human SAGE libraries used were: GSM1498, GSM693, GSM765, GSM670, GSM671, GSM755, GSM731, GSM678, GSM686, GSM757, GSM761, GSM676, GSM728, GSM708, GSM668, GSM709, GSM785, GSM762, GSM719, GSM716, and GSM784. Excel analysis was used to determine the union/intersection of the 21 pairwise statistical tests. Monte Carlo simulation was also carried out to compare the HES3 and HES4 SAGE libraries using the SAGE2000 software.
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RESULTS |
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Few early markers of differentiation were detected in our human ES SAGE libraries. Tags for early ectodermal markers of differentiation like SOX1, NESTIN, and ßIII–TUBULIN; early endodermal markers like PDX1, MIXER, and SOX17b; and mesodermal markers like cardiac ACTIN and ß-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.
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). 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 [33] 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 [35, 36], 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.
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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.
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, 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 [37]. 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.
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. 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.
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). 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 [26].
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). 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.
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). 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 [38, 39]. 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 [40]. 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 |
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REX1 Is not Expressed in HES4
No SAGE tag for REX1 was detected in HES4, and this was confirmed by quantitative and semiquantitative RT-PCR. It is tempting to speculate that this could account for some of the differential gene expression between HES3 and HES4. The lack of REX1 expression in HES4 is surprising because it has been serially propagated for over 100 passages and is capable of forming teratomas in SCID mice. In the mouse, Rex1 is a direct downstream target of Pou5f1 [41] and its promoter is functional in human ES cells [42]. The exact involvement of Rex1 in the self-renewal of mouse ES cells is still unclear. However, F9 cells induced to differentiate along the visceral endoderm pathway showed increased Rex1 mRNA levels and F9 Rex1-/- cells; however, they do not form primitive and visceral endoderm upon retinoic acid-induced differentiation [43]. Taken together, these findings suggest that REX1 expression may not be essential for self-renewal in human ES cells. However, we cannot rule out if REX1 has a role in the establishment of the ICM or in specific differentiation pathways. The confirmation that HES4 carries a null allele of REX1 might have practical implications on its directed differentiation into specific cell types. It would also be prudent to determine REX1 expression in the other human ES cell lines, several of which share a similar ethnic background and source with HES4.
Comparison of the Human and Mouse ES Transcriptomes by SAGE
Some basic similarities in the SAGE profiles of human and mouse ES cells exist. Highly expressed genes in both of these mammalian ES cell types include metabolic enzymes, ribosomal proteins, and cytoskeletal proteins (TUBB2, TMSB10, PFN1). However, there are significant differences between the mouse and human ES cell transcriptomes. Transcription factors with a defined role in the maintenance of pluripotency and whose expression is downregulated upon differentiation, including SOX2, HESX1, UTF1, and REX1 [18, 41, 44, 45], are consistently expressed at higher levels in human ES cells, with POU5F1 expression reaching ~10-fold higher. In contrast, members of the leukemia inhibitory factor (LIF) signaling pathway (STAT3, LIF, LIFR, and IL6ST), FGF4, and TDGF1 are very highly expressed in mouse ES cells only. Galanin and sialoadhesin, which are highly expressed in mouse ES cells [26], are expressed at lower levels in human ES cells (Table 5). Conversely, genes that are differentially expressed in human ES cells are expressed at much lower levels in mouse ES cells. The absolute difference in the expression levels of these ES-restricted transcription factors, coupled with an inactive LIF signaling pathway, indicate there are fundamental differences in the regulatory networks that control pluripotency and self-renewal in human and mouse ES cells.
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Stemness Phenotype of Human ES Cells
A list of candidate human ES cell marker genes responsible for stemness in human ES cells is presented in Table 6. All of these genes were present in our list of 192 upregulated transcripts. Five of them, POU5F1, SOX2, REX1, NANOG, and FLJ10713, have been previously identified in mouse ES cells [10, 15, 20, 21, 26], and eight of them, including TGIF, TDGF1, CHEK2, GDF3, GJA1, and FLJ21837, have been identified as upregulated in a recent microarray study of the human ES cell transcriptome [47]. None of the remaining genes have been previously implicated to be important for human ES cells. These candidate human ES marker genes are either very highly expressed in human ES cells (GJA1, CLDN6, CKS1B, ERH, HMGA1) or show highly restricted expression patterns (LIN28, DNMT3B, FLJ14549, FLJ21837, TNFRSF6, NPM1, OC90). In addition, some of these new marker genes (LIN28, DNMT3B, FLJ14549, OC90, HESX1) were strongly downregulated during ES cell differentiation. Besides these known genes, we have also identified eight orphan SAGE tags that are both highly expressed and restricted to human ES cells. These genes should also prove to be extremely useful as markers for undifferentiated human ES cells.
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Besides the identification of putative transcription factors and signaling pathways that are important for the maintenance of pluripotency and self-renewal in human ES cells, a huge amount of potentially important hypothetical genes, ESTs, and novel transcripts have been uncovered. The presence of many potentially novel transcripts has partially validated our decision to rely on SAGE for the profiling of the human ES cell transcriptome. Despite past failure to identify transcripts that are exclusively restricted to ES cells, some of these orphan SAGE tags are detected only for the first time in human ES cells, indicating that ES-specific genes might exist. The next phase would be to convert these short 10-bp tags to longer cDNA sequences for gene identification purposes and the subsequent evaluation of these genes as key regulators of stemness phenotype. The identification and cloning of the large number of rare human ES cell transcripts will remain a formidable challenge. The enrichment of human ES cells, by cell lineage marking or the erasure of differentiating human ES cells, in combination with single-cell transcript analysis or a micro-cDNA libraries-based approach, may help to quickly refine and identify important human ES-specific genes [48–50]. Single-cell gene expression profiling might be able to confirm if there are functional subsets of human ES cells [51].
While our results have helped to confirm many of the essential attributes of stemness proposed previously [15], we have been unable to demonstrate the involvement of certain key signaling molecules such as FGF-4 and LIF, which are central to the concept of stemness in mouse ES cells. Since these studies [14, 15, 26] have employed LIF to suppress mouse ES cell differentiation, we are inclined to believe that some of these differences might be attributed to an active LIF pathway in mouse ES cells. Nevertheless, these human ES genes that we have identified, in combination with what has been reported earlier for mouse ES cells and other adult stem cells, will remain extremely useful for the dissection of the key molecular pathways involved in the maintenance of pluripotency, self-renewal, and perhaps, even the mechanism used by human ES cells to suppress differentiation.
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ACKNOWLEDGMENT |
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FOOTNOTES |
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 A. Polesskaya, S. Cuvellier, I. Naguibneva, A. Duquet, E. G. Moss, and A. Harel-Bellan Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency Genes & Dev., May 1, 2007; 21(9): 1125 - 1138. [Abstract] [Full Text] [PDF]
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 Z.-X. Wang, C. H.-L. Teh, J. L. L. Kueh, T. Lufkin, P. Robson, and L. W. Stanton Oct4 and Sox2 Directly Regulate Expression of Another Pluripotency Transcription Factor, Zfp206, in Embryonic Stem Cells J. Biol. Chem., April 27, 2007; 282(17): 12822 - 12830. [Abstract] [Full Text] [PDF]
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S. Assou, T. Le Carrour, S. Tondeur, S. Strom, A. Gabelle, S. Marty, L. Nadal, V. Pantesco, T. Reme, J.-P. Hugnot, et al. A Meta-Analysis of Human Embryonic Stem Cells Transcriptome Integrated into a Web-Based Expression Atlas Stem Cells, April 1, 2007; 25(4): 961 - 973. [Abstract] [Full Text] [PDF]
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 C. Allegrucci and L.E. Young Differences between human embryonic stem cell lines Hum. Reprod. Update, March 1, 2007; 13(2): 103 - 120. [Abstract] [Full Text] [PDF]
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W. M. Schopperle and W. C. DeWolf The TRA-1-60 and TRA-1-81 Human Pluripotent Stem Cell Markers Are Expressed on Podocalyxin in Embryonal Carcinoma Stem Cells, March 1, 2007; 25(3): 723 - 730. [Abstract] [Full Text] [PDF]
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S. N. Brimble, E. S. Sherrer, E. W. Uhl, E. Wang, S. Kelly, A. H. Merrill Jr., A. J. Robins, and T. C. Schulz The Cell Surface Glycosphingolipids SSEA-3 and SSEA-4 Are Not Essential for Human ESC Pluripotency Stem Cells, January 1, 2007; 25(1): 54 - 62. [Abstract] [Full Text] [PDF]
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G. Cauffman, I. Liebaers, A. Van Steirteghem, and H. Van de Velde POU5F1 Isoforms Show Different Expression Patterns in Human Embryonic Stem Cells and Preimplantation Embryos Stem Cells, December 1, 2006; 24(12): 2685 - 2691. [Abstract] [Full Text] [PDF]
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K. Hasegawa, T. Fujioka, Y. Nakamura, N. Nakatsuji, and H. Suemori A Method for the Selection of Human Embryonic Stem Cell Sublines with High Replating Efficiency After Single-Cell Dissociation Stem Cells, December 1, 2006; 24(12): 2649 - 2660. [Abstract] [Full Text] [PDF]
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M. Mimeault and S. K. Batra Concise Review: Recent Advances on the Significance of Stem Cells in Tissue Regeneration and Cancer Therapies Stem Cells, November 1, 2006; 24(11): 2319 - 2345. [Abstract] [Full Text] [PDF]
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R. Araki, R. Fukumura, N. Sasaki, Y. Kasama, N. Suzuki, H. Takahashi, Y. Tabata, T. Saito, and M. Abe More Than 40,000 Transcripts, Including Novel and Noncoding Transcripts, in Mouse Embryonic Stem Cells Stem Cells, November 1, 2006; 24(11): 2522 - 2528. [Abstract] [Full Text] [PDF]
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S. Mitalipov, H.-C. Kuo, J. Byrne, L. Clepper, L. Meisner, J. Johnson, R. Zeier, and D. Wolf Isolation and Characterization of Novel Rhesus Monkey Embryonic Stem Cell Lines Stem Cells, October 1, 2006; 24(10): 2177 - 2186. [Abstract] [Full Text] [PDF]
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 S.-y. Yasuda, N. Tsuneyoshi, T. Sumi, K. Hasegawa, T. Tada, N. Nakatsuji, and H. Suemori NANOG maintains self-renewal of primate ES cells in the absence of a feeder layer. Genes Cells, September 1, 2006; 11(9): 1115 - 1123. [Abstract] [Full Text] [PDF]
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S. Tabibzadeh and A. Hemmati-Brivanlou Lefty at the Crossroads of "Stemness" and Differentiative Events Stem Cells, September 1, 2006; 24(9): 1998 - 2006. [Abstract] [Full Text] [PDF]
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J. E. Huettner, A. Lu, Y. Qu, Y. Wu, M. Kim, and J. W. McDonald Gap Junctions and Connexon Hemichannels in Human Embryonic Stem Cells Stem Cells, July 1, 2006; 24(7): 1654 - 1667. [Abstract] [Full Text] [PDF]
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L. Armstrong, O. Hughes, S. Yung, L. Hyslop, R. Stewart, I. Wappler, H. Peters, T. Walter, P. Stojkovic, J. Evans, et al. The role of PI3K/AKT, MAPK/ERK and NF{kappa}{beta} signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis Hum. Mol. Genet., June 1, 2006; 15(11): 1894 - 1913. [Abstract] [Full Text] [PDF]
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M. Richards, S.-P. Tan, W.-K. Chan, and A. Bongso Reverse Serial Analysis of Gene Expression (SAGE) Characterization of Orphan SAGE Tags from Human Embryonic Stem Cells Identifies the Presence of Novel Transcripts and Antisense Transcription of Key Pluripotency Genes Stem Cells, May 1, 2006; 24(5): 1162 - 1173. [Abstract] [Full Text] [PDF]
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 A. Trounson The Production and Directed Differentiation of Human Embryonic Stem Cells Endocr. Rev., April 1, 2006; 27(2): 208 - 219. [Abstract] [Full Text] [PDF]
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 J. D. Raman, N. P. Mongan, L. Liu, S. K. Tickoo, D. M. Nanus, D. S. Scherr, and L. J. Gudas Decreased expression of the human stem cell marker, Rex-1 (zfp-42), in renal cell carcinoma Carcinogenesis, March 1, 2006; 27(3): 499 - 507. [Abstract] [Full Text] [PDF]
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T. Jaatinen, H. Hemmoranta, S. Hautaniemi, J. Niemi, D. Nicorici, J. Laine, O. Yli-Harja, and J. Partanen Global Gene Expression Profile of Human Cord Blood-Derived CD133+ Cells Stem Cells, March 1, 2006; 24(3): 631 - 641. [Abstract] [Full Text] [PDF]
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J. Cai, J. Chen, Y. Liu, T. Miura, Y. Luo, J. F. Loring, W. J. Freed, M. S. Rao, and X. Zeng Assessing Self-Renewal and Differentiation in Human Embryonic Stem Cell Lines Stem Cells, March 1, 2006; 24(3): 516 - 530. [Abstract] [Full Text] [PDF]
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 A. L. Olsen, D. L. Stachura, and M. J. Weiss Designer blood: creating hematopoietic lineages from embryonic stem cells Blood, February 15, 2006; 107(4): 1265 - 1275. [Abstract] [Full Text] [PDF]
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I. Mateizel, N. De Temmerman, U. Ullmann, G. Cauffman, K. Sermon, H. Van de Velde, M. De Rycke, E. Degreef, P. Devroey, I. Liebaers, et al. Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders Hum. Reprod., February 1, 2006; 21(2): 503 - 511. [Abstract] [Full Text] [PDF]
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 L. G. Puente, D. J. Borris, J.-F. Carriere, J. F. Kelly, and L. A. Megeney Identification of Candidate Regulators of Embryonic Stem Cell Differentiation by Comparative Phosphoprotein Affinity Profiling Mol. Cell. Proteomics, January 1, 2006; 5(1): 57 - 67. [Abstract] [Full Text] [PDF]
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T. Enver, S. Soneji, C. Joshi, J. Brown, F. Iborra, T. Orntoft, T. Thykjaer, E. Maltby, K. Smith, R. A. Dawud, et al. Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells Hum. Mol. Genet., November 1, 2005; 14(21): 3129 - 3140. [Abstract] [Full Text] [PDF]
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J. Adjaye, J. Huntriss, R. Herwig, A. BenKahla, T. C. Brink, C. Wierling, C. Hultschig, D. Groth, M.-L. Yaspo, H. M Picton, et al. Primary Differentiation in the Human Blastocyst: Comparative Molecular Portraits of Inner Cell Mass and Trophectoderm Cells Stem Cells, October 1, 2005; 23(10): 1514 - 1525. [Abstract] [Full Text] [PDF]
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K. Noaksson, N. Zoric, X. Zeng, M. S. Rao, J. Hyllner, H. Semb, M. Kubista, and P. Sartipy Monitoring Differentiation of Human Embryonic Stem Cells Using Real-Time PCR Stem Cells, October 1, 2005; 23(10): 1460 - 1467. [Abstract] [Full Text] [PDF]
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H. Skottman, M. Mikkola, K. Lundin, C. Olsson, A.-M. Stromberg, T. Tuuri, T. Otonkoski, O. Hovatta, and R. Lahesmaa Gene Expression Signatures of Seven Individual Human Embryonic Stem Cell Lines Stem Cells, September 1, 2005; 23(9): 1343 - 1356. [Abstract] [Full Text] [PDF]
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C. A. Cowan, J. Atienza, D. A. Melton, and K. Eggan Nuclear Reprogramming of Somatic Cells After Fusion with Human Embryonic Stem Cells Science, August 26, 2005; 309(5739): 1369 - 1373. [Abstract] [Full Text] [PDF]
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 A Vats, N S Tolley, A E Bishop, and J M Polak Embryonic stem cells and tissue engineering: delivering stem cells to the clinic J R Soc Med, August 1, 2005; 98(8): 346 - 350. [Full Text] [PDF]
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G. Keller Embryonic stem cell differentiation: emergence of a new era in biology and medicine Genes & Dev., May 15, 2005; 19(10): 1129 - 1155. [Abstract] [Full Text] [PDF]
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J. Cai, D. Ash, L. E. Kotch, E. W. Jabs, T. Attie-Bitach, J. Auge, G. Mattei, H. Etchevers, M. Vekemans, Y. Korshunova, et al. Gene expression in pharyngeal arch 1 during human embryonic development Hum. Mol. Genet., April 1, 2005; 14(7): 903 - 912. [Abstract] [Full Text] [PDF]
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 A. M. Wobus and K. R. Boheler Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy Physiol Rev, April 1, 2005; 85(2): 635 - 678. [Abstract] [Full Text] [PDF]
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C. L. Wei, T. Miura, P. Robson, S.-K. Lim, X.-Q. Xu, M. Y.-C. Lee, S. Gupta, L. Stanton, Y. Luo, J. Schmitt, et al. Transcriptome Profiling of Human and Murine ESCs Identifies Divergent Paths Required to Maintain the Stem Cell State Stem Cells, February 1, 2005; 23(2): 166 - 185. [Abstract] [Full Text] [PDF]
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T. Dvash, Y. Mayshar, H. Darr, M. McElhaney, D. Barker, O. Yanuka, K. J. Kotkow, L. L. Rubin, N. Benvenisty, and R. Eiges Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies Hum. Reprod., December 1, 2004; 19(12): 2875 - 2883. [Abstract] [Full Text] [PDF]
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R. R. Rao and S. L. Stice Gene Expression Profiling of Embryonic Stem Cells Leads to Greater Understanding of Pluripotency and Early Developmental Events Biol Reprod, December 1, 2004; 71(6): 1772 - 1778. [Abstract] [Full Text] [PDF]
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 M. F. Pera and A. O. Trounson Human embryonic stem cells: prospects for development Development, November 15, 2004; 131(22): 5515 - 5525. [Abstract] [Full Text] [PDF]
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M. Richards, C.-Y. Fong, S. Tan, W.-K. Chan, and A. Bongso An Efficient and Safe Xeno-Free Cryopreservation Method for the Storage of Human Embryonic Stem Cells Stem Cells, September 1, 2004; 22(5): 779 - 789. [Abstract] [Full Text] [PDF]
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T. Sumi, Y. Fujimoto, N. Nakatsuji, and H. Suemori STAT3 Is Dispensable for Maintenance of Self-Renewal in Nonhuman Primate Embryonic Stem Cells Stem Cells, September 1, 2004; 22(5): 861 - 872. [Abstract] [Full Text] [PDF]
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D. S. Kaufman Unique genetics of human embryonic stem cells Blood, April 15, 2004; 103(8): 2872 - 2872. [Full Text] [PDF]
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