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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

Derivation of a Xeno-Free Human Embryonic Stem Cell Line

^aCellartis AB, Göteborg, Sweden;
^bStem Cell Center, Lund University, Lund, Sweden;
^cReproductive Medicine, Department of Obstetrics and Gynaecology, Institute for Health of Women and Children, Sahlgrenska Academy, Göteborg, Sweden

Key Words. Human embryonic stem cell • Human serum • Human feeders • Clinical therapies

Correspondence: Henrik Semb, Ph.D., Stem Cell Center, Lund University, BMC, B10, SE-221-84 Lund, Sweden. Telephone: 46-46-222-31-59; Fax: 46-46-222-36-00; e-mail: henrik.semb{at}med.lu.se

Received March 6, 2006; accepted for publication May 23, 2006.
First published online in STEM CELLS EXPRESS   June 1, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [STEM CELLS 2006;24:221–229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611.

	INTRODUCTION

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Human embryonic stem cells (hESCs) hold great promise for future clinical cell therapies in the fields of, for example, diabetes, cardiac infarction, and neurodegenerative diseases, because of their unique potential to differentiate into all cell types found in the human body. In addition, it has recently been revealed that hESCs and their differentiated derivatives are less susceptible to immune rejection than adult cells [1], which is encouraging news for future clinical application. However, the majority of hESC lines available to date have been directly or indirectly exposed to animal material during their derivation and/or propagation in vitro. Transplanting xeno-contaminated hESCs to patients will increase the risk of graft rejection [2] and transfer of nonhuman pathogens, suggesting that the existing hESC lines are unsuitable for clinical applications. Closer inspection of previous attempts to establish xeno-free hESC lines reveals, in each case, that not all animal products had been replaced. For example, several recent hESC derivation reports [3–5] used Knock-Out serum replacement (SR), which contains animal protein and is a source of nonhuman sialic acid [2]. Although Richards et al. [6] used human serum (HS) and human feeder cells, and Ludwig et al. [7] recently presented the derivation of two human ES cell lines (WA15 and WA16) in a chemically defined cell culture system, they both used immunosurgery to isolate the inner cell mass (ICM). Immunosurgery is a method commonly used to remove the outer trophectoderm epithelial cell layer from the blastocyst by incubation in polyclonal rabbit anti-human whole-serum antibodies and guinea pig complement. Consequently, although these culture systems may be free of animal products, the derivation procedure is not, and therefore, these hESC lines should be considered potentially xeno-contaminated.

To obtain a truly xeno-free system for the derivation and maintenance of hESCs, three major sources of xeno-contamination must be eliminated. First, stable long-term maintenance of self-renewing and pluripotent hESCs traditionally involves the use of feeder cells. Most commonly used feeder cells are either of animal origin, such as mouse embryonic fibroblasts (MEFs), or have been exposed to animal proteins (e.g., by the use of fetal bovine serum [FBS]) during their isolation and culture procedure. Second, the medium supplements necessary for the cultivation of hESCs, such as FBS or SR, contain various animal proteins. Third, trophectoderm removal to isolate the ICM is traditionally performed by immunosurgery as described above. Several groups have attempted to exclude individual animal components by using feeder-free matrices [3, 7, 8], feeder cells of human origin [6, 9–12], or defined xeno-free media [7]. However, to date, it has not been possible to fully eliminate all animal material during both derivation and culturing of hESCs in order to create a completely xeno-free system.

To completely avoid exposure of hESCs to animal products, we developed a xeno-free protocol for the derivation and culture of new hESC lines. All animal-derived and animal-exposed components have been meticulously replaced by tested human-derived or recombinant/synthetic substances. Initially, a xeno-free culture environment based on a human feeder layer in combination with a HS-supplemented medium was designed and tested extensively. The culture system proved suitable to support the stable maintenance of self-renewal and pluripotency of previously established hESCs for more than 50 passages. To replace the xeno-contaminated commercially available human feeder cells, we then established new primary cultures of human foreskin fibroblast (HFF) feeders under xeno-free conditions. Finally, to derive new xeno-free hESCs, immunosurgery was replaced with a xeno-free alternative method.

We now report the successful derivation of the first xeno-free hESC line, which has been established and cultured under truly animal protein-free conditions. To date, the xeno-free hESC line SA611 has been maintained in culture for over 30 consecutive passages. Characterization of SA611 shows that the cell line is genetically normal and exhibits the characteristics of undifferentiated pluripotent hESCs.

	MATERIALS AND METHODS

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Human Material
Human blood from healthy donors and surplus human embryos from clinical in vitro fertilization (IVF) treatment were donated after informed consent and approval of the local ethics committee at Göteborg University.

Preparation of HS
HS was prepared as previously described [13]. For the preparation of each batch of serum, human blood was collected from 15 healthy blood donors at the hospital's blood center. All donors were from the general public and belonged to the regular registered blood donors at the hospital. The blood was collected in a transfusion bag (Dry Pack; JMS, Singapore, http://www.jmss.com) blended, and stored overnight at 4–8°C before centrifugation at 1,000g for 8 minutes. Serum was pooled, sterile-filtered, and frozen until use. Superior quality human serum was repeatedly produced in our laboratory from donated human blood, which was tested for standard pathogens (hepatitis B and C, HIV, human T-cell leukemia virus type 1, and syphilis) at the hospital's blood center.

Culture of Commercial Non-Xeno-Free HFF Feeders
Commercially available non-xeno-free HFFs were obtained from the American Type Culture Collection (CRL-2429; American Type Culture Collection, Manassas, VA, http://www.atcc.org). After expansion, confluent monolayers of HFFs were treated with mitomycin (Mutamycin; Bristol-Myers Squibb, Princeton, NJ http://www.bms.com)-C (Sigma-Aldrich, Stockholm, Sweden, http://www.sigmaaldrich.com) and plated on 0.1% gelatin (Sigma-Aldrich)-coated IVF wells (200,000 cells per 2.89 cm²) in the xeno-free medium based on knockout-Dulbecco's modified Eagle's medium (KO-DMEM) (Gibco, Paisley, Scotland, http://www.invitrogen.com) supplemented with 20% HS, 4 ng/ml human recombinant (hrb) fibroblast growth factor (FGF) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol/l ß-mercaptoethanol, and 1% nonessential amino acids (all reagents from Invitrogen).

Culture of hESCs on Commercial HFFs in HS-Supplemented Medium
The hESC line SA121 [14] (Cellartis, Göteborg, Sweden, http://www.cellartis.com), which originally was established and maintained on MEF feeders in VitroHES medium (Vitrolife AB, Kungsbacka, Sweden, http://www.vitrolife.com), was transferred onto mitotically inactivated commercial HFFs (CRL-2429) in KO-DMEM (Invitrogen) supplemented with 10% HS, 4 ng/ml hrbFGF (Invitrogen), 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol/l ß-mercaptoethanol and 1% nonessential amino acids (all reagents from Invitrogen). The hESCs were mechanically passaged to new culture dishes with fresh feeders every 5–7 days using a Stem Cell Tool (Swemed Lab International AB, Billdal, Sweden, http://www.swemed.com).

Derivation and Culture of Xeno-Free HFF Feeders
Under Swedish legislation, the use of nontraceable human surplus material that would otherwise be discarded does not require human subjects approval from the ethics committee. Human foreskin from circumcised infant boys was aseptically collected in Iscove's modified Dulbecco's medium (IMDM) (Invitrogen) + 1% penicillin-streptomycin. The skin explants were cut into small pieces using a sterile scalpel and placed into 25-cm² tissue culture flasks containing 4 ml of IMDM with 1% penicillin-streptomycin (Invitrogen) and 10% HS. After approximately 10 days, a confluent monolayer of primary HFFs was established. For subsequent expansion, the HFF cells were dissociated to single cells using a recombinant animal protein-free enzyme, TrypLE Select (Invitrogen) and passaged into 75-cm² flasks. Confluent monolayers of HFFs were treated with mitomycin-C (Sigma-Aldrich) and plated on 0.1% hrb gelatin (FibroGen, CA, http://www.fibrogen.com)-coated IVF wells (200,000 cells per 2.89 cm²).

Establishment and Culture of Xeno-Free hESCs
Donated embryos were cultured to the blastocyst stage as described previously [14]. To eliminate the zona pellucida and to damage the trophectoderm, blastocysts were incubated in acid Tyrode's solution (Medicult, Mollehaven, Denmark, http://www.medicult.com). Subsequently, zona-removed blastocysts were placed onto inactivated xeno-free HFFs. The culture medium consisted of KO-DMEM (Invitrogen) supplemented with 20% HS, 10 ng/ml hrbFGF (Invitrogen), 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol/l ß-mercaptoethanol, and 1% nonessential amino acids (all reagents from Invitrogen). Fifty percent of the medium was renewed every 2–3 days. After 10 days, the cells were mechanically passaged to fresh feeders. At passage 2, the hESCs (SA611) were enzymatically dissociated once using TrypLE Select in order to release the growing hESCs from an overgrowth of differentiated cells. Earlier attempts to perform this release by mechanical manipulation had been unsuccessful. From passage 3 on, SA611 was mechanically passaged approximately once a week. To eliminate the risk of cross-contamination the xeno-free HFFs, donated blastocysts, and the derived xeno-free hESC line SA611 were cultured and handled physically separated from all non-xeno-free cultures and components. Eleven fresh blastocysts that could not be used in infertility treatment were treated and cultured under identical conditions.

Immunohistochemical and Histochemical Analysis of hESCs
hESC cultures were fixed in 4% paraformaldehyde for 15 minutes, permeabilized for 5 minutes in 0.5% Triton X-100 solution (Sigma-Aldrich), and blocked in 5% FBS in phosphate-buffered saline (Invitrogen). The cells were incubated with primary antibodies (Oct-4, TRA-1-60, TRA-1-81, SSEA-1, SSEA-3, and SSEA-4; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.southernbiotech.com) overnight at 4°C, and visualized by incubation in fluorescein isothiocyanate (FITC)- or Cy3-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) for 60 minutes at room temperature. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich). The activity of alkaline phosphatase was determined using an alkaline phosphatase activity detection kit according to the manufacturer's instructions (Sigma-Aldrich). Stainings were evaluated and documented using a Nikon Eclipse TE-2000 U fluorescence microscope (Nikon, Tokyo, http://www.nikon.com).

Genetic Characterization
For karyotype analysis, the hESCs were incubated in the presence of colcemid, trypsinized, fixed, and mounted on glass slides. The chromosomes were visualized by DAPI staining, arranged, and documented using an inverted microscope equipped with appropriate filters and software (CytoVision; Applied Imaging, Santa Clara, CA, http://www.appliedimagingcorp.com). SA121 was analyzed after passage 30 (eight metaphases screened) and at passage 50 (19 metaphases screened). SA611 was analyzed at passage 9 (15 metaphases screened) and at passage 22 (13 metaphases screened).

For fluorescence in situ hybridization (FISH) analysis, the commercially available preimplantation genetic testing (includes chromosomes 13, 18, 21, and X and Y) multiprobe panel and chromosome enumerating probe (for chromosomes 12, 17, and 20) kits (Vysis, Downers Grove, IL, http://www.vysis.com) containing probes for chromosomes 12, 13, 17, 18, 21, X, and Y were used with minor modifications. The slides were analyzed in an inverted microscope equipped with appropriate filters and software (CytoVision). For each probe, a minimum of 100 nuclei were analyzed. In most cases, 200 nuclei were analyzed. Analysis of SA121 was performed after 30 and 50 passages. SA611 was analyzed at passages 9 and 22.

Analysis of Pluripotency In Vitro
Pluripotency was tested in vitro by spontaneous differentiation of hESCs on the feeder layer or after transfer to Matrigel plates. After 2–4 weeks, fixed and differentiated colonies were analyzed immunohistochemically to identify cells derived from the three germ layers. The following primary antibodies were used: Foxa2 (Santa Cruz Biotechnology), smooth-muscle actin (Chemicon, Temecula, CA, http://www.chemicon.com), and ß-tubulin III (Sigma-Aldrich). Alexa (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) and FITC-conjugated secondary antibodies (Jackson Immunoresearch Laboratories) were used for detection. Fixation and incubation was performed as described above.

Analysis of Pluripotency In Vivo
Pluripotency in vivo was assessed by teratoma formation in severe combined immunodeficient (SCID) mice as described earlier [14]. In brief, undifferentiated hESC colonies were mechanically cut into 200-µm x 200-µm pieces and surgically placed under the kidney capsule of SCID mice (C.B-17/lcrCrl-scidBR; Charles River Laboratories, Sulzfeld, Germany, http://www.criver.com). The mice were sacrificed after 8 weeks, and tumors were excised and fixed in 4% paraformaldehyde. Hematoxylin and eosin-stained paraffin sections were evaluated histologically for the presence of differentiated human tissue derived from all three embryonic germ layers, such as neuroectoderm, cartilage, and gut-like epithelium.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Validation of a hESC Culture System Based on HFF Feeders and HS-Supplemented Medium
To culture hESCs in the absence of animal cells and protein, we used a culture system based on HFF feeders and HS-supplemented culture medium. To validate the stability of such culture environment, colonies from hESC line SA121 [14] were transferred from the traditional culture system (MEF feeders in VitroHES medium) [14] to the new culture system, to test its ability to support maintenance of undifferentiated pluripotent hESCs for more than 50 consecutive passages. Based on the morphological appearance of the colonies, the hESCs required 3–5 passages to adjust to the new environment after the initial transfer. At regular intervals throughout the validation study (>50 passages), the cultures were evaluated morphologically, genetically, by marker expression analysis, and by teratoma formation. The results are summarized in Tables 1 and 2. At all passages analyzed, the cells expressed the expected stem cell markers Oct-4 (Fig. 1B; Table 1), TRA1-60, TRA1-81, SSEA-3, and SSEA-4 (Table 1), whereas they were negative for SSEA-1 (Table 1), a marker of differentiated hESCs. In addition, the colonies showed positive staining for alkaline phosphate activity (Table 1). At all passages analyzed throughout the validation study, hESCs maintained a stable diploid normal karyotype (46 XY) (Fig. 1C; Table 2). FISH analysis on selected chromosomes confirmed this finding (Table 2). Pluripotency was tested after 33 passages, demonstrating that the new culture environment supported growth of pluripotent hESCs; that is, histochemical analysis of teratomas revealed the presence of derivatives of endoderm (gut-like epithelium; Fig. 1D), mesoderm (cartilage; Fig. 1E), and ectoderm (neuroectoderm; Fig. 1F; Table 2).

View larger version (83K): [in this window] [in a new window]   Figure 1. Long-term validation of a culture system for human embryonic stem cells (hESCs) (SA121) based on human foreskin fibroblast and human serum. (A): Morphology of hESC line SA121 after 40 passages. Inset, x2 detail magnification. (B): Immunofluorescence staining of undifferentiated SA121 cells after >40 passages with anti-Oct4 antibodies. (C): Diploid normal karyotype of SA121 after >50 passages. (D–F): Histological analysis of teratomas derived from SA121 after 33 passages (D). Secretory epithelium (endoderm). (E): Cartilage (mesoderm). (F): Neuroectoderm (ectoderm). Scale bars = 100 µm (A, B), 50 µm (D–F).

Development of a Xeno-Free Alternative for the Isolation of the ICM
To prevent xeno-contamination during the establishment procedure, we replaced the commonly used combination of pronase digestion of the zona pellucida and immunosurgical removal of the trophectoderm by an approach that uses acid Tyrode's solution to dissolve the zona pellucida and to damage the trophectoderm. To avoid damage to the ICM while achieving complete removal of the zona pellucida, as well as partial destruction of the underlying trophectodermal cell layer, in a single step, exposure to the acidic solution (pH of 2.5–3) was carefully optimized by incubation for increasing periods of time, while the morphology of the zona pellucida, trophectoderm, and ICM were monitored by visual microscopical inspection. The optimal incubation time was found to be in the range of 30–45 seconds.

Establishment, Expansion, and Cryopreservation of a Xeno-Free hESC Line
To derive a new hESC line under xeno-free conditions, donated fresh blastocysts (Fig. 2A) were treated with acid Tyrode's solution under visual inspection and placed onto xeno-free HFFs. Initially, outgrowth of hESC-like cells from the ICM was accompanied by the appearance of differentiated cells, presumably representing primitive endoderm and trophectoderm-derived cell types. During subsequent mechanical passaging of hESC-like cells, the differentiated cells disappeared. The new hESC cell line SA611 forms distinct colonies with clearly defined borders, and the cells exhibit the characteristic morphology of hESCs, that is, densely packed cells with a high nucleus-to-cytoplasm ratio (Fig. 2B). To date, line SA611 has been in culture for over 8 months and has been propagated for more than 30 passages under xeno-free culture conditions. At passages 6, 7, 20, and 25, SA611 was cryopreserved in completely sealed straws by vitrification. Validation of the process by subsequent thawing of individual straws resulted in efficient recovery of viable, undifferentiated hESCs.

View larger version (60K): [in this window] [in a new window]   Figure 2. Morphological and immunohistochemical characterization of the xeno-free human embryonic stem cell (hESC) line SA611. (A): The blastocyst of SA611 (scale bar = 25 µm). (B): Morphology of hESC line SA611 after 12 passages under xeno-free conditions (scale bar = 100 µm). (C–H): Immunofluorescence stainings of undifferentiated SA611 cells after 12 passages using Oct-4 (C), SSEA-1 (D), TRA1-60 (E), TRA1-81 (F), SSEA-3 (G), and SSEA-4 (H) antibodies. Note that images in (C) and (E) are images of a double staining. Scale bars = 50 µm (C–E, G), 100 µm (F, H).

Karyotype analyses at passages 9 and 22 showed that SA611 maintained a stable diploid normal karyotype (46 XY) (Fig. 3A). FISH analyses at passages 9 and 22 on selected chromosomes (12, 13, 17, 18, 21, X, and Y) confirmed this finding (Fig. 3B, 3C).

View larger version (8K): [in this window] [in a new window]   Figure 3. Genetic characterization of the xeno-free human embryonic stem cell (hESC) line SA611 (passage 9). (A): The chromosomes from SA611 were diploid normal. The figure shows a representative karyotype. (B, C): Fluorescence in situ hybridization analysis of selected chromosomes from SA611 demonstrated that the cells were XY and diploid normal for chromosomes chromosome 12 and 17 (B) and for X (blue), Y (gold), 13 (red), 18 (aqua), and 21 (green) (C).

View larger version (99K): [in this window] [in a new window]   Figure 4. Confirmation of pluripotency of the xeno-free human embryonic stem cell (hESC) line SA611 in vivo (A, C, E) and in vitro (B, D, F). Histological analysis of teratomas from SA611 after 11 passages under xeno-free conditions was as follows. (A): Neuroectoderm (ectoderm); (C): Cartilage (mesoderm); (E): Secretory epithelium (endoderm). In vitro-differentiated SA611 cells were analyzed by immunofluorescence 2–4 weeks after passaging. (B): ß-III-tubulin-positive neurons (ectoderm); (D): ASMA-positive smooth muscle actin (mesoderm); (F): HNF3ß (Foxa2)-positive cells (endoderm). Scale bars = 50 µm (A, B, D–F), 100 µm (C).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
To date, all reported hESC lines have been exposed to animal material at some point during their derivation [e.g., 6, 7] or cultivation [e.g., 2]. To develop a reproducible xeno-free procedure for deriving and culturing hESCs, it is necessary to first show that all steps in the derivation, passaging, and culturing of hESCs are completely free of animal products. The second issue, which relates to the stability of the derivation and culture system, is to maintain the cells under the tested defined conditions long enough to ensure that the system is phenotypically and genetically stable.

The major sources of xeno-contamination are the presence of animal feeders and the use of either FBS or SR in the culture medium [2]. Several groups have previously reported successful culturing of hESCs on HFFs and other human feeders [4–6, 9–12]. Primary HFFs are isolated from infant foreskin, which can easily be obtained on a regular basis. The level of effort for derivation and culture of HFF is low compared with MEFs, as the isolation of the primary line from a tissue sample is simple and the primary cells can be easily expanded, whereas MEFs are generally only used in passages 2–3.

Previous attempts to use HS as a medium supplement failed due to problems with spontaneous differentiation, resulting in failure to maintain hESCs beyond passage 11 [6, 9, 15]. In contrast, we show that the use of HS as a medium supplement in combination with HFFs supports genetically stable (>50 passages) maintenance of self-renewal and pluripotency of hESCs.

The methods for preparing HS are not stated in any of the previous reports [6, 9, 15], but it is known that different methods of serum preparation may yield HS of different quality, with various effects on cell growth [13]. This could be an explanation for the divergent results. Alternatively, the discrepancy may be explained by the fact that we used a higher concentration of FGF (10 ng/ml) than previous attempts (4–8 ng/ml) [9, 15]. FGF is known to be an important factor in promoting hESC self-renewal [16, 17] in vitro. Having shown that xeno-free HS-supplemented medium, together with HFF feeders, supports maintenance of genetically and phenotypically stable hESCs, we next derived new HFF feeders under rigorously xeno-free conditions.

The final obstacle to overcome was the elimination of all animal components normally used during the isolation of the ICM. This procedure is traditionally performed by immunosurgery, which involves the use of rabbit polyclonal antibodies and guinea pig serum. Together with others, we recently reported [14, 18] that the immunosurgery procedure can be excluded, thus allowing ICM isolation without exposure to animal components. We chose to use acid Tyrode's solution for ICM isolation under xeno-free conditions, as it is used in IVF units for assisted hatching procedures [19] and has also been used to remove the zona pellucida prior to immunosurgery [20]. ICM isolation is also possible by mechanically opening up the zona pellucida to allow natural hatching, but that method requires the use of a micromanipulation system.

By optimizing the incubation time in acid Tyrode's solution, the zona pellucida was efficiently removed at the same time as the trophectodermal cell layer was damaged. As this treatment could not be used to destroy the trophectoderm completely without damaging the ICM, the initial outgrowth from the treated blastocysts was composed of a heterogenous cell population. However, with time, areas of morphologically distinct hESCs appeared, which could be handpicked and transferred to fresh plates. The fact that already at passage 5, colonies from line SA611 homogenously expressed Oct-4 and morphologically resembled undifferentiated hESCs, with a small cytoplasm-to-nucleus ratio, demonstrates that immunosurgery is not necessary for derivation of hESCs.

In summary, by developing alternative xeno-free procedures both for deriving and growing hESCs, we successfully established and characterized the first xeno-free hESC line, demonstrating that animal components are not necessary for the establishment and culture of hESCs. The hESC line SA611 can be used as a source of xeno-free cells for future applications in basic research and regenerative medicine. Of course, there are additional issues that should be addressed prior to clinical application of hESCs. One remaining concern is the possible introduction of unknown human pathogens to the hESC cultures by the blastocysts, feeders, or human serum. This concern is not limited to hESC culture but is to be generally considered in all allogenic transplantations in today's clinical reality. Therefore, improved pathogen screening technology, as well as more defined synthetic supplements, should be considered.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
H.S. owns stock in, has acted as a consultant for, and has served as an officer or member of the board for Cellartis AB within the last 2 years.

	ACKNOWLEDGMENTS

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This work was supported by the Juvenile Diabetes Research Foundation, the Swedish Research Council, and Cellartis AB. We thank G. Caisander and K. Emanuelsson for excellent work regarding conducting the karyotype analysis and teratoma experiments. We especially acknowledge the support of Dr. L. Rydberg, Head of the Blood Center Göteborg, as well as Drs. A. Lindahl and P. Borenstein.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 2006-0130v1 24/10/2170    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ellerström, C. Articles by Semb, H. Search for Related Content PubMed PubMed Citation Articles by Ellerström, C. Articles by Semb, H.