1 Departments of Clinical Science, 2 Clinical Research Centre, Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, 3 Medical Biochemistry and Biophysics, Laboratory of Molecular Neurobiology and 4 Centre for Molecular Medicine, Karolinska Institutet, S-171 77, Stockholm, Sweden
5 To whom correspondence should be addressed. e-mail: Outi.Hovatta{at}klinvet.ki.se
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Abstract |
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Key words: blastocyst/culture/feeder cells/fibroblasts/human embryonic stem cells
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Introduction |
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On the basis of experience with mouse ES cells (Evans and Kaufman, 1981; Martin, 1981
) these first reported hES cell lines were cultured with mitotically inactivated fetal mouse fibroblast feeder cells, which also kept the human ES cells undifferentiated. In contrast to the situation with mouse ES cells (Smith, 1988
), leukaemia inhibitory factor (LIF) alone did not keep human ES cells undifferentiated (Thomson et al., 1998
). The use of mouse fibroblast feeder cells to a large extent inhibits the spontaneous differentiation of human ES cells in vitro and removal of the feeder cells leads to markedly enhanced differentiation (Reubinoff et al., 2000
). This development can be partially guided by adding growth factors to the medium (Schuldiner et al., 2000
).
The use of non-human materials bears a risk of transmitting pathogens, and they are not optimal in cultures aimed at cell transplantation in humans. Hence, cultures have been developed using extracellular matrix, Matrigel- or laminin-coated dishes and conditioned mouse feeder cell medium (Xu et al., 2001). Successful culture of hES cells with human fetal muscle and skin cells and adult Fallopian tube epithelial cells as feeders has recently been described (Richards et al., 2002
).
With permission from the Ethics Committee of the Karolinska Institutet, we began to derive human embryonic stem cell lines from supernumerary blastocysts from our IVF unit. In the initial experiments, we used mouse embryo fibroblasts as feeder cells. Eleven cultures of ICM were kept proliferating for 10 days to 3 months before being cryopreserved. Six such ICM cultures were derived under conditions that fulfilled the criteria for stem cell registry at the National Institutes of Health (http://escr.nih.gov/).
In order to avoid using mouse feeder cells with human fetal cells, and to obtain a culture system which would be simple and feasible for IVF units, we developed the use of post-natal human fibroblasts, which have been used earlier as substrate cells for several cell types (Muggleton-Harris and Aroian, 1982; Muggleton-Harris and Findlay, 1991
), in our derivation and culture of human ES cells.
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Materials and methods |
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Isolation of the ICM was carried out by first removing the zona pellucida using 0.5% Pronase (Sigma, USA). The trophectoderm was removed by immunosurgery as described earlier (Solter and Knowles, 1975), using rabbit antihuman whole serum (Sigma) and guinea-pig complement serum (Sigma).
As substrate cells, we decided to use commercially available human foreskin fibroblasts (CRL-2429; ATCC Mananas, USA). The fibroblasts, with an anticipated life span of 61 doublings, were used in our experiments after 925 doublings. The cells were grown to form a confluent monolayer, and irradiated (35 Gy) before being used as substrate cells. The medium used in culture of these feeder cells was Iscovs medium (GibcoBRL, Life Technologies, Sweden) supplemented (10%) with fetal calf serum (FCS; SDS, Sweden).
The ICM were transferred to culture dishes containing the feeder cells. The dishes used were plastic dishes tested for use in embryo culture (Falcon; Becton Dickinson, USA). The culture medium used for derivation and culture of the hES cells was Knockout D-MEM 1x (GibcoBRL, Life Technologies), supplemented with 2 nmol/l L-glutamine, 20% FCS (R&D, Sweden), 0.1 mmol/l -mercaptoethanol (Gibco), 1% non-essential amino acids (Gibco), and recombinant human LIF, 1 µl/ml (Chemicon, UK). The FCS batch used in these experiments had been selected from a large number of tested batches based on the criteria of strong proliferation and induction of a minimum amount of differentiation, in two different mouse ES cell lines (D3 and GS-1). The same selection criteria are routinely used for selection of FCS for mouse ES cell cultures in our core facility for the generation of knock-out mice.
After an initial growth period of 919 days, the cell aggregates were removed from the dish by using a mild dispase solution (10 mg/ml; Gibco BRL, Life Technologies) and mechanical slicing using glass capillaries. The cell aggregates were transferred to new plates containing similar feeder cells. After the first splitting, the new growing aggregates were again split and transferred to new dishes every 5 days. Undifferentiated cells, as judged by morphology, were chosen for each further passage.
For freezing of the cells, vitrification in pulled open straws, using ethylene glycol, dimethylsulphoxide (20% each) and 1 mol/l sucrose as cryoprotectants, was carried out as described by Reubinoff et al. (2001).
The growing cells were characterized immunohistochemically by using antibodies against markers characteristic of non-differentiated ES cells. The antibody for Oct-4 was a kind gift from Dr G.A.Schulz (University of Calgary, Canada), that for TRA-1-60 was a kind gift from Dr P.Andrews (University of Sheffield, UK), and the antibody for SSEA-4 was from the Developmental Studies Hybridoma Bank, University of Iowa, USA. The expression of alkaline phosphatase was shown by using a Vector Blue/Red substrate kit (Vector Laboratories, USA). The cells were fixed with 4% paraformaldehyde. Fluorescent secondary antibodies, Cy3 (Jackson Immuno Research, USA) or AlexaFluor 488 (Molecular Probes Inc., USA) were used. P19 and TERA-2 cell lines and human foreskin fibroblasts were used as controls. Non-immune serum was also used as a negative control for the human ES cell line in question. The karyotype was determined by standard G-banding.To test pluripotency, 103 cells at passage 20 were injected to the right testis of SCID-beige mice (C.B.-17/GbmsTac-scid-bgDF N7 from M&B, Denmark). The teratoma formation was followed up by palpation and the resulting tumours were fixed, embedded in paraffin and processed for histology.
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Results |
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The first line, HS181, has been in continuous culture for 41 weeks, with a doubling time of 2436 h (Figure 1). This line has been enzymatically and mechanically split and transferred to new dishes by the time the aggregate has been formed by
2.56.4x103 cells. The size of the transferred aggregates has been
12x103 cells. Spontaneous differentiation was observed in 1020% of the cells, in one out of 20 colonies during the 5 day culture period. High-density cultures regularly showed spontaneous development into multinuclear aggregates containing both undifferentiated and differentiated cells, including neural tube-like cells and structures, and beating cells.
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Discussion |
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For IVF units working without animal facilities, the use of human cells is more convenient than the use of mouse cells as feeders. In addition, the quality control systems of most of the IVF units do not allow culture of mouse cells in the same facility.
Finding optimal techniques to culture human ES cells without a feeder layer, and culturing them without FCS, is important for cell transplantation. Xu et al. (2001) have already been successful with non-feeder cultures, but the conditioned medium from mouse cell cultures is not optimal as regards human transplantation. The risk of transmitting pathogens still exists when conditioned medium from mouse fibroblast cultures is used. In the present study we used FCS in the cultures, and the next step is to replace it with human serum, and find serum-free conditions with synthetic serum supplements. The use of human serum has already been successful when using other feeder cells (Richards et al., 2002
). Optimally, also the feeder cells should also be cultured in serum-free medium, which has been tested (Amit et al., 2000
). The serum-free system is hence already well established for the culture of human ES cells. On the other hand, FCS has been accepted as an ingredient of culture media for transplantable adult stem cells. To obtain transplantable cells, good manufacturing practice (GMP) conditions are needed, and at our hospital such a facility is being built.
The hES cell characteristics of the line 181 cells were supported by the expression of the surface markers, Oct-4, SSEA-4 and Tra-1-60. In addition, after injection of the 181 cells into immunodeficient mice, teratomas containing tissue components consistent with all the three embryonic layers were found in the recipient mice, as a sign of pluripotency.
Also when cultured at high density, spontaneous differentiation occurred.
We added rLIF to the cultures in spite of earlier reports that it does not support the growth of human ES cells without feeders (Thomson et al., 1998).
We could not obtain any really good quality blastocysts for our stem cell cultures, because all such embryos are either transferred to the subject or frozen for the couples infertility treatment in the future. None of our blastocysts had a good cell number in the ICM. Nevertheless, we obtained cell lines from ICM with low numbers of cells. The line HS207 was actually derived from a blastocyst with very few cells in the ICM, although it did not grow as well as the line HS181, and its pluripotency has not been verified. Nevertheless, this is encouraging when thinking of an hES cell bank with a large enough number of different genotypes for cell transplantation in the future. Creating good quality embryos for research might be an excellent option, but it bears ethical problems (McLaren, 2001), which may be avoided by using supernumerary low-quality embryos.
We intend to use this method to establish new ES cell lines for the new cell bank supported by the Swedish Medical Research Council and Juvenile Diabetes Research Foundation, to provide cells for academic collaborators.
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Acknowledgements |
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References |
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Submitted on November 7, 2002; resubmitted on January 30, 2003; accepted on March 31, 2003.