1 Department of Genetics, The Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel and 2 Curis Incorporation, 45 Moulton Street, Cambridge, Massachusetts 02138, USA
3 To whom correspondence should be addressed. or Email: rachela{at}mail.ls.huji.ac.il
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Abstract |
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Key words: differentiation/ES cells/gene expression/micro-arrays/pluripotency
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Introduction |
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While it is obvious that human ES cells could become a useful system to study the differentiation of human embryonic cells, presently little is known about the genes they express. Human ES cells do express several markers suggested to reflect human ICM cells (Henderson et al., 2002). Recently, mouse ES cells were compared to hematopoietic and neuronal stem cells using DNA micro-arrays (Ivanova et al., 2002
; Ramalho-Santos et al., 2002
). These analyses showed that embryonic stem cells have some features in common with adult stem cells, allowing the identification of a cluster of genes which is associated with the stemness character of all stem cells (Ivanova et al., 2002
; Ramalho-Santos et al., 2002
). Global transcription profiles for undifferentiated human ES cells were independently published by several groups (Sato et al., 2003
; Sperger et al., 2003
; Richards et al., 2004
). In the present study we chose to carry out a large-scale transcription analysis to profile human ES cells at different stages during their differentiation in vitro, as an initial step towards understanding the genetic control of human embryonic differentiation.
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Materials and methods |
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DNA micro-array analysis
Total RNA was extracted from populations of undifferentiated (confirmed by REX1 and OCT4 expression) and differentiated cell derivatives of human ES cells. RNA extraction was performed according to the manufacturer's protocol (Affymetrix). Hybridization to the DNA micro-arrays, washing and scanning were performed according to the manufacturer's protocols, and compared for expression pattern using Affymetrix U133 DNA chip micro-arrays. Affymetrix DNA micro arrays are provided with human specific probes each of which consists of 20 different sequence combinations, in order to overcome hybridization efficiency differences. Moreover, we have normalized signal value for each probe through dividing by the average signal of the hybridization in each experiment, to reduce differences in signal levels between experiments. Analysis of the results was performed using the GENE SPRING and GO ANNOTATION programs.
The U133 GeneChip human micro-array, contains nearly 45 000 probe sets (representing 33 000 human genes). This DNA micro-array was used to compare the global expression of H9 human ES cells and 2, 10 and 30-day old EBs. Each differentiation stage was assayed in three independent experiments to determine the variability of the system.
ES specific gene expression analysis
Average signal value was calculated for each probe in ES cells and EBs, and ordered according to the ratio of ES/EB value. Complementary (cDNA) probes in which expression was at least as high as the average signal level of the chip (value of 100 following normalization) and for which the ratio between ES and EBs was >20 (total of 50 sequences: see our website: http://www.ls.huji.ac.il/nissimb/gene_profiling.html) were further examined for tissue distribution by searching in databanks (dbESTs-NCBI and Source-USCS).
Databases
We have used the following databases: Entrez (NCBI)the text-based search system used at NCBI for its major databases. Unigene (NCBI)a database which partitions GeneBank sequences into a non-redundant set of gene-oriented clusters, each of which contains sequences that represent a unique gene and its related information i.e. map location and tissues where it is expressed. Blast (NCBI)a sequence similarity search tool. LocusLink (NCBI)provides a descriptive information regarding genetic loci (e.g. information on official nomenclature, aliases and sequence accession numbers etc.) and AceView (NCBI)offers an integrated view of the human genes as reconstructed by alignment of all publicly available mRNAs and Expressed Sequence Tags (ESTs) on the genome sequence. SOURCE (Stanford University)collects and compiles data about genetics and molecular biology of genes from human and other species genomes. BLAT (UCSC)contains reference sequences for human and other species and integrate map location and various types of annotation.
RTPCR analysis
Total RNA was extracted as described (Chomczynski and Sacchi, 1987) and 1 µg of RNA was reverse transcribed by random hexamer priming using EZ-First Strand cDNA Synthesis Kit (Biological Industries). Amplification was performed on the cDNA using Takara Ex TaqTM, in the presence of X1 Ex TaqTM Buffer, 200 µM dNTPs each, and 2.5 mM Mg2 + . PCR conditions include a first step of 3 min at 94°C, a second step of 2030 cycles of 30 s at 94°C, 30 s annealing step at 6064°C, and 45 s at 72°C, and a final step of 5 min at 72°C. Several markers were examined: OCT4 as a marker for undifferentiated ES cells, GAPDH as a house keeping gene, LEFTY A as a transiently expressed gene and NODAL, LEFTY B and PITX2 as genes related to the LEFTY A pathway. A full description of primers, annealing temperature and size of final products is described in Table I. Final products were assessed by gel electrophoresis on 2% agarose ethidium-bromide stained gels and their identity was verified by direct sequencing.
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Results |
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Discussion |
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Profiling human ES cells
Hierarchical cluster analysis with different ES cell lines or different cultures of the same line show high similarity. Variation is most probably due to spontaneous differentiation that occurs during ES cell propagation. The difference between ES cells and fully matured EBs is dramatically larger than the variance between two different ES cell lines, representing transcriptional changes that would be expected to accompany differentiation.
As part of our attempt to establish a genetic bar code for ES cells at the transcriptional level, we characterized cytokine receptors and their related growth factors. Of 74 receptors examined, 28 are expressed in ES cells, with a relatively high expression of members of the PTP, FGF, IGF and BMP, activin and TNF receptor families. TGFR, MET and gp130 seem to be present at a very low level. These results may explain why leukemia inhibitory factor (LIF), which is based on the activation of STAT3 by the gp130-LIF receptor pathway, may not play a role in sustaining undifferentiated growth in human ES cells, as it does in mouse ES cells (Thomson et al., 1998
; Reubinoff et al., 2000
). In addition, the high expression level of FGF receptors in ES cells may support the need of bFGF in the growth media (Thomson et al., 1998
). Since a good correlation exists between the relative level of the receptors and their associated ligands, it should be interesting to investigate the role of these factors in human ES cells.
Pluripotent specific genes
There is presently little information regarding genes that are directly associated with pluripotency and self-renewal. A short list of molecular markers that rapidly down-regulate upon differentiation is available in mouse (for a review, see Eiges and Benvenisty, 2002). Most of these are transcription factors that are also expressed by the ICM. However none are exclusively expressed by pluripotent cells. Oct4 is the only gene that has been shown to be directly involved in the maintenance of the undifferentiated state of the cells in vivo (Nichols et al., 1998
). By comparing the expression profile of human ES cells to their differentiated derivatives, we identified a list of candidate genes which may be associated with the pluripotent nature of the cells. Sequences that are highly expressed in ES cells and down regulated upon differentiation are predicted to be good marker genes for determining the state of differentiation of ES cells in vitro. However, most are expressed by other differentiated cell types. Since the DNA micro-array analysis contains probes of only previously cloned sequences, it is not possible to isolate unique sequences, which are truly ES-specific. Under the assumption that genes associated with pluripotency should be expressed by both early embryonic cells and germ cells, we examined the tissue distribution of highly expressed genes in databanks and identified genes expressed exclusively in ES cells and germ cells (enriched in ovary, teratoma, testis and pure populations of germ cells). Using these constraints, five sequences were defined as ES-specific: OCT4 and four other uncharacterized ESTs. RTPCR showed down-regulation of all five sequences upon differentiation of ES cells and most have DNA-binding motifs, suggesting a role as transcription factors. The presence of OCT4 strongly supports the strategy used to find these genes. The function of these genes and their targets remains to be determined. In addition, it should be interesting to examine their expression in the ICM of human blastocyst-stage embryos.
Recently, the transcriptional programs in mouse embryonic and adult stem cells were compared to identify transcripts enriched in embryonic, neuronal and hematopoietic stem cells (Ivanova et al., 2002; Ramalho-Santos et al., 2002
). These transcribed genes may characterize the stemness of the different types of murine stem cells. We wished to examine whether orthologs of these mouse genes are also enriched in undifferentiated human ES cells, and whether our selected genes appear among them. Although the annotation of probe sets now allows a fairly comprehensive comparison, there are technical limitations (resulting from the construction of different probe sets in the two DNA chips), which prevent us from drawing definitive conclusions. Nonetheless, this analysis suggests that genes implicated in mouse stemness of embryonic and adult cells differ from the set of genes we have identified as enriched in undifferentiated human ES cells. We suggest that these two sets of genes may complement one another. In the present study we compared the profile of gene expression in ES cells to that of EBs, their immediate differentiated cell derivatives. These EBs are composed of mature stem cells, progenitor cells and fully differentiated cells. Thus, we aim to identify genes specific to the pluripotent state of human ES cells. This is in contrast to previous studies searching for "stemness genes" in which genes unique, but common, to different types of stem cells (embryonic and adult) were identified. It would be interesting to evaluate in the future the hierarchy and inter-relationship between these two sets of genes.
Human EB differentiation as a model for early development
It is possible to trigger differentiation of ES cells in vitro by growth in suspension culture, resulting in formation of EBs. The EBs, which contain mesodermal, ectodermal and endodermal cells, undergo spontaneous differentiation. This is accompanied by morphological alterations which, similar to mouse, include cavitation (beginning by day 5) and continuous expansion, resulting in a fluid-filled cystic EB (by 2 weeks) (Itskovitz-Eldor et al., 2000). It was proposed that expanding EBs mimic, to some extent, early embryonic development. Indeed, in mice it has been shown that some temporal and spatial relationships between developmentally regulated genes which exist in vivo, are recapitulated in vitro (Leahy et al., 1999
). Thus, we wished to use DNA micro-array analysis in order to characterize and follow the differentiation program of growing human EBs. A detailed analysis shows that in developing EBs some expressed genes are temporally expressed and may be classified into three distinct groups: early, mid and late expressed genes.
A more refined analysis shows that these three groups (ES, early/mid EBs and late EBs) may be further subdivided into: early, early-mid, mid, mid-late, and late expressed genes. Perhaps these subgroups represent sequential stages in embryo development: (i) blastocyst and ICM specific genes, (ii) ICM and primitive ectoderm, (iii) gastrulation, (iv) early organogenesis and (v) late organogenesis. Indeed, LECTIN (galactoside binding, soluble 1) may be a marker for ICM cells; OCT4 is known to be expressed in the mouse in the ICM and the primitive ectoderm (Pelton et al., 2002); LEFTY A is a marker for gastrulation (Hamada et al., 2002
);
-FETOPROTEIN is expressed in early organogenesis (Gillespie and Uversky, 2000
); and SURFACTANT-D is expressed in late organogenesis (Crouch, 1998
) (Figure 4B). Furthermore, such analysis may allow the isolation of new developmentally regulated genes and could serve as an in vitro model for studying aspects of human gastrulation and organogenesis. Nevertheless, since differentiation in EBs is largely disorganized, it remains impractical to study pattern formation in vitro.
Finally, we demonstrate that it is possible to follow differentiation by studying the temporal expression pattern of a cascade of genes that are involved in a given pathway but are active in succession. As an attempt to determine how well in vitro differentiation of EBs correlates with early embryogenesis, we have studied the expression pattern of the Nodal signaling pathway, which holds a major role in the determination of embryonic axes. In the mouse, Nodal induces the expression of Lefty A and Lefty B, which restrict its expression and its downstream target, PITX2, to the left side of the embryo by acting as midline barriers and feedback inhibitors (Hamada et al., 2002). By comparing the expression level of NODAL, LEFTY A, LEFTY B and PITX2, in early, mid and fully matured human EBs (2, 10 and 30-day old EBs), we were able to demonstrate their transient expression at different time courses during EB formation, and recover the molecular pathway at the cellular level. These results support the impression that human EBs may model, at least to some extent, early human embryogenesis and encourages us to believe that large-scale cDNA comparisons can provide new insights into the stages of normal human embryo development, which are otherwise inaccessible for research.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 5, 2004; resubmitted on June 7, 2004; accepted on August 26, 2004.