1 Department of Obstetrics and Gynecology, Rambam Medical Center, POB 9602, Haifa 31096, 2 Biotechnology-Interdisciplinary Unit and 3 Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
4 To whom correspondence should be sent. e-mail: Itskovitz{at}rambam.health.gov.il
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Abstract |
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Key words: aneuploid zygote/derivation/differentiation/embryonic stem cells
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Introduction |
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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, 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.
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Materials and methods |
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hESC derivation and cultivation
After zona pellucida digestion by Tyrodes acidic solution (Sigma, St Louis, MO), whole blastocysts were placed on mitotically inactivated mouse embryonic fibroblasts (MEFs). Cells were grown, passaged, 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-specific 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.
Immunofluorescence and confocal microscopy
Cultured colonies or attached EBs were fixed 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-specific embryonic antigen 3 (SSEA3) or 4 (SSEA4), anti-tumour rejection antigen (TRA) 160 and 181, all kindly provided by Professor P.Andrews, University of Sheffield, UK), anti-CD31 (Dako, Denmark) and anti-cytokeratin 17 and anti-tubulin III isoform (both from Chemicon International, Temecula CA). Cells were then rinsed three times with PBS (Gibco) and incubated for 30 min with a suitable fluorescein isothiocyanate (FITC)- or Cy3-conjugated secondary antibody (Sigma). 4',6-Diamidino-2-phenylindole (DAPI) or propidium iodide (PI) (Sigma) was added (1:1000) to the last rinse. The immunolabelled cells were examined using either fluorescence microscopy (Carl Zeiss, Jena, Germany) or a confocal laser scanning system (BioRad Laboratories Ltd, Hertfotdshire, UK).
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Results |
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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.
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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).
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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 specific MLD mutation was also performed. DNA was extracted at passages 10 and 24. The line was found to be heterozygous to the disease.
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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-160 and TRA-181 (Figure 4), and weakly positive for SSEA3.
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EB formation in vitro. Similarly to other hESCs, once removed from its feeder layer and cultured in suspension (Itskovitz-Eldor 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).
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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).
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Discussion |
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Acknowledgements |
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References |
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Submitted on May 6, 2003; resubmitted on August 14, 2003; accepted on December 4, 2003.