Similar biological characteristics of human embryonic stem cell lines with normal and abnormal karyotypes.
ABSTRACT Human embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar.
Seven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines.
All seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines.
The present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research.
- [show abstract] [hide abstract]
ABSTRACT: The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and the inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methylation-specific polymerase chain reaction (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the maternal inactive X to the paternal inactive X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X by using specific primers for the unmethylated allele. The latter pattern was complementary to the former pattern, and a combination of these patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had non-random inactivation patterns (>80:20 or <20:80). Four patients with an X; autosome translocation showed extremely non-random patterns, and these results were consistent with those obtained by previous molecular/cytogenetic studies. We conclude that M-PCR provides an accurate assay for X-inactivation and that it can be performed on various DNA samples unsuitable for restriction digestion.Human Genetics 01/1999; 104(1):49-55. · 4.63 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: 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.Human Molecular Genetics 02/2006; 15(1):65-75. · 7.69 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A deletion of 15q11-q13 and uniparental disomy 15 lead to Prader-Labhart-Willi syndrome (PWS) or Angelman syndrome (AS) because this region contains genes expressed exclusively from the paternal (PWS) or maternal (AS) chromosome, respectively. DNA methylation plays a role in the control of imprinted gene expression, but so far only a few 5'-CG-3' dinucleotides within the recognition sites of the methylation sensitive enzymes have been studied. As part of a study on DNA methylation patterns in the human genome, we have applied the bisulfite protocol of genomic sequencing to study all 5'-CG-3' dinucleotides around exon 1 of SNRPN and at the D15S63 locus, which contains a start site for alternative SNRPN transcripts possibly involved in imprint switching during gametogenesis. At least 17 PCR products derived from single chromosomes of normal individuals as well as PWS and AS patients have been sequenced. We have found that cytosine residues outside 5'-CG-3' dinucleotides are always unmethylated. However, > 96% of all of the 23 5'-CG-3' dinucleotides around SNRPN exon 1 are methylated on the maternal chromosome and completely devoid of methylation on the paternal chromosome. This finding is in contrast to the D15S63 locus, where only the two Cfol/Hhal sites are methylated on the maternal chromosome at the same frequency as seen for the SNRPN segment. At the other five 5'-CG-3' dinucleotides, differential methylation is less pronounced, i.e. 45-70% on the maternal chromosome and 5-14% on the paternal chromosome. The differences between SNRPN and D15S63 methylation may reflect different biological functions of the alternative SNRPN transcripts. The systematic investigation of 5'-CG-3' methylation patterns as reported here will provide the basis for a PCR-based methylation test to diagnose PWS and AS.Human Molecular Genetics 04/1997; 6(3):387-95. · 7.69 Impact Factor
Similar biological characteristics of human embryonic
stem cell lines with normal and abnormal karyotypes
Xiaofang Sun1, Xiaolin Long, Yifei Yin, Yonghua Jiang, Xinjie Chen, Weiqiang Liu, Wenhong
Zhang, Hongzi Du, Shaoying Li, Yuhong Zheng, Shu Kong, Qianying Pang, Yu Shi, Yulin
Huang, Shengchan Huang, Baoping Liao, Guohong Xiao and Weihua Wang
Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road, Guangzhou,
People’s Republic of China
1Correspondence address. Tel: þ86-20-81292202; Fax: þ86-20-81292013; E-mail: email@example.com
BACKGROUND: Human embryonic stem cell (hESC) lines derived from poor quality embryos usually have either
normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar.
METHODS: Seven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype,
one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological character-
istics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and
X chromosome inactivation status were compared between different cell lines. RESULTS: All seven hESC lines
had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression
of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcrip-
tion factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline
phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methyl-
ation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell
lines. CONCLUSIONS: The present results suggest that hESC lines with abnormal karyotype are also useful exper-
imental materials for cell therapy, developmental biology and genetic research.
Keywords: human embryonic stem cell lines; characterization; karyotype; methylation; X-inactivation
Human embryonic stem cell (hESC) research is one of the most
rapidly growing areas in cell biology and medicine. Recent evi-
dence has indicated that hESC can be cultured in the labora-
tory, unlimitedly passed from generation to generation
(Thomson et al., 1998; Stojkovic et al., 2004; Oh et al.,
2005; Peura et al., 2007) and induced to differentiate into all
kinds of somatic cells under appropriate conditions. These dif-
ferentiated cells can be used to restore damaged tissues and to
treat some kinds of diseases (Assady et al., 2001; Kehat et al.,
2001; Wang et al., 2005; Lim et al., 2006).
Since the first hESC line was established in 1998 (Thomson
et al., 1998), more than 400 hESC lines have been established
in 20 countries and some of them have been registered in the
National Institutes of Health (http:escr.nih.gov/) (Guhr et al.,
2006). To establish new hESC lines, human embryos are
required. However, it is difficult to obtain good quality
human embryos for research purposes and it is not permitted
to use human embryos for research in some countries. Hence,
most researchers use discarded human embryos from IVF
clinics. Indeed, in IVF clinics, many poor quality human
embryos have been discarded because they showed no survival
characteristics at the end of culture.
Hardarson et al. (2003) found that 58% of the embryos
produced by IVF had chromosomal abnormalities at blastocyst
stage. These abnormal embryos can be used to derive hESC
lines (Baharvand et al., 2006). However, it is still unknown
whether hESC lines with abnormal karyotypes have similar
biological characteristics and functions to those with normal
karyotypes. Therefore, in the present study, we used the
discarded embryos to establish hESC lines and then compare
the biological characteristics, imprinted gene expression,
DNA methylation and X chromosome between the hESC
lines with normal and abnormal karyotypes.
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Human Reproduction Vol.23, No.10 pp. 2185–2193, 2008
Advance Access publication on July 8, 2008
Materials and Methods
Preparation of feeder layers
The feeder layers of murine embryonic fibroblasts (MEF) were pre-
paredfrom Day13.5 post-coitumfetuses of Kunming miceas previous
described (Li et al., 2004).
Culture of human embryos
This research was approved by the ethics committee of Guangzhou
Medical College. Human embryos from IVF centers were donated
on Day 3 after the patients signed the consent. The embryos were cul-
tured in G2.3 medium (Vitrolife, Gothenburg, Sweden) until Day 5
(Kim et al., 2005). On Day 5, early blastocysts were cultured for
additional 2 days in a blastocyst optimum culture medium, which is
G2.3 medium supplemented with 2000 U/ml of human recombinant
leukemia inhibitory factor (hLIF; Chemicon, Temecula, CA, USA)
and 10 ng/ml of human basic fibroblast growth factor (bFGF;
Isolation of inner cell mass
Day 7 expanded blastocysts and hatched blastocysts were used to
derive the ESC lines. Zona pellucida of expanded blastocyst was
removed by treatment with 0.1% pronase (Sigma). The inner cell
mass (ICM) of blastocysts were isolated by immunosurgery or mech-
anical method. Isolated ICMs were then placed on mitomycin
C-treated MEF feeder layers for further culture.
Culture of hESCs
After the ICMs were seeded on the feeder layer, the formation of dome
structure was examined after 8–9 days of culture. The ICMs were then
mechanically broken down into 2–3 small clumps using a small
pipette and the ICM clumps were transferred to a freshly prepared
feeder layer. These cells were again mechanically dissociated during
the initial five passages. After five passages, they were incubated in
1 mg/ml collagenase IV (Invitrogen) for 20–25 min at 378C before
further culture on freshly prepared feeders. The cells were routinely
passed every 4–5 days, and the medium was changed every day.
The hESC culture medium is knockout-Dulbecco’s modified Eagle’s
medium (Gibco) supplemented with 15% serum replacement
(GIBCO), 5% defined fetal bovine serum (Hyclone), 2 mM glutamine,
0.1 mM b-mercaptolethanol, 0.1 mM non-essential amino acids,
100 U/ml penicillin, 100 mg/ml streptomycin, 4 ng/ml bFGF (Invi-
trogen) and 2000 U/ml hLIF. After 10 passages, hLIF was not
added in the culture medium.
For karyotype analysis, ESCs at passages 12, 22 and 32 were incu-
bated in the culture medium with 0.25 mg/ml colcemid (Gibco) for
4 h, then with 0.4% sodium citrate and 0.4% chloratum Kaliumat
(1:1, v/v) at 378C for 5 min, and finally were fixed in methanol:acetic
acid (3:1, v/v) solution. After Giemsa staining, at least 20 cells were
examined in each group for the karyotype analysis.
Fluorescence in situ hybridization
For fluorescence in situ hybridization (FISH) analysis, ESC suspen-
sions were dropped onto wet slides, dried at 638C overnight and
then dehydrated with ethanol in sequential concentrations of 70%,
85% and 100% before hybridization. FISH was performed using
Vysis MultiVysionwPGT Multi-color Probe set (Vysis Inc., No.
32–131080), which includes five probes for chromosomes of 13, 18,
21, X and Y. The samples were stained according to recommended
FISH protocols from manufacturer and examined under a fluorescence
microscope. At least, 10 cells were examined in each cell line at each
time of examination.
Staining for ESC markers
Human ESC marker staining was performed after 20 passages. To
detect alkaline phosphatase (AP) activity, ESC colonies were fixed
with 90% alcohol for 2 min, washed three times with Tween-BST sol-
ution [phosphate-buffered saline (PBS) with 1% bovine serum
albumin and 0.2% Tween-20], and then stained with BCIP/NBT
(AP substrate solution, Maxim Biotech Inc., USA.) for 30 min. To
detect the hESC stage-specific embryonic markers, ESCs were fixed
with 4% paraformaldehyde for 30 min and then incubated with 4%
goat serum for 1 h before ESC marker staining. Primary antibodies
were stage-specific embryonic antigens (SSEA)-4, SSEA-1, tumor-
rejection antigen (TRA)-1-81 and TRA-1-60 (Chemicon). All anti-
bodies were diluted 1:50 with PBS and the cells were incubated
with antibody solution at room temperature for 1 h. The cells were
washed three times with Tween-BST solution for 5 min and then incu-
bated with the secondary antibody [goat anti-mouse immunoglobulin
(Ig)G and goat anti-mouse IgM, both 1:100 dilution] conjugated to flu-
orescein isothiocyanate for 30 min. Negative controls were carried out
without the addition of the primary antibodies. Hoechst 33342 was
used for nuclear staining. The cells were then washed again and exam-
ined under a fluorescence microscope or confocal microscope.
Total RNA was purified using Trizol Kit (Invitrogen) and RT–PCR
reaction was carried out using Qiagen One Step for RT–PCR Kit
Octamer binding protein 4 (Oct-4) primers were used (Table I).
RT–PCR was carried out by reverse transcription for 30 min at
508C, initial PCR activation for 15 min at 958C, followed by 30
cycles of denaturation for 1 min at 948C, annealing for 1 min at
548C and finally extension for 1 min at 728C. The PCR amplified
Table I. RT–PCR and methylation-specific PCR primer sequences.
Gene Primer forward 50-30
ACATCAAACATC TCC AACAACCA
Oct-4, octamer binding protein 4; IGF2: insulin-like growth factor; SNRPN, small nuclear ribonucleoprotein polypeptide N; GAPD, glyceraldehyde-3-phosphate
dehydrogenase; SNRPN-M, used to analyze methylated status; SNRPN-P, used to analyze unmethylated sites.
Sun et al.
products were analyzed on 1.5% agarose gel and visualized by ethi-
dium bromide (Invitrogen) staining.
DNA fingerprinting and HLA typing
Total DNA was extracted using Qiagen DNeasy Tissue Kit (Qiagen)
according to manufacturer’s instructions. Extracted DNA was ampli-
fied for 16 different genetic loci using the Promega PowerPlex 16
System kit (Promega, USA). Capillary electrophoresis was carried
out on an automated ABI 3100 Genetic Analyzer (Applied Biosys-
tems). The 16 short tandem repeat (STR) loci were D3S1358, TH01,
D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539,
CSF1PO, Penta D, amelogenin, vWA, D8S1179, TPOX and FGA.
HLA typing was performed by PCR with sequence specific primers
(Biotest, Landsteinerstr, Dreieich Germany, Biotest HLA SSP Kit
http://www.biotest.de). The products were identified using agarose
gel electrophoresis followed by the detection of the DNA bands in
UV light with the aid of the Biotest SSP typing software to determine
the HLA-A, HLA-B and HLA-DR loci. All manipulations were per-
formed according to manufacturer’s recommendations.
Differentiation assessment in vitro
The ESC colonies were dissociated with 1 mg/ml collagenase IV and
cultured in culture plates to prevent attachment of the cells. After
culture for 3 days, the cells were transferred to a new culture plate.
Seven days after culture, the formation of embryoid bodies (EBs)
was examined. EBs were transferred to 0.1% gelatin-coated culture
dish for spontaneous differentiation. The differentiated cells were
stained with antibodies against human smooth muscle actin, cardiac
troponin I, alpha fetoprotein and nestin (Chemicon). The EB culture
medium was the same as hESC culture medium but without bFGF
Differentiation assessment in vivo
The ESC colonies of passage 15 or beyond were harvested and were
broken down into 300–400 small ESC colony suspension. The colo-
nies were injected into inguinal groove of 6-week-old male severe
combined immunodeficiency (SCID) mice. Twelve weeks later, the
resultant tumors were removed, fixed in 4% paraformaldehyde and
embedded in paraffin. Sections were prepared, stained with hemat-
oxylin and eosin, and examined for the presence of tissues derived
from the three germ layers.
Analysis of imprinted genes in undifferentiated hESCs
In order to identify the imprinted gene expression in undifferentiated
hES cells, total RNA was extracted from different hESC lines. Gene
expression pattern in undifferentiated cells was profiled using the
QIAGENE one step RT–PCR kit. Selected imprinted genes were
H19, insulin-like growth factor (IGF)2, small nuclear ribonucleopro-
tein polypeptide N (SNRPN) and the conditional gene GNAS
(Table I). The PCR was performed using 508C for 30 min, 958C for
15 min and followed by 948C for 30 s, 558C for 30 s and 728C for
45 s for 45 cycles and 728C for 5 min (Sun et al., 2006). The PCR pro-
ducts were analyzed by 2% polyacrylamide gel electrophoresis,
stained with ethidium bromide and documented using the BioImaging
system (UVP, Upland, CA, USA). Glyceraldehyde-3-phosphate
dehydrogenase served as a ubiquitously expressed control. Genomic
contamination was ruled out by including an RT-negative sample in
each PCR set as a control.
DNA methylation analysis
Methylation patterns of the imprint control (IC) region of the human
SNRPN-gene (Table I) were studied in the undifferentiated hESCs.
Four hESC lines, FY-hESC-1 (46, XY), -5 (unbalanced Robertsonian
translocations), -8 (46, XX) and FY-3PN (69, XXX), were analyzed.
Prader–Willi syndrome (PWS) and Angelman syndrome (AS)
patients as well as normal DNA samples were also analyzed by
using methylation-specific PCR (MSP) assay (Kubota et al., 1997).
Genomic DNA was extracted according to the manufacturer’s
instructions (QIAamp DNA Blood Mini Kit). The PCR products
were analyzed by 7% polyacrylamide gel electrophoresis.
X chromosome inactivation status
Human androgen receptor gene contains a highly polymorphic trinu-
cleotide repeat in the first exon. It has been found that the methylation
of HpaII and HhaI sites ,100 bp away from this polymorphic STR
correlates with X inactivation. MSP was used to determine the methyl-
ation status of the selected sample with XX chromosome (FY-hES-5,
-7, -8 and FY-3PN). Genomic DNA was extracted from the XX hESC
lines. Two sets of PCR were prepared. One was for methylated X
alleles and the other was for unmethylated alleles. MSP primers
were Primer ARM-F 50-GCG AGC GTA GTA TTT TTC GGC-30,
Primer ARM-R 50-AAC CAA ATA ACC TAT AAA ACC TCT
ACG-30, Primer ARU-F 50-GTT GTG AGT GTA GTA TTT TTT
GGT-30and Primer ARU-R 50-CAA ATA ACC TAT AAA ACC
TCT ACA-30. Amplification and gel analysis were performed as man-
ufacturer’s instruction. Bisulfite-converted CpGenome Universal
bisulfite-converted female human blood DNA were used as positive
controls. Sample tubeswere loaded to Genetic Analyzer 310 for analy-
sis of fragmentation. The size of PCR products of the androgen recep-
tor gene was between 177 and 221 bp (Kubota et al., 1999).
Derivation of hESC lines
In this study, 265 donated embryos were used and 42 (15.8%)
developed to early blastocysts on Day 5 (Fig. 1I, A). When
optimum culture medium for another 2 days, 36 developed to
expanded blastocysts and 6 to hatched blastocysts. A total of
42 ICMs (Fig. 1I, B) were isolated using immunosurgery (19
ICM) or mechanical method (23 ICM). All ICMs were
seeded on MEF feeder layer (Fig. 1I, C-H).
Seven hESC lines have been established in our laboratory
(16.7% of the blastocysts or 2.6% of Day 3 embryos). After
nine passages, cells at various passages were frozen and
thawed to examine the survival status and were found to
survive in the subsequent cultures. FY-hES-1 has been in con-
tinuous culture for 1 year and 76 passages, whereas FY-3PN,
FY-hES-3, -4, -5, -7 and -8 have been in continuous cultures
for 44, 38, 30, 27, 20 and 15 passages, respectively (Table II).
Characterization and identification of hESC lines
Cells in all seven lines showed a high level of AP activity and
(Fig. 1II, A, D, E and C, respectively) but not SSEA-1 proteins
(Fig. 1II, B). Oct-4 mRNA expression was observed in all
seven hESC lines (Fig. 2). Sixteen STR loci were analyzed
for hESC lines and each cell line showed distinct STR loci indi-
cating that they were derived from different embryos (Fig. 3A–
G). HLA typing also showed that the seven lines have different
HLA-A and DBR loci (Table III).
Characteristics of human embryonic stem cells
Karyotypes of the hESC lines
Chromosome analysis and FISH examination showed that
FY-hES-1, -3 and -4 had normal 46, XY karyotypes,
FY-hES-7 and -8 had normal 46, XX karyotypes, FY-3PN
had 69, XXX karyotype (Fig. 4A–F), whereas FY-hES-5 was
an unbalanced Robertsonian translocations with 46, XX,þ13,
der(13;13)(q10;q10) (Fig. 4G) irrespective of the passages
12, 22 and 32. As shown in Fig. 4H, FISH images of
FY-hES-5 at the 22 passage showed three chromosome 13
(red signals), 2 chromosome 18 (aqua), 2 chromosome 21
(green), 2 X chromosome (blue) and no Y chromosome after
five probe staining, which was the same as examined in other
passages. All cells in other cell lines also maintained the
same karyotypes as original chromosomal analysis. Embryo
donor for FY-hES-5 had normal karyotype.
Differentiation of hESC lines
The cells of the hESC lines were cultured in suspension on
Petri dishes, simple EBs were formed on Day 3 and cystic
EBs on Dasy 6–7. On Day 8, these EBs were transferred to
0.1% gelatin-coated plates for further culture to examine cell
differentiation. After 4 days culture, these cells were positively
stained by antibodies against human smooth muscle actin
(mesoderm), alpha fetoprotein (endoderm) and nestin (ecto-
derm) (data not shown). Hence, all hESC lines were able to
differentiate into three germ layers in vitro. When hESCs
were injected into SCID mice, teratomas were first observed
at 4–5 weeks and the size of teratomas reached 25 ? 30 mm
after 12 weeks. After the teratomas were excised and sectioned
for examination, three embryonic germ layers including endo-
derm (gut epithelium), mesoderm (cartilage and muscle) and
ectoderm (squamous epithelium, neuroectoderm and neural
ganglia) were identified (Fig. 5A–D).
Analysis of imprinted genes in undifferentiated hES cells
In order to assess if there are any differences in gene expression
between normal and abnormal karyotype hESC lines, we
examined maternal expressed imprinted geneH19, paternal
expressed imprinted gene IGF2 and SNRPN and conditional
gene GNAS as disrupted expression of these genes is
associated with human genetic diseases, such as PWS and
AS. We found that gene expression patterns in all four of
these cell lines were similar. These results indicate that
expression of H19, IGF2, SNRPN and GNAS in the abnormal
karyotype hESC lines were regulated in a similar way as in the
normal ESC lines (Fig. 6).
The SNRPN is a paternally expressed imprinted gene that is
located on chromosome 15q11–13, a region related to PWS
and AS. The IC-region of the SNRPN-gene showed 23
CpG-sites that are methylated on the maternal chromosome
and unmethylated on the paternal chromosome (Zeschnigk
et al., 1997). Genomic sequencing of the SNRPN region after
bisulfite treatment has revealed that .96% of all CpG dinu-
cleotides are methylated on the maternal chromosome, but
none on the paternal chromosome. A normal person or
normal hESCs have both methylated and unmethylated
SNRPN gene sites. In this study, two pairs of primers were
used to analyze SNRPN gene methylation status. Primer
SNRPN-M was used to analyze methylated status (174 bp
band) and primer SNRPN-P was used to analyze unmethylated
sites (100 bp band). Normal and abnormal karyotype hESC
lines showed both 100 and 174 bp bands. MSP analysis
Figure 1: Derivation of hESC lines and characteristics.
(I) Human embryos and isolated ICM. (A) A Day 5 blastocyst cultured
in G2.3 blastocyst medium. Note the small ICM. (B) A Day 7 blasto-
cyst that has been cultured in the blastocyst optimum medium for
another 2 days. The ICM became clearer. (C) An ICM isolated by
mechanical method. (D) A round ICM colony surrounded by a
group of residual trophectoderm after mechanical isolation. (E) An
ICM isolated by immunosurgery. (F) A dome-like structure on the
feeder layer formed after 8 days culture of ICM isolated with immu-
nosurgery. Arrows in C to F indicate ICMs. (G) Typical round
hESC colonies with very clear boundary at low magnification. (H)
hESC morphology in a colony, showing a high nucleus–cytoplasm
ratio; note the presence of nucleoli and typical intercellular spaces
at high magnification. Bar ¼ 25 mm in A, B, C and D and
bar ¼ 100 mm in E, F and G. (II) Immunocytochemical staining of
undifferentiated hESC colonies after 32 passages in four cell lines
(A) AP, (B) SSEA-1, (C) SSEA-4, (D) TRA-1-60 and (E)
TRA-1-81. Bar ¼ 100 mm.
Sun et al.
demonstrated that all of the hESC lines have a normal SNRPN
methylation status (Fig. 6).
Determination of X-inactivation pattern
X-inactivation means that one of the X chromosomes is
silenced in XX female mammals. Initiation of this process
during early development is controlled by the X-inactivation
centre, a complex locus that determines how many and
which X chromosomes will be inactivated. In order to
analyze DNA methylation in XX hESC lines, MSP was used
to observe the X-inactivation status in these ESC lines
In a random X chromosome inactivation pattern, the XX
hESC should have two active alleles and two inactive alleles.
The peak area ratio in both active and inactive allele should
be 50:50 (the peak area ratio of the small allele to the larger
allele). However, in the present study, we found that all of
the XX hESC lines have both active and inactive X chromo-
somes with non-random inactivation patterns of either
.80:20 or ,20:80 (Fig. 7). FY-hES-5 and -8 had almost
complete non-random X chromosome inactivation patterns
with only one inactive X chromosome (194 bp in FY-hES-5
and 191 bp in FY-hES-8). The inactivation ratio in the
FY-3PN was also non-random (data not shown). In contrast,
in the normal female blood DNA samples (as a control),
there were two active (194 and 207 bp) and two inactive
(194 and 207 bp) X chromosomes with a random X chromo-
some inactivation pattern of 50:50. The XY hESC line
(FY-hES-1) always had one active X chromosome (197 bp)
but did not have inactive X chromosome (Fig. 7).
Table II. Human embryonic stem cell (hESC) lines and their characteristics.
hESC linesFY-hES-1 FY-hES-3FY-hES-4FY-hES-5 FY-hES-7FY-hES-8 FY-3PN
Karyotypes 46, XY 46, XY46, XY46, XX, þ13,der
46, XX46, XX 69,XXX
Embryoid in vitro
Teratomas in vivo
X chromosome status
No. of Passages
AP, alkaline phosphatase; SSEA, stage-specific embryonic antigen; TRA, tumor-rejection antigen; STR, short tandem repeat; FISH, fluorescence in-situ
hybridization; “p” indicates that the samples were analyzed; N/A, not applicable (not analyzed).
Figure 2: Octamer binding protein 4 expression by RT–PCR.
Lanes 3–9 represent FY-hES-1, -3, -4, -5, -7, -8 and FY-3PN, respec-
tively. Lane 1 is a negative control and lane 2 is human b-actin
Figure 3: DNA fingerprinting of FY-hES-1 (A), -3 (B), -4 (C), -5 (D), -7 (E), -8 (F) and -3PN (G), respectively.
Characteristics of human embryonic stem cells