High-Resolution Analysis of the Subtelomeric Regions of Human Embryonic Stem Cells
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《干细胞学杂志》
a Department of Clinical Genetics, Sahlgrenska University Hospital/East, G?teborg, Sweden;
b Cellartis AB, G?teborg, Sweden
Key Words. Human embryonic stem cells ? Chromosomes ? Multiplex ligation-dependent probe amplification analysis ? Subtelomeric regions
Correspondence: Peter Sartipy, Ph.D., Cellartis AB, Arvid Wallgrens Backe 20, 41346 G?teborg, Sweden. Telephone: 46-31-7580930; Fax: 46-31-7580910; e-mail: peter.sartipy@cellartis.com; and Catarina Darnfors, Ph.D., Department of Clinical Genetics, Sahlgrenska University Hospital/East, 416 #85 G?teborg, Sweden. Telephone: 46-31-3434123; Fax: 46-31-842160; e-mail: catarina.darnfors@vgregion.se
ABSTRACT
Human embryonic stem cells (hESCs) can be isolated from the inner cell mass of blastocysts and represent a population of pluripotent stem cells . These cells have the potential to develop into cell types representing the three embryonic germ layers in vitro and in vivo, and they are capable of unlimited, undifferentiated proliferation in vitro. Based on these fundamental properties, hESCs hold great promise to prove useful for future cell replacement therapies, but they are also likely to revolutionize the drug discovery process and provide novel possibilities for in vitro toxicology .
Several independent investigators have successfully established hESC lines using a variety of experimental protocols . In most studies, the hESC lines seemed to maintain their pluripotency and normal karyotypes during long-term culture in vitro. However, a recent study indicated that hESCs may be subject to genomic distortions . In support of this observation, an isodicentric X chromosome was detected in another hESC line after long-term culture in vitro . Furthermore, a moderate frequency of aneuploidy was observed in hESC lines subjected to long-term feeder-free culture, although none of these changes resulted in increased proliferation rate for the hESC, and no significant difference in aneuploidy frequency was observed between early- and late-passage cultures . Contrary to these reports, a separate study performed using six other hESC lines demonstrated that these cells were indeed chromosomally stable during extended in vitro culture . Based on the limited data available and the conflicting reports in the literature, it is at this point impossible to assess how common chromosomal alterations in hESCs are. Additional studies are needed to resolve this critical issue and also, more important, to establish the biological significance of any detected alteration.
The telomeric regions of the chromosomes consist of TG-rich repeats (TTAGGG)n, and adjacent to these are complex families of repetitive chromosome-specific DNA sequences. Next to these, along the chromosome, are very gene rich areas with high transcriptional and recombinational activities . The homology between subtelomeric regions of nonhomologous chromosomes may cause crosstalk between these regions during meiosis, which leads to subtelomeric rearrangements. Abnormalities in subtelomeric sequences in patients cause mental retardation and malformations , indicating that these aberrations have direct effects on the developmental potential of the affected cells. It is not yet understood whether deletions of all chromosome ends are associated with specific phenotypes besides mental retardation, but some end deletions cause recognizable syndromes .
In this study we used multiplex ligation-dependent probe amplification (MLPA) analysis to investigate the possible occurrence of subtelomeric deletions or duplications in eight different hESC lines and in one clonally derived hESC line. The recently developed MLPA method is based on the polymerase chain reaction (PCR) amplification of probe pairs that have been hybridized to genomic DNA and subsequently ligated. Presently 41 telomere regions can be screened in just one multiplex reaction . The hESCs were analyzed in early and late passages and after freeze/ thaw cycles to determine if in vitro manipulation of hESCs could result in subtelomeric deletions. We did not detect any deletions in any of the cell lines analyzed in this study. Taken together, these data support the conclusion that hESCs can be cultured in vitro for extended time periods while retaining genomic stability, which is a prerequisite for many downstream applications.
MATERIALS AND METHODS
All of the cell lines included in this study were established and cultured on MEF feeder layers using VitroHES medium, and the cells were dissociated and passaged by mechanical cutting every 4–5 days. The hESC lines have been extensively characterized, and they display the typical markers and properties of undifferentiated hESCs (Cellartis, unpublished results) and have been subjected to freeze/thaw cycles. Importantly, data from karyotyping and FISH analyses of hESC lines SA002, AS034, AS034.1, AS038, SA121, and SA181 were recently published and demonstrated that the cell lines present with normal karyotypes, except for SA002, which display trisomy 13 . The remaining three cell lines included in this study (SA001, SA240, and SA461) have also been analyzed using the same techniques; the cells display normal stable karyotypes, and no large-scale genomic aberrations have been detected (Fig. 1, Table 1).
Figure 1. Karyotype of human embryonic stem cell (hESC) line SA001. (A): Representative normal karyotype from hESC line SA001 (passage 26). (B): Signals obtained from fluorescent in situ hybridization analysis of hESC line SA001 (passage 32) using probes for chromosomes 13, 18, 21, X, and Y.
Table 1. Summary of the results from karyotyping and fluorescent in situ hybridization (FISH) analysis of human embryonic stem cell lines SA001, SA240, and SA461
For the purpose of this study, undifferentiated hESCs were harvested in early and late passages, as indicated in Table 2, and analyzed using MLPA. With two exceptions (see below), the MLPA telomere P019/P020 analyses did not detect any subtelomeric abnormalities in the hESCs. Each MLPA P019/P020 analysis was performed in duplicate. In some cases (Table 2), these analyses were also confirmed using a more recently developed complementary MLPA telomere P036 assay. Except for one probe, all sequences detected by probes in the P019/P020 probe mixes are different from the probes in the P036 mix. The significant number of individual hESC lines analyzed and the long-term in vitro culture of several of these lines support the conclusion that undifferentiated hESCs can be propagated extensively while maintaining genomic stability.
Table 2. Summary of the results from the multiplex ligation-dependent probe amplification (MLPA) telomere P019/P020 analyses of undifferentiated human embryonic stem cells (hESCs)
As indicated above, one hESC line included in this study, SA002, displays trisomy 13 . The MLPA analyses of this hESC line were performed using cells in passage 14 and 144. Without prior knowledge about the trisomy, the analyst detected this chromosome abnormality in hESC line SA002. Figure 2 illustrates the different peak patterns obtained from a normal XY hESC line (SA001, passage 49) (panel A) and from the trisomic XX hESC line SA002 (passage 144) (panel B) using the MLPA P020 probe combination that covers chromosomes 12–22 and Y. The peak area of each amplification product reflects the relative copy number of that target sequence, making the identification of deleted or duplicated sequences possible.
Figure 2. Representative peak patterns obtained from the multiplex ligation-dependent probe amplification telomere P020 analyses of undifferentiated human embryonic stem cells (hESCs). The P020 mix contains probes for chromosomes 12–22 and Y. (A): Normal XY hESC line (SA001, passage 49) with two specific Y peaks. (B): Trisomic XX hESC line SA002 (passage 144) in which all three chromosome 13–specific peaks show an approximate 1.5-fold increase and the Y specific peaks are absent. The arrows indicate the chromosome 13–specific peaks.
In another hESC line, SA121 (passages 73 and 117), an apparent 6p-deletion, was detected by MLPA analysis using the P019/P020 probe sets (Table 2, Fig. 3B). However, in subsequent analyses, this observed deletion could not be confirmed by FISH analysis using metaphase spreads from the same cell line (passage 81) using 6ptel and 6qtel probes (Fig. 4) nor by the MLPA telomere P036 assay using SA121 cells in passage 117 (Table 2). Taken together, these results indicate that the apparent 6p deletion in SA121 most likely represents a polymorphism at the binding site of the probe, a finding also reported by others (personal communication, Dr. J.P. Schouten, MRC-Holland).
Figure 3. Representative peak patterns obtained from the multiplex ligation-dependent probe amplification telomere P019 analyses of undifferentiated human embryonic stem cells (hESCs). The P019 mix contains probes for chromosomes 1-11 and X. (A): Normal XY hESC line (AS034, passage 11). (B): Apparent 6p-deletion observed in SA121 (passage 73). The arrows indicate the 6p-specific peaks.
Figure 4. Representative illustration of a fluorescent in situ hybridization analysis using 6p/6q subtelomere probes performed on meta-phase spreads prepared from undifferentiated human embryonic stem cells (SA121) in passage 81.
DISCUSSION
In this study, we have provided high-resolution experimental data supporting the conclusion that it is possible to maintain hESCs in culture for extended time periods without compromising their genomic integrity. Although these results are promising and provide confidence and support for the therapeutic potential of hESCs, additional studies are necessary to safely move hESC research from bench to bed.
ACKNOWLEDGMENTS
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
McNeish J. Embryonic stem cells in drug discovery. Nat Rev Drug Discov 2004;3:70–80.
Davila JC, Cezar GG, Thiede M et al. Use and application of stem cells in toxicology. Toxicol Sci 2004;79:214–223.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.
Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.
Heins N, Englund MC, Sjoblom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. STEM CELLS 2004;22:367–376.
Draper JS, Smith K, Gokhale P et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 2004;22:53–54.
Inzunza J, Sahlen S, Holmberg K et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol Hum Reprod 2004;10:461–466.
Rosler ES, Fisk GJ, Ares X et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 2004;229:259–274.
Buzzard JJ, Gough NM, Crook JM et al. Karyotype of human ES cells during extended culture. Nat Biotechnol 2004;22:381–382.
Saccone S, De Sario A, Della Valle G et al. The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes. Proc Natl Acad Sci U S A 1992;89:4913–4917.
Knight SJ, Regan R, Nicod A et al. Subtle chromosomal rearrangements in children with unexplained mental retardation. Lancet 1999;354:1676–1681.
De Vries BB, Winter R, Schinzel A et al. Telomeres: a diagnosis at the end of the chromosomes. J Med Genet 2003;40:385–398.
Schouten JP, McElgunn CJ, Waaijer R et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57.
Rooms L, Reyniers E, van Luijk R et al. Subtelomeric deletions detected in patients with idiopathic mental retardation using multiplex ligation-dependent probe amplification (MLPA). Hum Mutat 2004;23:17–21.(Catarina Darnforsa, Anna )
b Cellartis AB, G?teborg, Sweden
Key Words. Human embryonic stem cells ? Chromosomes ? Multiplex ligation-dependent probe amplification analysis ? Subtelomeric regions
Correspondence: Peter Sartipy, Ph.D., Cellartis AB, Arvid Wallgrens Backe 20, 41346 G?teborg, Sweden. Telephone: 46-31-7580930; Fax: 46-31-7580910; e-mail: peter.sartipy@cellartis.com; and Catarina Darnfors, Ph.D., Department of Clinical Genetics, Sahlgrenska University Hospital/East, 416 #85 G?teborg, Sweden. Telephone: 46-31-3434123; Fax: 46-31-842160; e-mail: catarina.darnfors@vgregion.se
ABSTRACT
Human embryonic stem cells (hESCs) can be isolated from the inner cell mass of blastocysts and represent a population of pluripotent stem cells . These cells have the potential to develop into cell types representing the three embryonic germ layers in vitro and in vivo, and they are capable of unlimited, undifferentiated proliferation in vitro. Based on these fundamental properties, hESCs hold great promise to prove useful for future cell replacement therapies, but they are also likely to revolutionize the drug discovery process and provide novel possibilities for in vitro toxicology .
Several independent investigators have successfully established hESC lines using a variety of experimental protocols . In most studies, the hESC lines seemed to maintain their pluripotency and normal karyotypes during long-term culture in vitro. However, a recent study indicated that hESCs may be subject to genomic distortions . In support of this observation, an isodicentric X chromosome was detected in another hESC line after long-term culture in vitro . Furthermore, a moderate frequency of aneuploidy was observed in hESC lines subjected to long-term feeder-free culture, although none of these changes resulted in increased proliferation rate for the hESC, and no significant difference in aneuploidy frequency was observed between early- and late-passage cultures . Contrary to these reports, a separate study performed using six other hESC lines demonstrated that these cells were indeed chromosomally stable during extended in vitro culture . Based on the limited data available and the conflicting reports in the literature, it is at this point impossible to assess how common chromosomal alterations in hESCs are. Additional studies are needed to resolve this critical issue and also, more important, to establish the biological significance of any detected alteration.
The telomeric regions of the chromosomes consist of TG-rich repeats (TTAGGG)n, and adjacent to these are complex families of repetitive chromosome-specific DNA sequences. Next to these, along the chromosome, are very gene rich areas with high transcriptional and recombinational activities . The homology between subtelomeric regions of nonhomologous chromosomes may cause crosstalk between these regions during meiosis, which leads to subtelomeric rearrangements. Abnormalities in subtelomeric sequences in patients cause mental retardation and malformations , indicating that these aberrations have direct effects on the developmental potential of the affected cells. It is not yet understood whether deletions of all chromosome ends are associated with specific phenotypes besides mental retardation, but some end deletions cause recognizable syndromes .
In this study we used multiplex ligation-dependent probe amplification (MLPA) analysis to investigate the possible occurrence of subtelomeric deletions or duplications in eight different hESC lines and in one clonally derived hESC line. The recently developed MLPA method is based on the polymerase chain reaction (PCR) amplification of probe pairs that have been hybridized to genomic DNA and subsequently ligated. Presently 41 telomere regions can be screened in just one multiplex reaction . The hESCs were analyzed in early and late passages and after freeze/ thaw cycles to determine if in vitro manipulation of hESCs could result in subtelomeric deletions. We did not detect any deletions in any of the cell lines analyzed in this study. Taken together, these data support the conclusion that hESCs can be cultured in vitro for extended time periods while retaining genomic stability, which is a prerequisite for many downstream applications.
MATERIALS AND METHODS
All of the cell lines included in this study were established and cultured on MEF feeder layers using VitroHES medium, and the cells were dissociated and passaged by mechanical cutting every 4–5 days. The hESC lines have been extensively characterized, and they display the typical markers and properties of undifferentiated hESCs (Cellartis, unpublished results) and have been subjected to freeze/thaw cycles. Importantly, data from karyotyping and FISH analyses of hESC lines SA002, AS034, AS034.1, AS038, SA121, and SA181 were recently published and demonstrated that the cell lines present with normal karyotypes, except for SA002, which display trisomy 13 . The remaining three cell lines included in this study (SA001, SA240, and SA461) have also been analyzed using the same techniques; the cells display normal stable karyotypes, and no large-scale genomic aberrations have been detected (Fig. 1, Table 1).
Figure 1. Karyotype of human embryonic stem cell (hESC) line SA001. (A): Representative normal karyotype from hESC line SA001 (passage 26). (B): Signals obtained from fluorescent in situ hybridization analysis of hESC line SA001 (passage 32) using probes for chromosomes 13, 18, 21, X, and Y.
Table 1. Summary of the results from karyotyping and fluorescent in situ hybridization (FISH) analysis of human embryonic stem cell lines SA001, SA240, and SA461
For the purpose of this study, undifferentiated hESCs were harvested in early and late passages, as indicated in Table 2, and analyzed using MLPA. With two exceptions (see below), the MLPA telomere P019/P020 analyses did not detect any subtelomeric abnormalities in the hESCs. Each MLPA P019/P020 analysis was performed in duplicate. In some cases (Table 2), these analyses were also confirmed using a more recently developed complementary MLPA telomere P036 assay. Except for one probe, all sequences detected by probes in the P019/P020 probe mixes are different from the probes in the P036 mix. The significant number of individual hESC lines analyzed and the long-term in vitro culture of several of these lines support the conclusion that undifferentiated hESCs can be propagated extensively while maintaining genomic stability.
Table 2. Summary of the results from the multiplex ligation-dependent probe amplification (MLPA) telomere P019/P020 analyses of undifferentiated human embryonic stem cells (hESCs)
As indicated above, one hESC line included in this study, SA002, displays trisomy 13 . The MLPA analyses of this hESC line were performed using cells in passage 14 and 144. Without prior knowledge about the trisomy, the analyst detected this chromosome abnormality in hESC line SA002. Figure 2 illustrates the different peak patterns obtained from a normal XY hESC line (SA001, passage 49) (panel A) and from the trisomic XX hESC line SA002 (passage 144) (panel B) using the MLPA P020 probe combination that covers chromosomes 12–22 and Y. The peak area of each amplification product reflects the relative copy number of that target sequence, making the identification of deleted or duplicated sequences possible.
Figure 2. Representative peak patterns obtained from the multiplex ligation-dependent probe amplification telomere P020 analyses of undifferentiated human embryonic stem cells (hESCs). The P020 mix contains probes for chromosomes 12–22 and Y. (A): Normal XY hESC line (SA001, passage 49) with two specific Y peaks. (B): Trisomic XX hESC line SA002 (passage 144) in which all three chromosome 13–specific peaks show an approximate 1.5-fold increase and the Y specific peaks are absent. The arrows indicate the chromosome 13–specific peaks.
In another hESC line, SA121 (passages 73 and 117), an apparent 6p-deletion, was detected by MLPA analysis using the P019/P020 probe sets (Table 2, Fig. 3B). However, in subsequent analyses, this observed deletion could not be confirmed by FISH analysis using metaphase spreads from the same cell line (passage 81) using 6ptel and 6qtel probes (Fig. 4) nor by the MLPA telomere P036 assay using SA121 cells in passage 117 (Table 2). Taken together, these results indicate that the apparent 6p deletion in SA121 most likely represents a polymorphism at the binding site of the probe, a finding also reported by others (personal communication, Dr. J.P. Schouten, MRC-Holland).
Figure 3. Representative peak patterns obtained from the multiplex ligation-dependent probe amplification telomere P019 analyses of undifferentiated human embryonic stem cells (hESCs). The P019 mix contains probes for chromosomes 1-11 and X. (A): Normal XY hESC line (AS034, passage 11). (B): Apparent 6p-deletion observed in SA121 (passage 73). The arrows indicate the 6p-specific peaks.
Figure 4. Representative illustration of a fluorescent in situ hybridization analysis using 6p/6q subtelomere probes performed on meta-phase spreads prepared from undifferentiated human embryonic stem cells (SA121) in passage 81.
DISCUSSION
In this study, we have provided high-resolution experimental data supporting the conclusion that it is possible to maintain hESCs in culture for extended time periods without compromising their genomic integrity. Although these results are promising and provide confidence and support for the therapeutic potential of hESCs, additional studies are necessary to safely move hESC research from bench to bed.
ACKNOWLEDGMENTS
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
McNeish J. Embryonic stem cells in drug discovery. Nat Rev Drug Discov 2004;3:70–80.
Davila JC, Cezar GG, Thiede M et al. Use and application of stem cells in toxicology. Toxicol Sci 2004;79:214–223.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.
Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.
Heins N, Englund MC, Sjoblom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. STEM CELLS 2004;22:367–376.
Draper JS, Smith K, Gokhale P et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 2004;22:53–54.
Inzunza J, Sahlen S, Holmberg K et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol Hum Reprod 2004;10:461–466.
Rosler ES, Fisk GJ, Ares X et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 2004;229:259–274.
Buzzard JJ, Gough NM, Crook JM et al. Karyotype of human ES cells during extended culture. Nat Biotechnol 2004;22:381–382.
Saccone S, De Sario A, Della Valle G et al. The highest gene concentrations in the human genome are in telomeric bands of metaphase chromosomes. Proc Natl Acad Sci U S A 1992;89:4913–4917.
Knight SJ, Regan R, Nicod A et al. Subtle chromosomal rearrangements in children with unexplained mental retardation. Lancet 1999;354:1676–1681.
De Vries BB, Winter R, Schinzel A et al. Telomeres: a diagnosis at the end of the chromosomes. J Med Genet 2003;40:385–398.
Schouten JP, McElgunn CJ, Waaijer R et al. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57.
Rooms L, Reyniers E, van Luijk R et al. Subtelomeric deletions detected in patients with idiopathic mental retardation using multiplex ligation-dependent probe amplification (MLPA). Hum Mutat 2004;23:17–21.(Catarina Darnforsa, Anna )