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Genomic and proteomic characterization of embryonic stem cells
Genomic and proteomic characterization of embryonic stem cells Lawrence W Stanton and Manjiri M Bakre Stem cell biology, like all areas of cell biology, has been significantly affected by the arrival of the genomics era. The rendering of the human and mouse genome sequences and the development of attendant technologies have made it possible to comprehensively explore embryonic stem cell biology at the molecular level. Recently, there has been emphasis on global characterization of the transcriptome, epigenome, and proteome of embryonic stem cells. These omic evaluations of embryonic stem cells are leading to improved methods for cell-based therapies and are advancing our basic understanding of early embryonic development. Addresses Stem Cell and Developmental Biology Program, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore Corresponding author: Stanton, Lawrence W ([email protected])
Current Opinion in Chemical Biology 2007, 11:399–404 This review comes from a themed issue on Chemical Biology and Stem Cells Edited by David Schaeffer Available online 23rd July 2007 1367-5931/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2007.05.029
Introduction The landscape of stem cell biology was altered substantially in 1998 with the first successful derivation of embryonic stem—human embryonic stem cell (hESC) lines from human blastocytsts [1]. Work done in early 1980s with mouse ESC (mESC) established that they have the defining characteristics of unlimited self-renewal and a capacity to give rise to all cell types of the developing and adult organism [2,3]. The most useful aspect of ESC derived from the mouse was that they provided a direct and efficient means to generate a genetically modified mammal [2]. The arrival of human-derived ESC lines has been heralded with great fanfare for other reasons [3,4]. One reason for the excitement is that we now have the opportunity to study the earliest stages of human embryology. However, it is the potential of hESC for use in regenerative medicine that receives the greatest attention from scientists, clinicians, patients, and society. To realize the clinical potential of hESC it is necessary to better understand the molecular pathways that regulate differentiation and harness that knowledge into efficient and reliable methods for directed differentiation of therapeutically useful cells. The www.sciencedirect.com
application of new tools that comprehensively characterize the genome and proteome of ESC is helping in this regard.
The ESC transcriptome Several high-throughput technologies are now available to comprehensively characterize all transcripts expressed in a cell population [5]. These include older sequencing-based approaches such as expressed sequence tags (EST) [6], serial analysis of gene expression (SAGE) [7], and massively parallel signature sequencing (MPSS) [8]. Numerous groups have used these methods to capture a detailed view of genes expressed in ESC [3]. One of the advantages of sequence-based methods is that novel transcripts can be identified. Indeed one consistent finding among the first groups that interrogated the transcriptomes of mESC and hESC was the large number of novel transcripts discovered. For example, Anisimov et al. [9] identified 16 000 (35%) potential novel tags by SAGE in mESC, and Brandenberger et al. [10] identified 16 000 (50%) potentially novel ESTs in hESC. Although many of these will prove to be artifacts, some are likely to be unique ESC transcripts representing alternative RNA from known genes or perhaps non-coding RNA. In one recent report novel transcripts were identified in mESC using a new, tagbased strategy for transcriptome characterization called ‘gene identification signature’ (GIS) [11]. Among more than 100 000 transcripts identified in mESC by this method 506 were novel transcripts, of which 3 were examples of unconventional fusion transcripts. For example, one fusion transcript derived from two genes, Set and Ppp2r4 is probably generated by a trans-splicing event between two independently expressed transcripts. The real workhorses for gene expression analyses today are DNA microarrays. Array-based technologies have advanced considerably and there are now several commercial suppliers of arrays, including Affymetrix, Agilent, and Illumina. Each of these vendors supplies high quality arrays for expression analysis of mouse and human cells covering more than 20 000 genes. Reductions in costs, technical improvements, and increasing probe content of these arrays have made them reliable and accessible to most molecular biologists. For ESC, arrays have been used to profile changes in gene expression that correlate with the state of differentiation [12–14] to compare adult and embryonic stem cells [15–17] and to compare independent ESC lines with one another [13,18,19]. A common goal of these studies was to generate molecular signatures of ESC that define the individual components and the pathways that regulate ESC self-renewal and differentiation. The transcriptome data have also the potential to identify molecular markers, which will be essential for rigorous Current Opinion in Chemical Biology 2007, 11:399–404
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quality control of ESC to be used in the clinic and will also mitigate inter-lab variability and intra-lab variability. Although the body of gene expression data is quite extensive, it is difficult to access and to compare results from different groups. Recently, deVos and his colleagues have produced a gene expression atlas that allows ready access to publicly available microarray data (http:// amazonia.montp.inserm.fr) [20]. Their analysis highlighted the extensive variability among the published data regarding the hESC transcriptome. In fact, they found that only a single gene, Oct4 (POU5F1), was commonly included in all 38 lists of hESC-specific genes. However, their statistical analysis of all public data now provides a compendium of genes that distinguish differentiated from undifferentiated hESC. The accumulating ESC transcriptome data are now being mined to select candidate genes for functional studies. In one recent noteworthy example, 65 genes were selected from microarray expression data and tested for their involvement in self-renewal of mESC [21]. This functional genomics approach identified Tbx3, Esrrb, Tcl1, Dppa4, and Mm.343880 as genes required for maintenance of mESC in an undifferentiated state. Thus, this study has added new players to the cadre of known ESC regulatory molecules, Oct4, Sox2, and Nanog. Recently, two additional transcriptional regulators of ESC pluripotency, Zic3, and Sall4, have been identified by functional genomics [22,23]. These transcription factors were selected for study based upon their expression patterns in ESC—they are expressed in undifferentiated ESC and repressed upon differentiation. Recent functional characterization of Zic3 and Sall4 indicates that they operate within a regulatory network that includes Oct4, Sox2, and Nanog to maintain ESC pluripotency. It has now been established by co-immunoprecipitation experiments that Sall4 and Nanog form a complex that binds specific sites on the genome to regulate transcription of target genes [24]. The importance of Sall4, like Nanog, in early development has been established since Sall4-knockout mice fail to form primitive endoderm [25,26]. Similarly, an early embryonic role for Zic3 is underscored by the failure of Zic3 null embryos to form visceral endoderm [27]. These recent examples indicate that our molecular understanding of ESC biology and early mammalian embryogenesis is growing as the field of stem cell genomics evolves to more functional genomic studies. For both practical and political reasons, the bulk of functional genomics work to date has been with mouse ESC. However, given the myriad of differences between mESC and hESC, more emphasis needs to be placed on working with human lines in order to advance cell-based therapies.
The ESC proteome Although transcript-mapping approaches provide genomewide coverage of the RNA, they do not necessarily capture Current Opinion in Chemical Biology 2007, 11:399–404
protein dynamics in a cell because of post-transcriptional events, such as stability, degradation, and modification. In recent years, efforts have been directed at improving technologies for comprehensive detection and analysis of proteins. However, proteomic analyses are filled with issues regarding detection sensitivity and difficulties due to post-translational modifications, size, solubility, stability, and accessibility of proteins. Most current technologies use liquid chromatography (LC) followed by mass spectrometry (MS) to specifically identify peptides, which are then mapped to proteins. LC–LC–MS/MS approaches are certainly superior to 2D gel-based approaches to identify proteins. New chemical approaches such as ICAT, SILAC, and iTRAQ have been added, which provide the much needed sensitivity [28,29]. In addition, protein microarrays are now available for high-throughput interrogation of protein abundance and function [30]. In spite of the limitations, proteomic analysis of ESC is providing new insights. Nagano et al. used an automated microscale 2D LC–MS/MS, which identified known markers of ESC, such as Oct4/Sox2, UTF1, and many other ESC-specific proteins [31]. Importantly, they were able to detect ESC-specific transcription factors of low abundance (104 to 105 copies/cell). New biological insight drawn from this study is that 36% of the total proteins were located in the nucleus, which agrees with the high nuclear to cytoplasmic ratio commonly observed in ESC colonies. Baharvand et al. also reported that the variety and total content of nuclear proteins were highest in human ESC proteome [32]. Additionally, Nagano et al. identified a number of proteins involved in signaling cascades characteristic of differentiated cell types [31]. This interesting and potentially useful finding was further confirmed by the same group in an elegant study using surface biotinylation and purification followed by 2D LC–MS/MS [33]. They showed that undifferentiated mESC express a variety of cell surface-bound kinase and phosphatase-associated receptors, GPCRs, transporters, integrins, cell adhesion molecules, matrix metalloproteases, CD antigens, and other signaling molecules, which may prove to play essential roles in maintaining pluripotency or can be utilized to induce lineage-specific differentiation. Phosphorylation is a key post-translational modification involved in activation/inactivation of a protein, and this modification is often missed during MS evaluation as the number of phospho peptides is low. Phosphoproteome analysis of undifferentiated and differentiated mESC using phosphoprotein affinity purification followed by 2DGE nano LC–MS/MS indicated that many chromatin-remodeling proteins are regulated by phosphorylation [34]. This is in accordance with the observation that epigenetic factors play a crucial role in maintaining pluripotency of ESC. Importantly, many of the proteins identified by this study had no significant changes in RNA expression, which stresses the value of proteomic analysis. www.sciencedirect.com
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A comparative analysis of proteomes from undifferentiated ESC and their differentiated counterparts identified 191 proteins exclusive to ESC [35]. The list includes not only several ‘gold standards’ but also many uncharacterized proteins that may be novel ESC-specific proteins with important biological activities. Sall4, Rif1, and Rnf2 from this list were independently found to be components of an interaction network involved in maintaining pluripotency of ESC [36]. In this insightful study several Nanog-associated proteins were identified, including Oct4, Dax1, Nac1, Zfp281, and Rex1 using affinity purification coupled with LC–MS/MS. Analysis of these proteins revealed that this protein interaction network is tightly regulated, interactive, linked to multiple co-repressors, and highly enriched for proteins that are necessary for regulating differentiation [36]. Proteomic studies can be informative in analyzing ESCspecific proteins but are plagued by inconsistencies. Too often these studies result in identification of proteins with unknown functions and hence more functional studies such as siRNA and western blots should be performed before assigning them as ESC-specific proteins [37]. Lack of crystal structure of the proteins and antibodies is typically a bottleneck in the process of protein characterization. Also, the proteomic approaches need to improve on sensitivity to quantify proteins from limited samples. Although most of the current proteomic methods are useful in establishing a proteome of ESC, methods that can analyze proteins for (a) differential interactions and localizations and (b) acting as an activator versus suppressor, will offer the cutting edge required to get inside the ‘master’ game plan. A combined approach of transcriptome and proteome analysis can be valuable to understand the dynamic ESC biology. However, such attempts can be time consuming and have the added burden of database incompatibility [29].
Transcriptional networks in ESC Recent studies have begun to elucidate transcriptional networks that operate to control ESC pluripotency by working from the foundation that three transcription factors, Oct4, Sox2, and Nanog, are essential for the maintenance of pluripotency in the early embryo and in ESC [38]. It is reasoned that some of the target genes regulated by these transcription factors would be additional components of a common regulatory network. Two groups have now performed chromatin immunoprecipitation (ChIP) experiments to identify genes that are directly regulated by Oct4, Sox2, and Nanog in hESC [39] and mESC [40], and HH Ng (unpublished). The two groups have applied newly developed ChIP methods that provide a genome-wide assessment of transcription factor binding sites. Both groups identified large numbers of genes that have binding sites for Oct4 (782, 623), Sox2 (750, 1271), and Nanog (1802, 1687) in mESC and hESC, respectively. One exciting result that emerged from these www.sciencedirect.com
studies was the high degree of overlap between the genes targeted by pairs or all three transcription factors indicating that Oct4, Sox2, and Nanog operate in concert as part of tightly interlocked network that controls differentiation. The large collection of identified target genes represents ideal candidates for functional analysis to see where they fit into the network and how they contribute to regulating multidimensional ESC differentiation.
The ESC epigenome ESC are proving to be an important model system to study epigenetic regulation of gene expression and its relationship to cell fate specification. The chromatin of pluripotent ESC undergoes genome-wide reorganization as the cells differentiate [41]. Pluripotent ESC have an ‘open’ chromatin architecture that is transcriptionally permissive, whereas differentiated cells contain many regions of ‘closed’ chromatin where transcription is repressed. The ‘open’ and ‘closed’ chromatin architectures are marked by differences in histone modifications, associations with chromatin-modifying proteins, and DNA methylation. In two recent reports, genes expressed at low levels or repressed in mES were found to have an unusual histone architecture—they had both transcription-permissive (acetylated H3K9 and methylated H3K4) and transcription-repressive (methylated H3K27) modifications [42,43]. These groups conclude that ESC maintain this bivalent chromatin structure to hold genes in primed states, poised for a rapid activation as required for lineage-specific differentiation. This transiently repressed state of developmentally important genes is regulated, partly, by polycomb repressive complexes (PRC) as demonstrated recently in mESC [44] and hESC [45]. PRCs are highly conserved in metazoans and promote gene silencing by localized modification of chromatin [46]. Using ChIP, the two groups have mapped PRC binding sites across the ESC genome and found that PRC is preferentially associated with transcriptionally repressed genes [44,45]. Quite interestingly, many of the PRC-bound genes were also co-occupied by Oct4, Sox2, and Nanog [45]. These results establish a strong molecular link between PRC-mediated gene silencing and transcription factors that regulate pluripotency.
Conclusion The availability of tools to carefully and systematically explore the transcriptome, epigenome, and proteome has already contributed significantly to our basic understanding of ESC biology. However, it is generally recognized that work on ESC has proceeded with a general lack of standards for adequate qualification of ESC [47]. This, in turn, has led to inconsistencies and difficulties in reproducing work from independent labs. These problems are particularly vexing for medically driven efforts with human ESC. An international consortium of scientists has begun to take on this problem. The International Stem Cell Forum has set a goal to compare 75 hESC lines Current Opinion in Chemical Biology 2007, 11:399–404
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for a number of molecular characteristics and to make these data readily available to the scientific community (http://www.stemcellforum.org.uk). It is essential to have a good set of molecular markers that can easily establish the quality and differentiation status of a cell line. The field has relied upon a handful of antibodies directed against a few cell surface antigens, which has been useful but not adequate. Transcriptome and proteome results are now providing many new and potentially useful markers. In addition, microarray profiling with well-established commercial systems can now be used to look at the entire transcriptome to assess the status of ESC at an unprecedented degree of sensitivity [48]. In addition, ESC lines can now be easily characterized with respect to genetic and epigenetic variations to determine if there are underlying differences inherent in lines or if there are changes attributed to long-term passage. DNA chips originally designed for genotyping can also provide a fingerprint of the genome and thus be used for comparison of all hESC lines [49]. By working together to establish standards that can be easily measured and compared, the field will be able to move ahead more quickly to realize the potential of embryonic stem cells.
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toward the endodermal lineage. Thus, Zic3 is another component of a transcriptional regulatory network that controls ESC pluripotency. 23. Zhang J, Tam WL, Tong GQ, Wu Q, Chan HY, Soh BS, Lou Y, Yang J, Ma Y, Chai L et al.: Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol 2006, 8:1114-1123. In this study, candidate genes that regulate pluripotency were selected from gene expression profiles and tested for their ability to influence Oct4 expression when knocked down by RNA interference. From this functional genomics screen, Sall4, a member of the spalt family of transcription factors, was identified and subsequently shown to directly activate Oct4 transcription by binding to its promoter region. The group showed that siRNA knockdown or knockout of one copy of Sall4 in ESC by homologous recombination reduced the pluripotential of the cells and drove them toward extraembryonic ectoderm. 24. Wu Q, Chen X, Zhang J, Loh YH, Low TY, Zhang W, Sze SK, Lim B, Ng HH: Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells. J Biol Chem 2006, 281:24090-24094. 25. Elling U, Klasen C, Eisenberger T, Anlag K, Treier M: Murine inner cell mass-derived lineages depend on Sall4 function. Proc Natl Acad Sci USA 2006, 103:16319-16324. 26. Sakaki-Yumoto M, Kobayashi C, Sato A, Fujimura S, Matsumoto Y, Takasato M, Kodama T, Aburatani H, Asashima M, Yoshida N et al.: The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development 2006, 133:3005-3013. 27. Ware SM, Harutyunyan KG, Belmont JW: Zic3 is critical for early embryonic patterning during gastrulation. Dev Dyn 2006, 235:776-785. 28. Unwin RD, Evans CA, Whetton AD: Relative quantification in proteomics: new approaches for biochemistry. Trends Biochem Sci 2006, 31:473-484. This review describes how transcriptomic and proteomic data can complement each other and the current issues in the analysis. Proteomic approaches are ‘open’ approaches and hence can detect any number of proteins, but increased proteomic penetration is essential. Transcriptomic approaches give information regarding ‘fixed’ number of genes but can give genome-wide coverage. The comparison of two methods to evaluate both mRNA and proteins is extremely time consuming and corelates poorly. Improvements in both technologies for more consistent data and resolving data base incompatibilities are the bottlenecks in the process. 29. Unwin RD, Whetton AD: Systematic proteome and transcriptome analysis of stem cell populations. Cell Cycle 2006, 5:1587-1591. 30. LaBaer J, Ramachandran N: Protein microarrays as tools for functional proteomics. Curr Opin Chem Biol 2005, 9:14-19. 31. Nagano K, Taoka M, Yamauchi Y, Itagaki C, Shinkawa T, Nunomura K, Okamura N, Takahashi N, Izumi T, Isobe T: Largescale identification of proteins expressed in mouse embryonic stem cells. Proteomics 2005, 5:1346-1361. 32. Baharvand H, Hajheidari M, Ashtiani SK, Salekdeh GH: Proteomic signature of human embryonic stem cells. Proteomics 2006, 6:3544-3549. 33. Nunomura K, Nagano K, Itagaki C, Taoka M, Okamura N, Yamauchi Y, Sugano S, Takahashi N, Izumi T, Isobe T: Cell surface labeling and mass spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol Cell Proteomics 2005, 4:1968-1976. 34. Puente LG, Borris DJ, Carriere JF, Kelly JF, Megeney LA: Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling. Mol Cell Proteomics 2006, 5:57-67. 35. Van Hoof D, Passier R, Ward-Van Oostwaard D, Pinkse MW, Heck AJ, Mummery CL, Krijgsveld J: A quest for human and mouse embryonic stem cell-specific proteins. Mol Cell Proteomics 2006, 5:1261-1273. www.sciencedirect.com
36. Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, Orkin SH: A protein interaction network for pluripotency of embryonic stem cells. Nature 2006, 444:364-368. This group utilized biotinylation/overexpression of individual proteins and proteomics approach to identify proteins associated with Nanog, Oct4, Dax1, Nac1, Zfp281 and Rex1 to build a protein interaction network involved in maintenance of pluripotency. Many interacting proteins were shared while some were exclusively interacting with a particular protein. The interactions have been validated by co-immunoprecipitations and RNA inhibition analysis. The network can function as a ‘cellular module’ controlling pluripotency as it is highly enriched for proteins that individually are critical for early development, and many genes (proteins) within the network are co-down regulated upon ESC differentiation and are direct transcriptional targets of its own members. In addition, the network involves multiple co-repressor pathways involved in transcriptional repression. 37. Van Hoof D, Mummery CL, Heck AJ, Krijgsveld J: Embryonic stem cell proteomics. Expert Rev Proteomics 2006, 3:427-437. 38. Boiani M, Scholer H: Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol 2005, 6:872-884. 39. Boyer L, Lee T, Cole M, Johnstone S, Levine S, Zucker J, Guenther M, Kumar R, Murray H, Jenner R et al.: Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005, 122:947-956. In this paper, binding sites for the pluripotency-regulating transcription factors Oct4, Sox2, and Nanog were mapped on the hESC genome. These authors utilized a new method that couples chromatin immunoprecipitation with DNA microarrays, colloquially known as ‘‘ChIP-onchip’’ or ‘‘ChIP-chip’’. The DNA arrays used did not permit genome-wide interrogation, but rather focused on the promoter regions (8kb to +2kb from the transcription start site) of 18,000 human genes. They found that all three transcription factors bind to hundreds of sites in hESC and, in many cases, these three transcription factors bind to regulatory sites within the same gene. These results have expanded our understanding of the regulatory network that controls ESC differentiation. 40. Loh Y, Wu Q, Chew J, Vega V, Zhang W, Chen X, Bourque G, George J, Leong B, Liu J et al.: The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 2006, 38:431-440. This group has made use of a new chromatin immunoprecipitation technology to look for Oct4, Sox, and Nanog binding sites, without bias, across the entire genome in mESC. The newly developed ChIP-PET method uses a sequence-based approach to identify binding sites. The ChIPed DNA is cloned and sequenced to produce paired-end-tags (PET) of each fragment. The PETs are then mapped back to the genome to determine where the binding sites are. Millions of tags are produced for each ChIP and thus provide comprehensive coverage of the genome. Surprisingly, there was only about a 15% overlap of the data produced in this paper and that by Boyer et al. [39]. It remains to be determined if these differences are technical or represent unique binding landscapes in mouse and human ESC. 41. Meshorer E, Misteli T: Chromatin in pluripotent embryonic stem cells and differentiation. Nat Rev Mol Cell Biol 2006, 7:540-546. 42. Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, Gouti M, Casanova M, Warnes G, Merkenschlager M et al.: Chromatin signatures of pluripotent cell lines. Nat Cell Biol 2006, 8:532-538. In this paper the group compared chromatin structure in and around the promoters of several genes in mESC. They used replication timing and chromatin immunoprecipitation to show that genes involved in lineage specification have a unique epigenetic profile that is likely to hold their expression in check. This unique chromatin organization may be set for quick response to signals that drive differentiation. 43. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K et al.: A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006, 125:315-326. Here chromatin immunoprecipitation technology (ChIP-chip) was used to examine histone modifications in highly conserved noncoding (HCNE) regions of the genome in mESC. The HCNE regions are enriched for genes that encode transcription factors that are developmentally regulated, such as the Hox clusters. Their ChIP-chip experiments found that these regions have a unique combination of transcriptionally active and inactive histone modifications, which they call ‘‘bivalent domains’’. Like Azuara et al. [42], they conclude that genes required for lineage specification are maintained in a repressed state, but are primed for immediate activation by this unique chromatin organization. Current Opinion in Chemical Biology 2007, 11:399–404
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44. Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS, Wernig M, Tajonar A, Ray MK et al.: Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 2006, 441:349-353. 45. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K et al.: Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 2006, 125:301-313. 46. Lund AH, van Lohuizen M: Polycomb complexes and silencing mechanisms. Curr Opin Cell Biol 2004, 16:239-246.
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Current Opinion in Chemical Biology 2007, 11:399–404 This review comes from a themed issue on Chemical Biology and Stem Cells Edited by David Schaeffer Available online 23rd July 2007 1367-5931/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2007.05.029
Introduction The landscape of stem cell biology was altered substantially in 1998 with the first successful derivation of embryonic stem—human embryonic stem cell (hESC) lines from human blastocytsts [1]. Work done in early 1980s with mouse ESC (mESC) established that they have the defining characteristics of unlimited self-renewal and a capacity to give rise to all cell types of the developing and adult organism [2,3]. The most useful aspect of ESC derived from the mouse was that they provided a direct and efficient means to generate a genetically modified mammal [2]. The arrival of human-derived ESC lines has been heralded with great fanfare for other reasons [3,4]. One reason for the excitement is that we now have the opportunity to study the earliest stages of human embryology. However, it is the potential of hESC for use in regenerative medicine that receives the greatest attention from scientists, clinicians, patients, and society. To realize the clinical potential of hESC it is necessary to better understand the molecular pathways that regulate differentiation and harness that knowledge into efficient and reliable methods for directed differentiation of therapeutically useful cells. The www.sciencedirect.com
application of new tools that comprehensively characterize the genome and proteome of ESC is helping in this regard.
The ESC transcriptome Several high-throughput technologies are now available to comprehensively characterize all transcripts expressed in a cell population [5]. These include older sequencing-based approaches such as expressed sequence tags (EST) [6], serial analysis of gene expression (SAGE) [7], and massively parallel signature sequencing (MPSS) [8]. Numerous groups have used these methods to capture a detailed view of genes expressed in ESC [3]. One of the advantages of sequence-based methods is that novel transcripts can be identified. Indeed one consistent finding among the first groups that interrogated the transcriptomes of mESC and hESC was the large number of novel transcripts discovered. For example, Anisimov et al. [9] identified 16 000 (35%) potential novel tags by SAGE in mESC, and Brandenberger et al. [10] identified 16 000 (50%) potentially novel ESTs in hESC. Although many of these will prove to be artifacts, some are likely to be unique ESC transcripts representing alternative RNA from known genes or perhaps non-coding RNA. In one recent report novel transcripts were identified in mESC using a new, tagbased strategy for transcriptome characterization called ‘gene identification signature’ (GIS) [11]. Among more than 100 000 transcripts identified in mESC by this method 506 were novel transcripts, of which 3 were examples of unconventional fusion transcripts. For example, one fusion transcript derived from two genes, Set and Ppp2r4 is probably generated by a trans-splicing event between two independently expressed transcripts. The real workhorses for gene expression analyses today are DNA microarrays. Array-based technologies have advanced considerably and there are now several commercial suppliers of arrays, including Affymetrix, Agilent, and Illumina. Each of these vendors supplies high quality arrays for expression analysis of mouse and human cells covering more than 20 000 genes. Reductions in costs, technical improvements, and increasing probe content of these arrays have made them reliable and accessible to most molecular biologists. For ESC, arrays have been used to profile changes in gene expression that correlate with the state of differentiation [12–14] to compare adult and embryonic stem cells [15–17] and to compare independent ESC lines with one another [13,18,19]. A common goal of these studies was to generate molecular signatures of ESC that define the individual components and the pathways that regulate ESC self-renewal and differentiation. The transcriptome data have also the potential to identify molecular markers, which will be essential for rigorous Current Opinion in Chemical Biology 2007, 11:399–404
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quality control of ESC to be used in the clinic and will also mitigate inter-lab variability and intra-lab variability. Although the body of gene expression data is quite extensive, it is difficult to access and to compare results from different groups. Recently, deVos and his colleagues have produced a gene expression atlas that allows ready access to publicly available microarray data (http:// amazonia.montp.inserm.fr) [20]. Their analysis highlighted the extensive variability among the published data regarding the hESC transcriptome. In fact, they found that only a single gene, Oct4 (POU5F1), was commonly included in all 38 lists of hESC-specific genes. However, their statistical analysis of all public data now provides a compendium of genes that distinguish differentiated from undifferentiated hESC. The accumulating ESC transcriptome data are now being mined to select candidate genes for functional studies. In one recent noteworthy example, 65 genes were selected from microarray expression data and tested for their involvement in self-renewal of mESC [21]. This functional genomics approach identified Tbx3, Esrrb, Tcl1, Dppa4, and Mm.343880 as genes required for maintenance of mESC in an undifferentiated state. Thus, this study has added new players to the cadre of known ESC regulatory molecules, Oct4, Sox2, and Nanog. Recently, two additional transcriptional regulators of ESC pluripotency, Zic3, and Sall4, have been identified by functional genomics [22,23]. These transcription factors were selected for study based upon their expression patterns in ESC—they are expressed in undifferentiated ESC and repressed upon differentiation. Recent functional characterization of Zic3 and Sall4 indicates that they operate within a regulatory network that includes Oct4, Sox2, and Nanog to maintain ESC pluripotency. It has now been established by co-immunoprecipitation experiments that Sall4 and Nanog form a complex that binds specific sites on the genome to regulate transcription of target genes [24]. The importance of Sall4, like Nanog, in early development has been established since Sall4-knockout mice fail to form primitive endoderm [25,26]. Similarly, an early embryonic role for Zic3 is underscored by the failure of Zic3 null embryos to form visceral endoderm [27]. These recent examples indicate that our molecular understanding of ESC biology and early mammalian embryogenesis is growing as the field of stem cell genomics evolves to more functional genomic studies. For both practical and political reasons, the bulk of functional genomics work to date has been with mouse ESC. However, given the myriad of differences between mESC and hESC, more emphasis needs to be placed on working with human lines in order to advance cell-based therapies.
The ESC proteome Although transcript-mapping approaches provide genomewide coverage of the RNA, they do not necessarily capture Current Opinion in Chemical Biology 2007, 11:399–404
protein dynamics in a cell because of post-transcriptional events, such as stability, degradation, and modification. In recent years, efforts have been directed at improving technologies for comprehensive detection and analysis of proteins. However, proteomic analyses are filled with issues regarding detection sensitivity and difficulties due to post-translational modifications, size, solubility, stability, and accessibility of proteins. Most current technologies use liquid chromatography (LC) followed by mass spectrometry (MS) to specifically identify peptides, which are then mapped to proteins. LC–LC–MS/MS approaches are certainly superior to 2D gel-based approaches to identify proteins. New chemical approaches such as ICAT, SILAC, and iTRAQ have been added, which provide the much needed sensitivity [28,29]. In addition, protein microarrays are now available for high-throughput interrogation of protein abundance and function [30]. In spite of the limitations, proteomic analysis of ESC is providing new insights. Nagano et al. used an automated microscale 2D LC–MS/MS, which identified known markers of ESC, such as Oct4/Sox2, UTF1, and many other ESC-specific proteins [31]. Importantly, they were able to detect ESC-specific transcription factors of low abundance (104 to 105 copies/cell). New biological insight drawn from this study is that 36% of the total proteins were located in the nucleus, which agrees with the high nuclear to cytoplasmic ratio commonly observed in ESC colonies. Baharvand et al. also reported that the variety and total content of nuclear proteins were highest in human ESC proteome [32]. Additionally, Nagano et al. identified a number of proteins involved in signaling cascades characteristic of differentiated cell types [31]. This interesting and potentially useful finding was further confirmed by the same group in an elegant study using surface biotinylation and purification followed by 2D LC–MS/MS [33]. They showed that undifferentiated mESC express a variety of cell surface-bound kinase and phosphatase-associated receptors, GPCRs, transporters, integrins, cell adhesion molecules, matrix metalloproteases, CD antigens, and other signaling molecules, which may prove to play essential roles in maintaining pluripotency or can be utilized to induce lineage-specific differentiation. Phosphorylation is a key post-translational modification involved in activation/inactivation of a protein, and this modification is often missed during MS evaluation as the number of phospho peptides is low. Phosphoproteome analysis of undifferentiated and differentiated mESC using phosphoprotein affinity purification followed by 2DGE nano LC–MS/MS indicated that many chromatin-remodeling proteins are regulated by phosphorylation [34]. This is in accordance with the observation that epigenetic factors play a crucial role in maintaining pluripotency of ESC. Importantly, many of the proteins identified by this study had no significant changes in RNA expression, which stresses the value of proteomic analysis. www.sciencedirect.com
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A comparative analysis of proteomes from undifferentiated ESC and their differentiated counterparts identified 191 proteins exclusive to ESC [35]. The list includes not only several ‘gold standards’ but also many uncharacterized proteins that may be novel ESC-specific proteins with important biological activities. Sall4, Rif1, and Rnf2 from this list were independently found to be components of an interaction network involved in maintaining pluripotency of ESC [36]. In this insightful study several Nanog-associated proteins were identified, including Oct4, Dax1, Nac1, Zfp281, and Rex1 using affinity purification coupled with LC–MS/MS. Analysis of these proteins revealed that this protein interaction network is tightly regulated, interactive, linked to multiple co-repressors, and highly enriched for proteins that are necessary for regulating differentiation [36]. Proteomic studies can be informative in analyzing ESCspecific proteins but are plagued by inconsistencies. Too often these studies result in identification of proteins with unknown functions and hence more functional studies such as siRNA and western blots should be performed before assigning them as ESC-specific proteins [37]. Lack of crystal structure of the proteins and antibodies is typically a bottleneck in the process of protein characterization. Also, the proteomic approaches need to improve on sensitivity to quantify proteins from limited samples. Although most of the current proteomic methods are useful in establishing a proteome of ESC, methods that can analyze proteins for (a) differential interactions and localizations and (b) acting as an activator versus suppressor, will offer the cutting edge required to get inside the ‘master’ game plan. A combined approach of transcriptome and proteome analysis can be valuable to understand the dynamic ESC biology. However, such attempts can be time consuming and have the added burden of database incompatibility [29].
Transcriptional networks in ESC Recent studies have begun to elucidate transcriptional networks that operate to control ESC pluripotency by working from the foundation that three transcription factors, Oct4, Sox2, and Nanog, are essential for the maintenance of pluripotency in the early embryo and in ESC [38]. It is reasoned that some of the target genes regulated by these transcription factors would be additional components of a common regulatory network. Two groups have now performed chromatin immunoprecipitation (ChIP) experiments to identify genes that are directly regulated by Oct4, Sox2, and Nanog in hESC [39] and mESC [40], and HH Ng (unpublished). The two groups have applied newly developed ChIP methods that provide a genome-wide assessment of transcription factor binding sites. Both groups identified large numbers of genes that have binding sites for Oct4 (782, 623), Sox2 (750, 1271), and Nanog (1802, 1687) in mESC and hESC, respectively. One exciting result that emerged from these www.sciencedirect.com
studies was the high degree of overlap between the genes targeted by pairs or all three transcription factors indicating that Oct4, Sox2, and Nanog operate in concert as part of tightly interlocked network that controls differentiation. The large collection of identified target genes represents ideal candidates for functional analysis to see where they fit into the network and how they contribute to regulating multidimensional ESC differentiation.
The ESC epigenome ESC are proving to be an important model system to study epigenetic regulation of gene expression and its relationship to cell fate specification. The chromatin of pluripotent ESC undergoes genome-wide reorganization as the cells differentiate [41]. Pluripotent ESC have an ‘open’ chromatin architecture that is transcriptionally permissive, whereas differentiated cells contain many regions of ‘closed’ chromatin where transcription is repressed. The ‘open’ and ‘closed’ chromatin architectures are marked by differences in histone modifications, associations with chromatin-modifying proteins, and DNA methylation. In two recent reports, genes expressed at low levels or repressed in mES were found to have an unusual histone architecture—they had both transcription-permissive (acetylated H3K9 and methylated H3K4) and transcription-repressive (methylated H3K27) modifications [42,43]. These groups conclude that ESC maintain this bivalent chromatin structure to hold genes in primed states, poised for a rapid activation as required for lineage-specific differentiation. This transiently repressed state of developmentally important genes is regulated, partly, by polycomb repressive complexes (PRC) as demonstrated recently in mESC [44] and hESC [45]. PRCs are highly conserved in metazoans and promote gene silencing by localized modification of chromatin [46]. Using ChIP, the two groups have mapped PRC binding sites across the ESC genome and found that PRC is preferentially associated with transcriptionally repressed genes [44,45]. Quite interestingly, many of the PRC-bound genes were also co-occupied by Oct4, Sox2, and Nanog [45]. These results establish a strong molecular link between PRC-mediated gene silencing and transcription factors that regulate pluripotency.
Conclusion The availability of tools to carefully and systematically explore the transcriptome, epigenome, and proteome has already contributed significantly to our basic understanding of ESC biology. However, it is generally recognized that work on ESC has proceeded with a general lack of standards for adequate qualification of ESC [47]. This, in turn, has led to inconsistencies and difficulties in reproducing work from independent labs. These problems are particularly vexing for medically driven efforts with human ESC. An international consortium of scientists has begun to take on this problem. The International Stem Cell Forum has set a goal to compare 75 hESC lines Current Opinion in Chemical Biology 2007, 11:399–404
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for a number of molecular characteristics and to make these data readily available to the scientific community (http://www.stemcellforum.org.uk). It is essential to have a good set of molecular markers that can easily establish the quality and differentiation status of a cell line. The field has relied upon a handful of antibodies directed against a few cell surface antigens, which has been useful but not adequate. Transcriptome and proteome results are now providing many new and potentially useful markers. In addition, microarray profiling with well-established commercial systems can now be used to look at the entire transcriptome to assess the status of ESC at an unprecedented degree of sensitivity [48]. In addition, ESC lines can now be easily characterized with respect to genetic and epigenetic variations to determine if there are underlying differences inherent in lines or if there are changes attributed to long-term passage. DNA chips originally designed for genotyping can also provide a fingerprint of the genome and thus be used for comparison of all hESC lines [49]. By working together to establish standards that can be easily measured and compared, the field will be able to move ahead more quickly to realize the potential of embryonic stem cells.
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toward the endodermal lineage. Thus, Zic3 is another component of a transcriptional regulatory network that controls ESC pluripotency. 23. Zhang J, Tam WL, Tong GQ, Wu Q, Chan HY, Soh BS, Lou Y, Yang J, Ma Y, Chai L et al.: Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol 2006, 8:1114-1123. In this study, candidate genes that regulate pluripotency were selected from gene expression profiles and tested for their ability to influence Oct4 expression when knocked down by RNA interference. From this functional genomics screen, Sall4, a member of the spalt family of transcription factors, was identified and subsequently shown to directly activate Oct4 transcription by binding to its promoter region. The group showed that siRNA knockdown or knockout of one copy of Sall4 in ESC by homologous recombination reduced the pluripotential of the cells and drove them toward extraembryonic ectoderm. 24. Wu Q, Chen X, Zhang J, Loh YH, Low TY, Zhang W, Sze SK, Lim B, Ng HH: Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells. J Biol Chem 2006, 281:24090-24094. 25. Elling U, Klasen C, Eisenberger T, Anlag K, Treier M: Murine inner cell mass-derived lineages depend on Sall4 function. Proc Natl Acad Sci USA 2006, 103:16319-16324. 26. Sakaki-Yumoto M, Kobayashi C, Sato A, Fujimura S, Matsumoto Y, Takasato M, Kodama T, Aburatani H, Asashima M, Yoshida N et al.: The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development 2006, 133:3005-3013. 27. Ware SM, Harutyunyan KG, Belmont JW: Zic3 is critical for early embryonic patterning during gastrulation. Dev Dyn 2006, 235:776-785. 28. Unwin RD, Evans CA, Whetton AD: Relative quantification in proteomics: new approaches for biochemistry. Trends Biochem Sci 2006, 31:473-484. This review describes how transcriptomic and proteomic data can complement each other and the current issues in the analysis. Proteomic approaches are ‘open’ approaches and hence can detect any number of proteins, but increased proteomic penetration is essential. Transcriptomic approaches give information regarding ‘fixed’ number of genes but can give genome-wide coverage. The comparison of two methods to evaluate both mRNA and proteins is extremely time consuming and corelates poorly. Improvements in both technologies for more consistent data and resolving data base incompatibilities are the bottlenecks in the process. 29. Unwin RD, Whetton AD: Systematic proteome and transcriptome analysis of stem cell populations. Cell Cycle 2006, 5:1587-1591. 30. LaBaer J, Ramachandran N: Protein microarrays as tools for functional proteomics. Curr Opin Chem Biol 2005, 9:14-19. 31. Nagano K, Taoka M, Yamauchi Y, Itagaki C, Shinkawa T, Nunomura K, Okamura N, Takahashi N, Izumi T, Isobe T: Largescale identification of proteins expressed in mouse embryonic stem cells. Proteomics 2005, 5:1346-1361. 32. Baharvand H, Hajheidari M, Ashtiani SK, Salekdeh GH: Proteomic signature of human embryonic stem cells. Proteomics 2006, 6:3544-3549. 33. Nunomura K, Nagano K, Itagaki C, Taoka M, Okamura N, Yamauchi Y, Sugano S, Takahashi N, Izumi T, Isobe T: Cell surface labeling and mass spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Mol Cell Proteomics 2005, 4:1968-1976. 34. Puente LG, Borris DJ, Carriere JF, Kelly JF, Megeney LA: Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling. Mol Cell Proteomics 2006, 5:57-67. 35. Van Hoof D, Passier R, Ward-Van Oostwaard D, Pinkse MW, Heck AJ, Mummery CL, Krijgsveld J: A quest for human and mouse embryonic stem cell-specific proteins. Mol Cell Proteomics 2006, 5:1261-1273. www.sciencedirect.com
36. Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, Orkin SH: A protein interaction network for pluripotency of embryonic stem cells. Nature 2006, 444:364-368. This group utilized biotinylation/overexpression of individual proteins and proteomics approach to identify proteins associated with Nanog, Oct4, Dax1, Nac1, Zfp281 and Rex1 to build a protein interaction network involved in maintenance of pluripotency. Many interacting proteins were shared while some were exclusively interacting with a particular protein. The interactions have been validated by co-immunoprecipitations and RNA inhibition analysis. The network can function as a ‘cellular module’ controlling pluripotency as it is highly enriched for proteins that individually are critical for early development, and many genes (proteins) within the network are co-down regulated upon ESC differentiation and are direct transcriptional targets of its own members. In addition, the network involves multiple co-repressor pathways involved in transcriptional repression. 37. Van Hoof D, Mummery CL, Heck AJ, Krijgsveld J: Embryonic stem cell proteomics. Expert Rev Proteomics 2006, 3:427-437. 38. Boiani M, Scholer H: Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol 2005, 6:872-884. 39. Boyer L, Lee T, Cole M, Johnstone S, Levine S, Zucker J, Guenther M, Kumar R, Murray H, Jenner R et al.: Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005, 122:947-956. In this paper, binding sites for the pluripotency-regulating transcription factors Oct4, Sox2, and Nanog were mapped on the hESC genome. These authors utilized a new method that couples chromatin immunoprecipitation with DNA microarrays, colloquially known as ‘‘ChIP-onchip’’ or ‘‘ChIP-chip’’. The DNA arrays used did not permit genome-wide interrogation, but rather focused on the promoter regions (8kb to +2kb from the transcription start site) of 18,000 human genes. They found that all three transcription factors bind to hundreds of sites in hESC and, in many cases, these three transcription factors bind to regulatory sites within the same gene. These results have expanded our understanding of the regulatory network that controls ESC differentiation. 40. Loh Y, Wu Q, Chew J, Vega V, Zhang W, Chen X, Bourque G, George J, Leong B, Liu J et al.: The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 2006, 38:431-440. This group has made use of a new chromatin immunoprecipitation technology to look for Oct4, Sox, and Nanog binding sites, without bias, across the entire genome in mESC. The newly developed ChIP-PET method uses a sequence-based approach to identify binding sites. The ChIPed DNA is cloned and sequenced to produce paired-end-tags (PET) of each fragment. The PETs are then mapped back to the genome to determine where the binding sites are. Millions of tags are produced for each ChIP and thus provide comprehensive coverage of the genome. Surprisingly, there was only about a 15% overlap of the data produced in this paper and that by Boyer et al. [39]. It remains to be determined if these differences are technical or represent unique binding landscapes in mouse and human ESC. 41. Meshorer E, Misteli T: Chromatin in pluripotent embryonic stem cells and differentiation. Nat Rev Mol Cell Biol 2006, 7:540-546. 42. Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, Gouti M, Casanova M, Warnes G, Merkenschlager M et al.: Chromatin signatures of pluripotent cell lines. Nat Cell Biol 2006, 8:532-538. In this paper the group compared chromatin structure in and around the promoters of several genes in mESC. They used replication timing and chromatin immunoprecipitation to show that genes involved in lineage specification have a unique epigenetic profile that is likely to hold their expression in check. This unique chromatin organization may be set for quick response to signals that drive differentiation. 43. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K et al.: A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006, 125:315-326. Here chromatin immunoprecipitation technology (ChIP-chip) was used to examine histone modifications in highly conserved noncoding (HCNE) regions of the genome in mESC. The HCNE regions are enriched for genes that encode transcription factors that are developmentally regulated, such as the Hox clusters. Their ChIP-chip experiments found that these regions have a unique combination of transcriptionally active and inactive histone modifications, which they call ‘‘bivalent domains’’. Like Azuara et al. [42], they conclude that genes required for lineage specification are maintained in a repressed state, but are primed for immediate activation by this unique chromatin organization. Current Opinion in Chemical Biology 2007, 11:399–404
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44. Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS, Wernig M, Tajonar A, Ray MK et al.: Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 2006, 441:349-353. 45. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K et al.: Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 2006, 125:301-313. 46. Lund AH, van Lohuizen M: Polycomb complexes and silencing mechanisms. Curr Opin Cell Biol 2004, 16:239-246.
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47. Loring JF, Rao MS: Establishing standards for the characterization of human embryonic stem cell lines. Stem Cells 2006, 24:145-150. 48. Josephson R, Sykes G, Liu Y, Ording C, Xu W, Zeng X, Shin S, Loring J, Maitra A, Rao MS et al.: A molecular scheme for improved characterization of human embryonic stem cell lines. BMC Biol 2006, 4:28. 49. Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, Sui G, Cutler DJ, Liu Y, Brimble SN et al.: Genomic alterations in cultured human embryonic stem cells. Nat Genet 2005, 37:1099-1103.
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