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Human Molecular Genetics Advance Access originally published online on November 30, 2005
Human Molecular Genetics 2006 15(1):65-75; doi:10.1093/hmg/ddi427
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Temporal and parental-specific expression of imprinted genes in a newly derived Chinese human embryonic stem cell line and embryoid bodies

Bo Wen Sun1,2, A. Cong Yang1,2, Yun Feng3, Yi Juan Sun3, Yu fei Zhu1, Yi Zhang1, Hua Jiang1, Chun Liang Li1, Fu Rong Gao1, Zhi Hong Zhang1, Wei Cheng Wang1,2, Xiang Yin Kong1, Gang Jin1, Shi Jun Fu1 and Ying Jin1,2,*

1Institute of Health Science, Shanghai JiaoTong University School of Medicine and Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai 200025, China, 2Department of Molecular Developmental Biology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China and 3Reproductive Medical Center, Ruijin Hospital, Shanghai, China

* To whom correspondence should be addressed at: Institute of Health Science, Room 607, Building 1, 225 South Chongqing Road, Shanghai 200025, China. Tel/Fax: +86 2163852591; Email: yjin{at}sibs.ac.cn

Received September 22, 2005; Accepted November 12, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Although the study of imprinted genes in human development is very important, little is known about their expression and regulation in the early differentiation of human tissues due to lack of an appropriate model. In this study, a Chinese human embryonic stem (hES) cell line, SHhES1, was derived and fully characterized. Expression profiles of human imprinted genes were determined by Affymetrix Oligo micro-array in undifferentiated SHhES1 cells and SHhES1-derived embryoid bodies (EBs) at day 3, 8, 13 and 18. Thirty-two known human imprinted genes were detected in undifferentiated ES cells. Significantly, differential expression was found in nine genes at different stages of EB formation. Expression profile changes were confirmed by quantitative real-time reverse transcriptase–polymerase chain reaction in SHhES1 cells as well as in another independently derived hES cell line, HUES-7. In addition, the monoallelic expressions of four imprinted genes were examined in three different passages of undifferentiated ES cells and EBs of both hES cell lines. The monoallelic expressions of imprinted genes, H19, PEG10, NDNL1 and KCNQ1 were maintained in both undifferentiated hES cells and derived EBs. More importantly, with the availability of maternal peripheral blood lymphocyte sample, we demonstrated that the maternal expression of KCNQ1 and the paternal expression of NDNL1 and PEG10 were maintained in SHhES1 cells. These data provide the first demonstration that the parental-specific expression of imprinted genes is stable in EBs after extensive differentiation, also indicating that in vitro fertilization protocol does not disrupt the parental monoallelic expression of the imprinted genes examined.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Imprinting is an epigenetic phenomenon that gives rise to differential expression of paternally and maternally inherited alleles of certain genes. It is established afresh in the germ line in each generation and stably inherited throughout the somatic cell division (1Go–3Go). Although imprinted genes comprise a small subset of the human genome, they have been shown to be essential to fetal, placental and behavioral development (4Go). Disruption of allele-specific expression of imprinted genes is associated with human genetic diseases, progression of certain cancers and a number of neurological disorders (5Go–7Go). Although imprinted genes play important roles in early human development, a comprehensive analysis of expression profiles of imprinted genes during early development has never been conducted in humans. Studies of imprinted genes in humans have been limited primarily to delineating the association of aberrant expression of a few imprinted genes with a number of diseases and cancers (1Go,8Go). Many questions regarding expression and regulation of imprinted genes in humans remain unanswered.

Experimental evidence in mice has established links between in vitro embryo culture and the disrupted expression of imprinted genes (9Go–11Go). However, a direct relationship between in vitro human embryonic development and the expression status of imprinted genes has not been established. Recently, safety questions have been raised when using assisted reproductive technology (ART) (12Go,13Go). Embryo manipulation in vitro, maturation of oocytes, ovarian hyper-stimulation and use of subfertile sperm might disturb the process of genomic imprinting and lead to related diseases. Therefore, there is a critical need for a better understanding of genomic imprinting in human embryogenesis as well as for a more rigorous monitoring expression of imprinted genes in conceptions resulting from ART.

The scarcity of human embryos available for research has severely hampered the study of imprinting regulation during the early stages of development. However, this problem can be circumvented to some extent using human embryonic stem (hES) cell lines derived from the inner cell mass (ICM) of human pre-implantation embryos (14Go,15Go). ES cells are the in vitro counterparts of blastocyst ICM cells. Their in vitro differentiation to embryoid bodies (EBs) mimics events that occur in vivo shortly before and after the embryonic implantation (16Go,17Go). It has been demonstrated that human EBs comprise the cells of the three embryonic germ layers and express embryonic-specific genes in a stage-specific manner (18Go,19Go). Importantly, ES cells are derived from pre-implantation embryos at a time when gametic methylation imprints must be retained, while most of the remainder of the genome is being stripped of its methylation (3Go). Therefore, the analysis of mRNA levels and parental-specific expression of imprinted genes during in vitro differentiation of hES cells could provide clues vis-á-vis the post-fertilization epigenetic events necessary for the establishment and maintenance of monoallelic expression during very early stages of development, for which, to date, there is no information. Furthermore, hES cell lines derived from surplus human blastocysts from clinical in vitro fertilization (IVF) could provide a cell model to evaluate the effects of (i) manipulation of embryos, (ii) cryopreservation and/or (iii) different culture media on imprinting gene expression. More importantly, hES cells are able to self-renew indefinitely in vitro and have the capacity to give rise to differentiated progeny representative of all three embryonic germ layers (20Go–24Go). Thus, these cells represent a potentially unlimited resource for cell replacement therapies in the treatment of human diseases. Recently, as a step towards using hES cells in the treatment of human diseases, patient-specific hES cell lines have been successfully established (25Go). For transplantation purposes, it is very important to monitor and maintain genetic and epigenetic stabilities in hES, especially after long periods of culture and differentiation. Studies in mouse ES cells show that stem cell-derived tissues and embryos cloned from ES cell nuclei often fail to maintain the epigenetic states of imprinted genes (26Go–28Go). The presence of cytogenetic abnormalities within hES cell culture has been reported in several laboratories (3Go,24Go,29Go,30Go). However, much less is known about the expression profile and the epigenetic status of imprinted genes in hES cell lines following extended culture and upon differentiation.

The aim of this study was to analyze temporal and parental-specific expression profiles of imprinted genes in hES cells in their development into human EBs. To this end, a novel Chinese hES cell line, SHhES1, was derived and a large-scale transcription analysis of imprinted genes in hES cells at different stages during their differentiation in vitro was carried out by Affymetrix Oligo micro-array GeneChip as an initial step towards (i) understanding the regulation of imprinted gene expression in early human development and (ii) estimating the risks of imprinting alteration after ART. We examined the monoallelic expressions of four imprinted genes in SHhES1 cells and of two imprinted genes in another independently derived hES cell line, HUES-7 (31Go), at different passages in the undifferentiated state and after extensive differentiation. More importantly, with the availability of maternal peripheral blood lymphocyte (PBL) samples, we are the first to demonstrate that parental-specific expression of three imprinted genes is maintained in SHhES1, indicating that manipulation of human embryos in vitro does not interfere with the establishment and maintenance of these imprinted genes. Our study established an in vitro model to investigate the imprinted genes in hES cells during cell proliferation and differentiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Derivation and characterization of hES cell line (SHhES1)
One hES cell line (SHhES1) was established using 33 surplus human blastocysts from clinical IVF on a mitotically inactivated mouse embryonic fibroblast feeder layer. After the zona pellucida was digested, the trophectoderm was removed from the ICM either by immunosurgery or by mechanical dissociation. A culture of the whole blastocyst was attempted without removal of the trophectoderm layer by placing the embryos on a fibroblast feeder layer. The primary blastocyst culture was successful in 10 of 33 cases. However, the cells displayed different growth capacities after mechanical splitting and only one line (SHhES1 which was derived from whole blastocyst culture) continued to proliferate. SHhES1 cells grew continuously more than 70 passages and survived freeze/thaw cycles. These cells have typical hES cell morphology, i.e. high nuclear-to-cytoplasm ratios, prominent nucleoli and distinct boundaries between cells (Fig. 1A and B). Similar to other established hES cell lines, SHhES1 cells exhibited high telomerase activities measured at passages 20 and 48, respectively (Fig. 1C). Karyotype analysis, carried out at passages 15 and 23, indicated a normal karyotype (46, XX) (Fig. 1D).



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  		Figure 1. Derivation and characterization of the SHhES1 cell line. (A) Colonies of hES cells; (B) higher magnification of a SHhES1 cell colony; (C) telomerase activities of SHhES1 cells, lane 1: negative control, lane 2: feeder layer, lane 3: positive control, lane 4: heated positive control, lane 5: SHhES1 cell sample and lane 6: heated SHhES1 cell sample. Solid arrow indicates 36 bp internal control band. Open arrow is the 50 nt band; (D) a normal chromosomal karyotype of SHhES1 cells.

 
Immunochemistry was used to categorize molecular markers characteristic of undifferentiated SHhES1 cells. The cells expressed high levels of alkaline phosphatase (AKP), TRA-1-81, TRA-1-60, SSEA-4 and SSEA-3 (Fig. 2A–G). The transcription factor OCT-4 was also highly expressed. However, SSEA-1 expression was undetectable. The reverse transcriptase–polymerase chain reaction (RT–PCR), used to probe for hES cell Class I molecular markers (32Go), revealed expression of all eight markers, including Thy-cell surface antigen, LEFTY A, OCT-4, SOX2, FGF4, TDGF1, REX-1 and NANOG (Fig. 2H).



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  		Figure 2. Expression of molecular markers in undifferentiated SHhES1 cells. (A) AKP; (B) TRA-1-81; (C) TRA-1-60; (D) SSEA-4; (E) SSEA-1; (F) SSEA-3; (G) OCT-4 and (H) representative RT–PCR result of expression of undifferentiated marker genes.

 
In vitro differentiation
Once removed from its feeder layer and cultured in suspension without basic fibroblast growth factor (bFGF), SHhES1 cells formed EBs, including simple and cystic EBs. In a few cases, a rhythmic beat was observed. The beat rate was 50±2 b.p.m. and lasted for ~1 month. The cells in the cystic EBs differentiated into various cell types after they became attached. Immunocytochemical staining was performed to assay the developmental potential of SHhES1 cells (Supplementary Material, Fig. S1A–D). Representative markers for the three germ layers were found, including neurofilament 70 and nestin (ectoderm), muscle actin (mesoderm) and cytokeratin 7 (endoderm). To further determine whether EBs derived from SHhES1 cells can serve as models for the study of early differentiation in development, mRNA from undifferentiated SHhES1 cells and from 3-, 8- and 13-day-old EBs were extracted and expression of known molecular markers for undifferentiated ES cells and three germ layers examined by quantitative real-time RT–PCR. As shown in Figure 3, mRNA level of OCT-4 was reduced significantly when ES cells differentiated into EBs. However, mRNA levels of marker genes for the three germ layers, including GATA-2 and KDR for mesoderm, AFP and TTR for endoderm, NCAM1 for ectoderm and MSX2 for trophoblast, were all increased significantly. These results indicate that SHhES1 cells have a pluripotent developmental potential in vitro and that differentiated EBs could be a useful model to study the gene regulation in early human development.



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  		Figure 3. Real-time quantitative RT–PCR analysis of marker gene expression in SHhES1 cells. Y-value is expressed as relative fold change in mRNA levels at different days of EB formation when compared with mRNA level of undifferentiated ES cells defined as 1.

 
In vivo differentiation
Undifferentiated SHhES1 cells were injected into the left hind limb muscle of SCID-beige mice. Teratomas formed 3 months later. Histochemical staining of the teratomas revealed cells and tissues from three germ layers (Fig. 4), including skin (ectoderm), fat, smooth muscle and cartilage (mesoderm), glandular tissues and gut epithelium (endoderm). The result demonstrates that the established SHhES1 cells are pluripotent in vivo.



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  		Figure 4. Histochemical staining of various tissues from SHhES1 cell-derived teratomas. (A) Ciliated columnar epithelium; (B) cartilage; (C) fat tissue; (D) smooth muscle; (E) skin and (F) glandular tissues.

 
Expression profile of imprinted genes in undifferentiated hES cells and EBs
In order to identify the imprinted genes expressed in undifferentiated hES cells and their differentiated progeny, total RNA was extracted from SHhES1 cells at passages 25, 38 and 40 and from cells of 3-, 8-, 13- and 18-day-old EBs, respectively. Gene expression pattern in undifferentiated SHhES1 cells was profiled using the Affymetrix Human Genome U133A plus 2.0 GeneChip, which included 22 283 probe sets. A total of 15 266 probe sets with at least one ‘present’ call were collected. Among these, 32 are known imprinted genes (Table 1). To determine temporal expression pattern of imprinted genes following hES cell differentiation in vitro, a pair-wise comparison of mRNA levels between undifferentiated ES cells and EBs of different stages (EBs at days 3, 8, 13 and 18, three independent experiments each) was made. Data were filtered by keeping the significance level at P<0.05 and maintaining 2-fold changes in at least one of the five stages. This strategy left nine imprinted genes, which varied significantly (Fig. 5A). These genes included up-regulated genes (IGF2, DCN, GNAS, PLAGL1 and CDKN1C) and down-regulated genes (GABRB3, IPW, PAWR and SNRPN). The remaining 23 imprinted genes, including MAGEL2/NDNL1 and IGF2R, did not change significantly during the differentiation process.


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  		Table 1. Human imprinted genes identified by GeneChip
 


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  		Figure 5. Determination of expression level of imprinted genes by GeneChip and real-time quantitative RT–PCR. (A) Clustering analysis of imprinted genes detected by GeneChip in undifferentiated SHhES1 cells and after EB formation for different days. (B) Real-time quantitative RT–PCR analysis for imprinted genes in SHhES1 cells. Y-value is expressed as relative fold change in mRNA levels at different stages of EB formation compared with that of undifferentiated ES cells defined as 1. Experiments were performed with two independently collected samples. The representative data were shown. (C) Real-time RT–PCR analysis for imprinted genes in HUES-7 cells same as in (B).

 
To further confirm the expression pattern of imprinted genes following differentiation detected by GeneChip, the temporal expression of some of the imprinted genes was quantitatively determined by real-time RT–PCR. As shown in Figure 5B, mRNA levels of IGF2, DCN and GNAS did, indeed, increase significantly with the formation of EBs. These data were consistent with those from GeneChip. In addition, expressions of PEG10 and H19, which were not detected in GeneChip, were also examined by real-time RT–PCR. Expression of H19 was dramatically up-regulated with differentiation of hES cells. Moreover, in order to test whether the differential expression of these imprinted genes detected during SHhES1 cell differentiation applies to other hES cell lines, the same experiment was performed with another independently derived hES cell line, HUES-7. As shown in Figure 5C, the expression patterns of imprinted genes were similar to those of SHhES1 cells. These results suggest that expression of IGF2, DCN, GABRB3, H19 and SNRPN was developmentally regulated.

Parental-specific or monoallelic expression of imprinted genes
Human chromosome 11p15 carries at least seven imprinted genes, including IPL, ORCTL2, KCNQ1, ASCL2, IGF2, CDKN1C and H19 (33Go). The Beckwith–Wiedemann syndrome (BWS), a somatic overgrowth disorder associated with an increased incidence of embryonal tumors, results from the aberrant expression of one or more of these imprinted loci. Therefore, the expression status of each known imprinted gene in this region was first examined. However, transcribed polymorphisms were only identified in KCNQ1 and H19. Sequencing of the cDNA product of the SHhES1 cells at passages 24, 33 and 47 showed monoallelic expression patterns in both H19 and KCNQ1 (Fig. 6A). The parental allele-specific gene expression was then characterized by direct sequencing of the RT–PCR products of the mother PBL sample. Only KCNQ1 expression was detected in mother PBLs, and direct sequencing showed maternal expression in all three samples chosen for this assay. Stable monoallelic expression of H19 was also found in cells from these three passages. Next, polymorphisms in the transcribed region for paternally expressed imprinted genes NDNL1 on chromosome 15 and PEG10 on chromosome 7q21 in SHhES1 cells were sought. Sequencing of the cDNA products of these two genes showed that monoallelic expressions of NDNL1 and PEG10 were maintained in all three passages examined. In addition, homozygous sequences of NDNL1 and PEG10 detected in maternal genomic DNA were different from those found in cDNA of SHhES1 cells, indicating that the expressed allele was paternal in origin (Fig. 6A). As a negative control, both genomic DNA and cDNA from feeder layer cells were isolated and sequencing performed to probe for these human imprinted genes. None was detected (data not shown). Furthermore, the biallelic expression pattern of a known non-imprinted gene, OSBPL5, located on chromosome 11p15, was found at passages 25 and 62, respectively (Fig. 6B). Next, in order to test whether the monoallelic expression of imprinted genes was maintained in other hES cell lines, genomic DNA and RNA were extracted from hES cells of the HUES-7 line. Direct sequencing of RT–PCR products containing heterozygous polymorphisms showed strict monoallelic expressions for maternally expressed H19 and paternally expressed PEG10 in samples from all three different passages of HUES-7 (Fig. 6C). This result indicates that monoallelic expression of these imprinted genes was maintained in both early and late passages in both hES cell lines. Lastly, whether differentiation affected the expression status of these imprinted genes was determined. Sequencing of the genomic DNA and cDNA products from cells of 28-day-old EBs derived from SHhES1 cells showed the same monoallelic expression patterns for H19, KCNQ1, PEG10, NDNL1 (Fig. 6D) and for H19 and PEG10 from cells of 18-day-old EBs of HUES-7 line (Fig. 6E), as did undifferentiated ES cells. Thus, the parental-specific expressions of KCNQ1, PEG10 and NDNL1 and monoallelic expression of H19 were maintained in both undifferentiated and differentiated SHhES1 cells. Furthermore, the monoallelic expressions of H19 and PEG10 were demonstrated in both undifferentiated and differentiated HUES-7 cells.



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  		Figure 6. Monoallelic expression of imprinted genes in SHhES1 and HUES-7 cells. (A) Sequencing of KCNQ1, H19, NDNL1 and PEG10 genes of genomic DNA and cDNA from mother PBL and SHhES1 cells; (B) sequencing of control gene, OSBPL5, for SHhES1 cells; (C) sequencing of H19 and PEG10 genes of genomic DNA and cDNA of HUES-7 cells; (D) sequencing of DNA from the differentiated SHhES1 cells and (E) sequencing of DNA from the differentiated HUES-7 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
This research establishes a new hES cell line, designated SHhES1, which fulfills the standards for hES cells, including (a) derivation from the early human embryo, (b) karyotypically normal, (c) proliferation for long periods of time in the undifferentiated state (more than 70 passages), (d) recovery after freezing and thawing and (e) differentiation into a variety of cell types in vitro and in vivo. In addition, the cells express the known Classes I and II molecular markers (32Go) for undifferentiated hES cells and have high telomerase activity. Importantly, the cell line maintains a stable parental-specific monoallelic expression of imprinted genes after prolonged culture and differentiation. The establishment of the SHhES1 cell line increases the availability of the hES cell line, which is vital for (i) facilitating basic research in human development, (ii) ART and (iii) therapeutic use of the cells for tissue replacement.

During development, genomic imprinting is erased in the primordial germ cells, reestablished during gametogensis and maintained in pre- or post-implantation embryonic development. Improper regulation and expression of imprinted genes can lead to human genetic disorders and may also be involved in carcinogenesis. For example, the imprinting domain on human chromosome 15q11–13 contains a large cluster of imprinted genes, including paternally expressed MAGEL2/NDNL1, NECDIN/NDN, SNRPN, PAR-5 and IPW and maternally expressed UBE3A and ATP10C. Improper regulation of imprinted genes in this cluster results in two independent imprinting disorders, the Prader-Willi (PWS) and Angelman syndromes, both of which exhibit a spectrum of developmental, neurological and behavioral phenotypes (8Go). Expression of different imprinted genes varies temporally and spatially (9Go). The analysis of these processes in humans has been limited by the scarcity of human embryos available for such study. The present study demonstrates that hES cells and EBs offer a powerful tool for studying the expression of imprinted genes shortly before and after the embryonic implantation. We have surveyed a large number of imprinted genes for temporal and monoallelic expression in undifferentiated SHhES1 cells and differentiated EBs in simple (3-day-old EBs), cavitated (8-day-old EBs) and cystic stages (13- and 18-day-old EBs). Using GeneChip, we detected the expression of 32 known human imprinted genes in undifferentiated SHhES1 cells. Abeyta et al. (34Go) have reported the transcriptional profiles of three independently derived hES cell lines, HSF-1, HSF-6 and H9 lines. In order to examine the similarity and difference in expression of imprinted genes in multiple hES cell lines, we analyzed expression profile of imprinted genes in these three lines and found 19, 23 and 22 known human imprinted genes expressed in HSF-1, HSF-6 and H9 hES cell lines, respectively. Comparison of expressed imprinted genes in these lines showed that 15 (15/19, 79%), 20 (20/23, 87%) and 19 (19/22, 86%) genes in HSF-1, HSF-6 and H9 lines, respectively, were common to the imprinted genes expressed in SHhES1 cell line. Of note, 19 imprinted genes were expressed in all three hES cell lines reported by Abeyta et al., whereas only 15 imprinted genes were common in all four hES cell lines. A possible explanation for more imprinted genes detected in the present study than that in Abeyta et al.'s data could be a less stringent criterion for collecting the original data in our study. More importantly, expression profiles could be influenced by differences in genetic background between embryos used for derivation of hES cells and by culture conditions employed by different laboratories. Nevertheless, the analysis indicates that the majority of imprinted genes expressed in HSF-1, HSF-6 and H9 cell lines overlap with those expressed in the SHhES1 cell line. Recently, Luedi et al. made a genome-wide prediction of imprinted murine genes by applying a statistical model based on DNA sequence characteristics (35Go). Of 23 788 annotated autosomal mouse genes, their model identified 600 (2.5%) genes to be potentially imprinted. Furthermore, the authors presented a set of human genes whose mouse homologs are predicted to be imprinted, which includes 27 additional potentially imprinted human genes. We analyzed the expression of 24 genes, out of 27, in four hES cell lines as mentioned earlier. Interestingly, 11 genes were expressed in SHhES1 cells. 8, 7 and 6 genes were detected in HSF-1, HSF-6 and H9 cell lines, respectively. In addition, 7 (7/8), 7 (7/7) and 4 (4/6) genes detected in HSF-1, HSF-6 and H9 cell lines were common to the genes detected in SHhES1 cells. Taken together, the expression profile of imprinted genes is similar in the four independently derived hES cell lines, although some of the imprinted genes are expressed uniquely in each cell line. Among 32 imprinted genes detected in undifferentiated SHhES1 cells, nine genes were expressed differentially after EB formation. Of note, IGF2, DCN and H19 were most significantly up-regulated during hES cell differentiation and EB formation. Paternally expressed insulin-like growth factor-2 (IGF2) and maternally transcribed H19 genes are closely linked on human chromosome 11. They have similar expression patterns during fetal development and both are involved in BWS, but appear to have opposite effects on growth (16Go,36Go,37Go). In the present study, the elevated expression of IGF2 and H19 during hES cell differentiation suggests that they play an important role at this stage of differentiation. Remarkably, the expression patterns of imprinted genes during differentiation from two independently derived hES cell lines were comparable, suggesting that the expression profile observed in our hES cells is applicable to other hES cell lines. This study can serve as a basic catalog of imprinted genes expressed in hES cells and during differentiation in vitro.

Monoallelic expression status of imprinted genes in hES cells after extended culture in vitro, especially after their differentiation, has remained obscure, although several studies suggest that imprinting is unstable in mouse ES cells (27Go,38Go). The deregulation of imprinted gene expression in ES cells may affect their developmental potential and give rise to aberrant phenotypes in ES cell-derived progenies. It is believed that disrupted imprinting in ES cells could result in an ES-derived fetus with abnormalities such as increased size and mass (26Go). Therefore, it is critical to examine the epigenetic stability of hES cells for their usefulness in transplantation. The results from this study show that all four imprinted genes examined, H19, KCNQ1, NDNL1 and PEG10, were monoallelically expressed in SHhES1 cells from all three selected passages. This result is in agreement with the experimental data from Rugg-Gunn et al. who recently reported monoallelic expression of six imprinted genes, including IGF2, IPW, KCNQ1OT1, H19, SLC22A18, NESP55 and TSSC4, along with the normal methylation patterns of three imprinting control regions in hES cells (39Go). The present study demonstrates stable monoallelic expression of additional imprinted genes in two independently derived hES cell lines. Furthermore, the analysis is extended by assessing the imprinting status in EBs that recapitulate early human development. These observations indicate that the epigenetic status of hES cells is undisturbed during prolonged culture and extensive differentiation. Most significantly, thanks to the availability of mother PBLs, the parental origin per se, which was not included in the study by Rugg-Gunn et al. (39Go), was determined in the present study. The observations demonstrate that the maternal expression of KCNQ1 gene and paternal expression of NDNL1 and PEG10 genes are established in human development at a stage when hES cells are being derived and remain stable during a long period of in vitro culture and after extensive differentiation.

The present study provides the first demonstration of the stability of parental-specific expression of imprinted genes in EBs after extensive differentiation. More importantly, the stable parental-specific expression of imprinted genes in these SHhES1 cells indicates that IVF and culture of early human embryos to the blastocyst stage do not disrupt the establishment and maintenance of imprinting, at least for the KCNQ1, PEG10 and NDNL1 genes examined in this study. The stable monoallelic expression of imprinting genes in hES cells from this study and that of Rugg-Gunn et al. contrasts epigenetic instability in mouse ES cells. The maintenance of allelic expression of imprinted genes in hES cells after prolonged culture and differentiation suggests that epigenetic regulation for imprinted genes is unique in humans and that imprinting may not be a major epigenetic obstacle for human stem cell transplantation. The difference in epigenetic stability between human and mouse could reflect the different time windows for both global and gene-specific methylation during early development between the two species. It has been documented by Onyango et al. (40Go) that three imprinted genes, TSSCS, H19 and SNRPN, showed monoallelic expression during in vitro differentiation in human embryonic germ cells. However, IGF2 and H19 had an abnormal imprinting pattern after differentiation in mouse embryonic germ cells. The authors stated that this relative stability may reflect differences between human and mouse cells. Also, the difference in epigenetic stability between human and mouse might stem from the differences in media and conditions used for the culture of embryos and ES cells. It is known that pre-implantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes (41Go). Although the precise mechanisms for epigenetic stability in hES cells remain obscure, the present study provides an overview of the temporal expression of human imprinted genes in hES cells and demonstrates that hES cells and EBs can serve as a useful model to (i) understand the epigenetic reprogramming in early development and (ii) monitor the procedures in both ART and somatic cell nuclear transfer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Derivation and culture of SHhES1 cell line
Surplus human embryos from clinical IVF for medically assisted induction of pregnancy were used. Human embryos, acquired after obtaining informed consent from patients and upon the approval from the Ethical Review Board of Ruijin Hospital, were employed for hES cell derivation. To isolate ICM without risking cell loss, embryos were treated with pronase (Sigma) to remove the zona pellucida and then placed onto mitomycin-C-treated mouse feeder layers. Within 9–10 days of plating, the central part of the cell clump was isolated mechanically and subcultured onto a fresh feeder layer. Basic culture medium for hES cell maintenance consisted of knockout-Dulbecco's modified Eagle's medium (Gibco) supplemented with 20% serum replacement (Invitrogen), 1 mM glutamine, 0.1 mM ß-mercaptoethanol, 0.1 mM non-essential amino acid, 50 U/ml penicillin plus 50 µg/ml streptomycin and 4 ng/ml bFGF (Invitrogen). For the initial culture of ICM cells, 20% fetal bovine serum (FBS, Hyclone) in the basic hES cell culture medium was used. During the early stage of hES cell cultivation, hES cell colonies were mechanically cut into pieces and transferred to a new culture plate with fresh feeder layers. When a four-well culture plate was replete with hES cell colonies, cells were passaged with 0.05% trypsin–EDTA (Invitrogen) for every 4–5 days. The SHhES1 cells were frozen in medium containing 90% FBS and 10% dimethylsulfoxide at a 1:8 split from a subconfluent 10 cm tissue culture dish.

Immunocytochemistry
Expression of stem cell markers was examined by immunocytochemistry using the ES cell characterization kit (Chemicon) as recommended by the manufacturer. The primary antibodies used were stage-specific embryonic antigens (SSEAs) -1, -4 and -3 and tumor recognition antigens (TRAs) -1–60 and -1–81 (1:20). AKP activity was detected using the Vector Blue Substrate kit (Vector laboratories).

RT–PCR analysis
The RT–PCR was performed to monitor the expression levels of genes from undifferentiated cells. Total RNA was isolated with TRIzol (Invitrogen) and transcribed into cDNA using oligo (dT)15 (Promega) and M-MLV RT (Promega). The primers used are listed in Supplementary Material, Table S1. PCR products were separated on a 2% agarose gel, stained with ethidium bromide, visualized and photographed on a UV transluminator.

Karyotype analysis
Karyotype analysis was carried out on the cells after passages 15 and 23. SHhES1 cell colonies were incubated with 0.05 µg/ml demecolcine (Sigma) for 3 h. Cells were then trypsinized, pelleted by centrifugation and resuspended in 0.075 M KCl for 30 min at 37°C. After being treated with hypotonic solution, cells were fixed in methanol:acetic acid (3 : 1) for 10 min at room temperature and dropped onto pre-cleaned slides. Chromosome spreads were Giemsa-banded and photographed. Approximately 20 metaphase spreads were counted and five metaphases analyzed for chromosomal rearrangements.

Telomerase activity
Telomerase activity of SHhES1 cells was measured using the TRAPeze Telomerase detection kit (Chemicon), according to the manufacturer's instructions.

In vitro differentiation
For the generation of EBs, undifferentiated SHhES1 cells were detached by treatment with collagenase IV (5 mg/ml, Invitrogen) for ~30 min at 37oC. EBs were formed from suspension cultures of hES cells in bacterial culture dishes, resulting in spontaneous differentiation, with hES cell culture medium (without bFGF) and with 20% FBS for ~15 days. The EBs thus formed were transferred to four-well plates and cultured for ~2–4 weeks before immunostaining. Differentiated cells were fixed in 4% paraformaldehyde (Sigma) and stained with antibodies against muscle actin (DAKO, 1 : 50), cytokeratin-7 (DAKO, 1 : 50), neurofilament-70 (Chemicon, 1 : 50) and nestin (Chemicon, 1 : 200).

Teratoma formation in vivo
All cells from a 10 cm dish were injected into the left rear leg muscle of 4-week-old male SCID-beige mice. An identical injection into the same muscle was administered 1 week later. Three months post-injections, the resulting teratoma was examined histologically using standard protocol.

GeneChip target preparation, hybridization and washing
Affymetrix Human Genome U133A gene chips were used for this study. All gene-chip experiments were performed and analyzed at the in-house Microarray Core of the Institute of Health Science. Three biological experiments of each time course were hybridized onto separate chips for statistical analyses. Hybridization probes were prepared according to the Affymetrix Technical Manual. The hybridization mixture was first heated at 99°C for 5 min, then at 45°C for 5 min and then centrifuged at 13 000g for 5 min. Gene chips were pre-hybridized with 200 µl of 1x hybridization buffer for 10 min at 45°C at 60 r.p.m. in the hybridization oven. Following removal of the pre-hybridization buffer, the gene chips were filled with 200 µl of the hybridization mixture and incubated for 16 h at 45°C at 60 r.p.m. The hybridization mixture was removed and stored at –70°C. Each chip was filled with 250 µl of non-stringent wash buffer (6x SSPE, 0.01% Tween-20). The signal was amplified by an additional treatment with goat IgG antibody (0.1 mg/ml) and with biotinylated antibody (3 µg/ml) and a second staining with SAPE. Chips were then scanned with an Affymetrix Scanner 3000 (Affymetrix, Santa Clara, CA, USA). The gene expression signal was collected using Affymetrix GCOS V1.1.1 software.

Quantitative RT–PCR
For real-time quantitative RT–PCR, two samples were collected at each time point. Primers were designed using Primer Express-2 software (Supplementary Material, Table S2). Real-time PCR was performed using water-blank negative controls, and each sample was analyzed in triplicate with GAPD as the inner control. The final PCR reaction volume of 10 µl contained 5 µl SYBR Green PCR Master Mix (ABI, CA, USA), 2 µl 1:4 diluted cDNA template and 3 µl primer mixture (final concentration, 250 nM of each primer). Thermal cycling was carried out with a 10 min denaturation step at 95°C, followed by 40 two-step cycles: 15 s at 95°C and 60 s at 60°C. Amplification data were collected by the ABI PRISM 7900 and analyzed by the Sequence Detection System 2.0 software (ABI).

Analysis of imprinted gene expression
Undifferentiated SHhES1and HUES-7 cells from three different passages and differentiated EBs from days 28 and 18, respectively, were used to analyze the imprinting status. Transcribed polymorphisms were identified by direct sequencing of sample genomic DNA. PCRs were carried out with the AmpliTaq Gold DNA Polymerase (Applied Biosystems). PCR analysis of the heterozygotes, performed to assess their imprinting status, was carried out as follows: initial denaturation at 95°C for 8 min, followed by 45 three-step cycles (95°C for 30 s, 58°C for 30 s and 72°C for 45 s) and a final cycle at 72°C for 5 min. Amplified DNA was purified using the PCR purification Kit (Watson) and sequenced using the BigDye Terminator Cycle Sequencing Kit and the ABI 3100 DNA analyzer (Applied Biosystems). Primers used are shown in Supplementary Material, Table S3.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Dr Melton for providing HUES-7 hES cells and Drs Peter Reinach and Kathryn Pokorny for their critical reading of the manuscript. The study was supported by Grants from Shanghai Science and Technology Developmental Foundations (Grant number: 03DJ14018 and No. 04DZ14006), and the National High Technology Research and Development Program of China (2000CB509900), Shanghai Jiaotong University School of Medicine and Shanghai Institutes for Biological Sciences, CAS.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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