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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

In vitro differentiation using embryoid bodies from hiPSCs generated and maintained in defined culture conditions.Immunohistochemistry of MAP2 (A), TUJ (B), FLK1 (C), VIMENTIN (D), and PDX1 (E) in the differentiated hiPSC line, UTA-SF-2-2, grown under hESF9a-based conditions. Differentiation was performed using embryoid body formation. The tissues in embryoid bodies were fixed and reacted with antibodies. Binding of these antibodies was visualized with AlexaFluor 488-conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bars are 50 µm.
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pone-0014099-g007: In vitro differentiation using embryoid bodies from hiPSCs generated and maintained in defined culture conditions.Immunohistochemistry of MAP2 (A), TUJ (B), FLK1 (C), VIMENTIN (D), and PDX1 (E) in the differentiated hiPSC line, UTA-SF-2-2, grown under hESF9a-based conditions. Differentiation was performed using embryoid body formation. The tissues in embryoid bodies were fixed and reacted with antibodies. Binding of these antibodies was visualized with AlexaFluor 488-conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bars are 50 µm.

Mentions: Next, we examined whether hiPSCs were generated from the reprogramming step under our feeder-free, defined culture conditions. For comparison, we used another defined xeno-free TeSR2 media, which was modified from mTeSR1 medium [13], for culturing human pluripotent stem cells. Adult human dermal fibroblasts (HDF) were infected with amphotropic retroviruses carrying the OCT4, SOX2, KLF4, and C-MYC genes and were cultured under hESF9a-based conditions or under TeSR2-based conditions (with TeSR2 medium on matrigel-coated dishes). After 26 days of culture, we detected hiPSC-like colonies by staining for ALP substrate or by their morphologies (Figure 5A). While no hiPSC-like colonies were detected under the TeSR2-based conditions, eight hiPSC-like colonies were detected under the hESF9a-based condition. We confirmed these results by independent multiple experiments (0 ALP-positive colonies/2 experiments in TeSR2-based conditions and 3 ALP-positive colonies/2 experiments in hESF9a-based conditions) (Figure 5B and C). In another experiment of the hiPSCs induction under hESF9a-based conditions, we picked up hiPS-like colonies for expansion under the same culture conditions. The ALP activity was maintained at 5 passages (Figure 5D). After 25–26 passages, we confirmed self-renewal marker expression and differentiation potential in these cell lines by immunocytochemistry (Figure 6, 7). One of the cell lines, designated UTA-SF2-2, retained proper proliferation rates for human pluripotent stem cells (Figure 8A; population doubling time: 28.2±4.9 h). Karyotype analysis revealed that UTA-SF2-2 cells at passage 31 was 46XX (Figure 8B). Finally, we examined Neu5Gc expression in the UTASF2-2 line by flow cytometry and showed that the levels of Neu5Gc in passage-30 cells were almost negative, as in negative control cells (Figure 9). Our established cell lines therefore showed little or no Neu5Gc contamination, suggesting that the hESF9a-based culture conditions generated hiPSCs steadily from reprogramming step using adult HDF and supported their pluripotency for long-term culture with less contamination of 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)

In vitro differentiation using embryoid bodies from hiPSCs generated and maintained in defined culture conditions.Immunohistochemistry of MAP2 (A), TUJ (B), FLK1 (C), VIMENTIN (D), and PDX1 (E) in the differentiated hiPSC line, UTA-SF-2-2, grown under hESF9a-based conditions. Differentiation was performed using embryoid body formation. The tissues in embryoid bodies were fixed and reacted with antibodies. Binding of these antibodies was visualized with AlexaFluor 488-conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bars are 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0014099-g007: In vitro differentiation using embryoid bodies from hiPSCs generated and maintained in defined culture conditions.Immunohistochemistry of MAP2 (A), TUJ (B), FLK1 (C), VIMENTIN (D), and PDX1 (E) in the differentiated hiPSC line, UTA-SF-2-2, grown under hESF9a-based conditions. Differentiation was performed using embryoid body formation. The tissues in embryoid bodies were fixed and reacted with antibodies. Binding of these antibodies was visualized with AlexaFluor 488-conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bars are 50 µm.
Mentions: Next, we examined whether hiPSCs were generated from the reprogramming step under our feeder-free, defined culture conditions. For comparison, we used another defined xeno-free TeSR2 media, which was modified from mTeSR1 medium [13], for culturing human pluripotent stem cells. Adult human dermal fibroblasts (HDF) were infected with amphotropic retroviruses carrying the OCT4, SOX2, KLF4, and C-MYC genes and were cultured under hESF9a-based conditions or under TeSR2-based conditions (with TeSR2 medium on matrigel-coated dishes). After 26 days of culture, we detected hiPSC-like colonies by staining for ALP substrate or by their morphologies (Figure 5A). While no hiPSC-like colonies were detected under the TeSR2-based conditions, eight hiPSC-like colonies were detected under the hESF9a-based condition. We confirmed these results by independent multiple experiments (0 ALP-positive colonies/2 experiments in TeSR2-based conditions and 3 ALP-positive colonies/2 experiments in hESF9a-based conditions) (Figure 5B and C). In another experiment of the hiPSCs induction under hESF9a-based conditions, we picked up hiPS-like colonies for expansion under the same culture conditions. The ALP activity was maintained at 5 passages (Figure 5D). After 25–26 passages, we confirmed self-renewal marker expression and differentiation potential in these cell lines by immunocytochemistry (Figure 6, 7). One of the cell lines, designated UTA-SF2-2, retained proper proliferation rates for human pluripotent stem cells (Figure 8A; population doubling time: 28.2±4.9 h). Karyotype analysis revealed that UTA-SF2-2 cells at passage 31 was 46XX (Figure 8B). Finally, we examined Neu5Gc expression in the UTASF2-2 line by flow cytometry and showed that the levels of Neu5Gc in passage-30 cells were almost negative, as in negative control cells (Figure 9). Our established cell lines therefore showed little or no Neu5Gc contamination, suggesting that the hESF9a-based culture conditions generated hiPSCs steadily from reprogramming step using adult HDF and supported their pluripotency for long-term culture with less contamination of 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