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Human Reproduction Vol.19, No.3 pp. 670±675, 2004
Advance Access publication 29 January, 2004
DOI: 10.1093/humrep/deh135
Derivation of a diploid human embryonic stem cell line
from a mononuclear zygote
Edith Suss-Toby1, S.Gerecht-Nir1,2, M.Amit1,3, D.Manor1 and J.Itskovitz-Eldor1,3,4
1
Department of Obstetrics and Gynecology, Rambam Medical Center, POB 9602, Haifa 31096, 2Biotechnology-Interdisciplinary
Unit and 3Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
4
To whom correspondence should be sent. E-mail: [email protected]
BACKGROUND: IVF occasionally produces aneuploid zygotes with one or three pronuclei (PN). Routinely, these
zygotes are discarded. The aim of this work was to establish human embryonic stem cell (hESC) lines from
blastocysts resulting from abnormal fertilization. METHODS: Abnormally fertilized zygotes were cultured to the
blastocyst stage and, following zona pellucida digestion, zona-free blastocysts were placed on a mouse feeder layer.
Culture of hESCs was carried out as described earlier. RESULTS: Six out of the nine developing blastocysts
attached to the feeder layer. One hESC line, originating from a mononuclear zygote following ICSI, was successfully
derived. This line displayed typical phenotype and embryonic surface markers, and exhibited the potential to
develop into all three embryonic germ layers both in vitro (by embryoid body formation) and in vivo (teratoma
generation). Genetic examination revealed normal diploid karyotype and heterozygotic appearance for
metachromatic leukodystrophy (MLD). CONCLUSION: This method, which requires neither immuno nor
mechanical removal of the trophectoderm, may facilitate the derivation of hESC lines in general, and those from
abnormal embryos in particular. Furthermore, it is shown that aneuploid zygotes can be used as a source for normal
hESC derivation and hold the potential to generate aneuploid hESC lines for research purposes.
Key words: aneuploid zygote/derivation/differentiation/embryonic stem cells
Introduction
Normal human zygotes consist of two pronuclei representing
each parent. However, IVF often results in abnormal zygotes
with one or three pronuclei (PN). The high percentage of
diploid embryos which was reported to develop from IVF 1PN
zygotes (Munne et al., 1993) drops to 10±30% following ICSI
(Sultan et al., 1995; Staessen and Van Steirteghem, 1997).
At the blastocyst stage, the embryo forms an inner cell mass
(ICM) capable of forming a wide range of cell types of the
body, and an outer trophectoderm, which is committed to form
part of the placenta (Winkel and Pedersen, 1998). Isolation and
seeding of the ICM on an appropriate feeder layer may
generate human embryonic stem cells (hESCs) (Thomson et al.,
1998; Reubinoff et al., 2000). Such isolation can be accomplished by removal of the trophectoderm either mechanically
(Amit and Itskovitz-Eldor, 2002) or by immunosurgery
(Thomson et al., 1998; Reubinoff et al., 2000; Lanzendorf
et al., 2001; Amit and Itskovitz-Eldor, 2002). All hESCs have
the ability to self-renew perpetually in culture and maintain
undifferentiated phenotype and normal karyotype. They are
pluripotent, i.e. capable of developing into all three primary
germ layer derivatives, namely ectoderm, mesoderm and
endoderm, both in vitro and in vivo [embryoid body (EB)
and teratoma formation, respectively] (Draper and Andrews,
670
2002). To date, blastocysts used for hESC derivation have been
obtained from donated normal embryos (Thomson et al., 1998;
Reubinoff et al., 2000; Lanzendorf et al., 2001; Amit and
Itskovitz-Eldor, 2002) or from poor-quality discarded embryos
(Mitalipova et al., 2003). This work aimed at deriving hESCs
from non-usable aneuploid zygotes. As it was reported that
mouse ES cells can be derived by simply culturing the
blastocyst on the feeder layer (Nagy et al., 2003), this was also
examined in the human.
Materials and methods
Blastocyst culture
Discarded zygotes were donated to this study by couples undergoing
IVF at Rambam Medical Center who signed consent forms. Zygotes
(n = 60) were cultured to the blastocyst stage according to our IVF
laboratory standard protocol: drops under oil using Cook media
(Cook, Queensland, Australia) for insemination, growth and
blastocyst stage (Cook IM, GM and BM, respectively).
hESC derivation and cultivation
After zona pellucida digestion by Tyrode's acidic solution (Sigma, St
Louis, MO), whole blastocysts were placed on mitotically inactivated
mouse embryonic ®broblasts (MEFs). Cells were grown, passaged,
Human Reproduction vol. 19 no. 3 ã European Society of Human Reproduction and Embryology 2004; all rights reserved
Normal human embryonic stem cell line
Figure 1. De novo hESC line derivation. (A) An abnormal blastocyst after zona pellucida digestion placed whole on an inactivated feeder
layer. (B) Attachment occurred within 24 h. (C) An hESC colony derived from the blastocyst. Bar = 100 mm.
frozen and thawed as previously described (Thomson et al., 1998;
Amit and Itskovitz-Eldor, 2002).
EB and teratoma formation, genetic characterization and
histological sections
All hESC characterizations were done as previously described (Amit
et al., 2003). Mutation of metachromatic leukodystrophy (MLD) was
detected using DNA which was isolated from cells of the newly
derived cell line, by mutation-speci®c PCR. The analysis was
performed by the Genetic Unit at Hadassah University Hospital,
Ein-Karem, Jerusalem, Israel and by the Developmental Biology Unit,
Department of Obstetrics and Gynaecology, Rambam Medical Center,
Haifa, Israel.
Immuno¯uorescence and confocal microscopy
Cultured colonies or attached EBs were ®xed in situ with 4%
paraformaldehyde (Sigma) in phosphate-buffered saline (PBS; Gibco,
San Diego, CA) for 30 min at room temperature. After blocking with
10% serum, the cells were stained with one of the following primary
antibodies: anti-stage-speci®c embryonic antigen 3 (SSEA3) or 4
(SSEA4), anti-tumour rejection antigen (TRA) 1±60 and 1±81, all
kindly provided by Professor P.Andrews, University of Shef®eld,
UK), anti-CD31 (Dako, Denmark) and anti-cytokeratin 17 and antitubulin bIII isoform (both from Chemicon International, Temecula
CA). Cells were then rinsed three times with PBS (Gibco) and
incubated for 30 min with a suitable ¯uorescein isothiocyanate
(FITC)- or Cy3-conjugated secondary antibody (Sigma). 4¢,6Diamidino-2-phenylindole (DAPI) or propidium iodide (PI) (Sigma)
was added (1:1000) to the last rinse. The immunolabelled cells were
examined using either ¯uorescence microscopy (Carl Zeiss, Jena,
Germany) or a confocal laser scanning system (BioRad Laboratories
Ltd, Hertfotdshire, UK).
Results
Blastocyst culture
Applying our standard protocol for blastocyst culture, most
aneuploid zygotes failed to develop into blastocysts. The nine
out of 60 zygotes that developed into blastocysts consisted of
seven 3PN and two 1PN. These zygotes showed either typical
blastocyst morphology or irregular blastocyst morphology, i.e.
large peripheral cells and an unclear ICM.
hESC line derivation and cultivation
Zona pellucida was digested and blastocysts were placed on a
mitotically inactivated mouse feeder layer (Figure 1A). Within
24 h, six out of the nine blastocysts attached to the feeder layer
(Figure 1B). The trophectoderm was immersed within the
feeder layer and ceased to proliferate. In contrast, after several
days, small tightly packed cells began to proliferate from four
clumps. Each of the small colonies was mechanically dissociated, divided into two and replaced on a fresh feeder layer.
A few days later, the same procedure was repeated. Most
isolated ICM clumps died out after two or three passages,
leaving a single ICM colony which continued to proliferate and
propagate. Consequently, one hESC line was established
(Figure 1C). The line, designated I9, exhibited normal growth
rate, was passaged for >40 passages and survived freeze and
thaw cycles.
hESC line characterization
Characterization of the derived line was conducted according
to the following parameters.
Morphology. The I9 line continued to proliferate and maintained a morphology typical of hESC colonies, i.e. round
colonies with sharp edges (Figure 2A), in which the spaces
between cells are clear (Figure 2B). Single cell morphology
showed a high nucleus to cytoplasm ratio with the presence of
at least two nucleoli (Figure 2C).
Genetic analyses. Karyotype analyses were performed on 66
cells from passages 27 and 29. Both analyses revealed normal
human karyotype (46,XX). Two examples of the examined
chromosomes are shown in Figure 3. As the line was donated
by a patient undergoing preimplantation genetic diagnostic
(PGD) treatment, analysis for the speci®c MLD mutation was
also performed. DNA was extracted at passages 10 and 24. The
line was found to be heterozygous to the disease.
Embryonic markers. The hESC line grown for 32 passages was
found to stain positive for typical primate ES cell surface
markers (Thomson et al., 1998) SSEA4, TRA-1±60 and TRA1±81 (Figure 4), and weakly positive for SSEA3.
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E.Suss-Toby et al.
Figure 3. Karyotype analyses. Analyses of separated chromosomes
of two representative single cells show a normal 46,XX karyotype.
(Figure 6A), neuron generation (Figure 6B) and epithelial cell
sheets expressing low molecular weight cytokeratins
(Figure 6C) were indicative of mesoderm, ectoderm and
endoderm, respectively.
Figure 2. hESC line morphology. (A) Typical hESC colonies are
round with sharp edges. (B) Clear spaces between cells were found
within the colony. (C) An ES cell with clear nucleoli. Bar = 100 mm.
Teratoma generation in vivo. Injection of undifferentiated I9
cells into the hind limb muscle of a SCID-beige mouse resulted
in the generation of a teratoma which possessed representatives
of all three germ layers (Figure 7).
Discussion
EB formation in vitro. Similarly to other hESCs, once removed
from its feeder layer and cultured in suspension (ItskovitzEldor et al., 2000) (Figure 5A), line I9 formed EBs, including
cystic ones (Figure 5B). Stem cells within these EBs differentiated into various cell types (Figure 5C).
The developmental potential of line I9 was examined by
expression of representatives of the three germ layers in 10- to
15-day-old EBs. Formation of blood vessel-like structures
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Blastocysts developed from aneuploid zygotes may exhibit
atypical morphology in which it is dif®cult to identify an ICM
zone, with a clear distinction between the ICM and
trophectoderm. Using a method where whole blastocysts
were placed on inactivated MEFs produced a successful
attachment rate (six out of nine) and the proliferation of
pluripotent clumps. This method has already been used in the
isolation of mouse ICM (Nagy et al., 2003), and is technically
simpler than immunosurgery or mechanical removal of the
trophoectoderm. It is feasible in human blastocysts and may
Normal human embryonic stem cell line
Figure 4. Embryonic surface markers. (A) (i) TRA-1±81 expressed by dense hESC colonies (red) while (ii) feeder layer cells stained only for
nuclei (blue). (iii) Distribution along a single hESC colony. (B) TRA-1±60 staining in two different colonies and (C) (i) SSEA4 in different
colonies and (ii) higher magni®cation showing staining in a large hESC colony. Bar = 100 mm.
facilitate the derivation of hESC lines from abnormal and
normal embryos. However, a more in-depth comparison
between the different techniques of ICM isolation should be
performed in order to be able to determine which is the most
ef®cient for the derivation of hESC lines. A new hESC line,
line I9, was successfully generated from a mononuclear zygote.
This line expressed speci®c embryonic markers and could
easily differentiate into derivatives of the three embryonic
germ layers. Although this line originated from a mononuclear
zygote, it was found to have normal diploid karyotype (46,XX).
It is well established that following ICSI, 10±30% of mono-
nuclear zygotes can develop into normal blastocysts, probably
due to normal fertilization followed by asynchronous formation of PN (Sultan et al., 1995; Staessen et al., 1997). Hence,
these otherwise discarded zygotes may be used as an available
source for the derivation of diploid hESC lines and may
generate aneuploid hESC lines for research.
Acknowledgements
We thank the IVF team at Rambam Medical Center and Dr Hanna
Segev for embryo and MLD analysis, Anna Ziskind and Ludmilla
Mazor for technical assistance, Ofer Shenker for confocal assistance,
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E.Suss-Toby et al.
Figure 5. Spontaneous EB formation. hESCs removed from their feeder and grown in suspension formed EBs. (A) Three-day-old EBs. (B) A
cyst developed in more mature EBs (9 days old). (C) A histological section stained with H&E revealed various cell shapes including clear
cysts (arrow) and other voids (arrowheads).
Figure 6. Developmental potential in vitro. (A) CD31+ cells show three-dimensional network formations, vascular-like channels within 11day-old EBs (3600). (B) Neuron networks were visible with the anti-tubulin bIII isoform (3800) (nuclei stained red with PI). (C)
Endodermal epithelium, stained with anti-cytokeratin 17, showed typical sheet organization with low molecular weight cytokeratin
distribution (3800).
and Hadas O'Neill for editing. We thank the Fund for Medical
Research and Development of Infrastructure and Health Services,
Rambam Medical Center and the Technion Research and
Development Foundation Ltd which supported this work.
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Normal human embryonic stem cell line
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Submitted on May 6, 2003; resubmitted on August 14, 2003; accepted on
December 4, 2003
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