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Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder- and serum-free defined conditions.

Hayashi Y, Chan T, Warashina M, Fukuda M, Ariizumi T, Okabayashi K, Takayama N, Otsu M, Eto K, Furue MK, Michiue T, Ohnuma K, Nakauchi H, Asashima M - PLoS ONE (2010)

Bottom Line: The successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine.Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions.This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Background: The successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine. In particular, hiPSCs are a promising source of cells for regenerative medicine and pharmacology. However, one of the major obstacles to such uses for hiPSCs is the risk of contamination from undefined pathogens in conventional culture conditions that use serum replacement and mouse embryonic fibroblasts as feeder cells.

Methodology/principal findings: Here we report a simple method for generating or culturing hiPSCs under feeder- and serum-free defined culture conditions that we developed previously for human embryonic stem cells. The defined culture condition comprises a basal medium with a minimal number of defined components including five highly purified proteins and fibronectin as a substrate. First, hiPSCs, which were generated using Yamanaka's four factors and conventional undefined culture conditions, adapted to the defined culture conditions. These adapted cells retained the property of self renewal as evaluated morphologically, the expression of self-renewal marker proteins, standard growth rates, and pluripotency as evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo (teratoma formation in immunodeficient mice). Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions. Second, we successfully generated hiPSCs using adult dermal fibroblast under the defined culture conditions from the reprogramming step. For a long therm culture, the generated cells also had the property of self renewal and pluripotency, they carried a normal karyotype, and they were Neu5Gc negative.

Conclusion/significance: This study suggested that generation or adaption culturing under defined culture conditions can eliminate the risk posed by undefined pathogens. This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

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Related in: MedlinePlus

Decreased expression of xenoantigen Neu5Gc in hiPSCs adapted under defined culture conditions.Flow cytometry analysis of Neu5Gc expression. CHO cells were grown in FCS-containing medium (A) and hiPSCs were grown under KSR-based conditions (B) or hESF9a-based conditions (C). The cells were exposed to anti-Neu5Gc antibody (red), control antibody (green), or blocking buffer (blue), and then stained with a secondary antibody for analysis by flow cytometry.
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pone-0014099-g004: Decreased expression of xenoantigen Neu5Gc in hiPSCs adapted under defined culture conditions.Flow cytometry analysis of Neu5Gc expression. CHO cells were grown in FCS-containing medium (A) and hiPSCs were grown under KSR-based conditions (B) or hESF9a-based conditions (C). The cells were exposed to anti-Neu5Gc antibody (red), control antibody (green), or blocking buffer (blue), and then stained with a secondary antibody for analysis by flow cytometry.

Mentions: Our data showed that hESF9a culture conditions maintain the pluripotency of hiPSCs. Conventional culture conditions currently use KSR for human ES/iPS cells, and it is accepted that these commercially supplied components may contain undefined animal-derived xenoantigens and pathogens. Because human cells cannot produce Neu5Gc genetically [4], it becomes a useful indicator of xenogenic contamination in human pluripotent stem cells [7]. We therefore examined the expression of Neu5Gc in UTA1 cells grown under KSR- and hESF9a-based conditions by flow cytometry using an antibody against Neu5Gc. The level of Neu5Gc was high in UTA1 cells cultured under the KSR-based conditions (23 passages) and was comparable with that in Chinese hamster ovary (CHO) cells cultured in fetal calf serum (FCS)-containing medium (Figure 4A, B). The Neu5Gc expression decreased almost to negative control levels (with control antibody or no primary antibody) in UTA1 cells cultured under the hESF9a-based conditions (18 passages in KSR-based condition and 27 passages in ESF9a-based condition) (Figure 4C). The conventional culture conditions contain animal-derived components such as MEF, FCS in which the MEFs were cultured, and porcine gelatin. Further, the KSR used for the conventional culture conditions also includes animal-derived components, such as lipid-enriched bovine serum albumin [9], [10], [11]. Hence, the UTA1 cells might metabolically incorporate substantial amounts of Neu5Gc from these factors. However, although the hESF9a-based conditions also contain animal-derived components such as bovine insulin, bovine serum albumin, porcine heparin, and bovine fibronectin, the UTA1 cells incorporate only low amounts of Neu5Gc. These results suggested that culturing of hiPSCs under the defined conditions with purified components could decrease the risk of xenogenic and human-derived pathogens.


Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder- and serum-free defined conditions.

Hayashi Y, Chan T, Warashina M, Fukuda M, Ariizumi T, Okabayashi K, Takayama N, Otsu M, Eto K, Furue MK, Michiue T, Ohnuma K, Nakauchi H, Asashima M - PLoS ONE (2010)

Decreased expression of xenoantigen Neu5Gc in hiPSCs adapted under defined culture conditions.Flow cytometry analysis of Neu5Gc expression. CHO cells were grown in FCS-containing medium (A) and hiPSCs were grown under KSR-based conditions (B) or hESF9a-based conditions (C). The cells were exposed to anti-Neu5Gc antibody (red), control antibody (green), or blocking buffer (blue), and then stained with a secondary antibody for analysis by flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990711&req=5

pone-0014099-g004: Decreased expression of xenoantigen Neu5Gc in hiPSCs adapted under defined culture conditions.Flow cytometry analysis of Neu5Gc expression. CHO cells were grown in FCS-containing medium (A) and hiPSCs were grown under KSR-based conditions (B) or hESF9a-based conditions (C). The cells were exposed to anti-Neu5Gc antibody (red), control antibody (green), or blocking buffer (blue), and then stained with a secondary antibody for analysis by flow cytometry.
Mentions: Our data showed that hESF9a culture conditions maintain the pluripotency of hiPSCs. Conventional culture conditions currently use KSR for human ES/iPS cells, and it is accepted that these commercially supplied components may contain undefined animal-derived xenoantigens and pathogens. Because human cells cannot produce Neu5Gc genetically [4], it becomes a useful indicator of xenogenic contamination in human pluripotent stem cells [7]. We therefore examined the expression of Neu5Gc in UTA1 cells grown under KSR- and hESF9a-based conditions by flow cytometry using an antibody against Neu5Gc. The level of Neu5Gc was high in UTA1 cells cultured under the KSR-based conditions (23 passages) and was comparable with that in Chinese hamster ovary (CHO) cells cultured in fetal calf serum (FCS)-containing medium (Figure 4A, B). The Neu5Gc expression decreased almost to negative control levels (with control antibody or no primary antibody) in UTA1 cells cultured under the hESF9a-based conditions (18 passages in KSR-based condition and 27 passages in ESF9a-based condition) (Figure 4C). The conventional culture conditions contain animal-derived components such as MEF, FCS in which the MEFs were cultured, and porcine gelatin. Further, the KSR used for the conventional culture conditions also includes animal-derived components, such as lipid-enriched bovine serum albumin [9], [10], [11]. Hence, the UTA1 cells might metabolically incorporate substantial amounts of Neu5Gc from these factors. However, although the hESF9a-based conditions also contain animal-derived components such as bovine insulin, bovine serum albumin, porcine heparin, and bovine fibronectin, the UTA1 cells incorporate only low amounts of Neu5Gc. These results suggested that culturing of hiPSCs under the defined conditions with purified components could decrease the risk of xenogenic and human-derived pathogens.

Bottom Line: The successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine.Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions.This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Background: The successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine. In particular, hiPSCs are a promising source of cells for regenerative medicine and pharmacology. However, one of the major obstacles to such uses for hiPSCs is the risk of contamination from undefined pathogens in conventional culture conditions that use serum replacement and mouse embryonic fibroblasts as feeder cells.

Methodology/principal findings: Here we report a simple method for generating or culturing hiPSCs under feeder- and serum-free defined culture conditions that we developed previously for human embryonic stem cells. The defined culture condition comprises a basal medium with a minimal number of defined components including five highly purified proteins and fibronectin as a substrate. First, hiPSCs, which were generated using Yamanaka's four factors and conventional undefined culture conditions, adapted to the defined culture conditions. These adapted cells retained the property of self renewal as evaluated morphologically, the expression of self-renewal marker proteins, standard growth rates, and pluripotency as evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo (teratoma formation in immunodeficient mice). Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions. Second, we successfully generated hiPSCs using adult dermal fibroblast under the defined culture conditions from the reprogramming step. For a long therm culture, the generated cells also had the property of self renewal and pluripotency, they carried a normal karyotype, and they were Neu5Gc negative.

Conclusion/significance: This study suggested that generation or adaption culturing under defined culture conditions can eliminate the risk posed by undefined pathogens. This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

Show MeSH
Related in: MedlinePlus