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Chromosomal Modification in Human Embryonic Stem Cells Cultured in a Feeder-Free Condition after Single Cell Dissociation using Accutase.

Kim YE, Park JA, Ha YW, Park SK, Kim HS, Oh SK, Lee Y - (2012)

Bottom Line: Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery.Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology.Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea ; Biotechnology Research Institute, Chungbuk National University, Cheongju 361-763, Korea.

ABSTRACT
Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.

No MeSH data available.


Comparison of stem cell marker expression in human ES cells passaged after single cell-dissociation with accutase and TrypLE. The H9 human ES cells were dissociated to single cells with accutase (A) or TrypLE (B) and then cultured in a feeder-free condition using MEF conditioned medium. Expression of stem cells markers was determined by FACS analysis.
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Figure 2: Comparison of stem cell marker expression in human ES cells passaged after single cell-dissociation with accutase and TrypLE. The H9 human ES cells were dissociated to single cells with accutase (A) or TrypLE (B) and then cultured in a feeder-free condition using MEF conditioned medium. Expression of stem cells markers was determined by FACS analysis.

Mentions: As we confirmed that human ES cells can be maintained by culture of single cells dissociated with accutase, we next investigated whether other enzymes such as TrypLE can be used in the same way to culture human ES cells as single cells. As shown in Fig. 2, expression of stem cell markers in TrypLE-passaged human ES cells was analyzed by FACS. Expression levels of stem cell markers such as Tra-1-60, SSEA-4, Nanog, Sox-2, and Klf-4 were similar in accutase-passaged cells (Fig. 2A) and TrypLE-passaged cells (Fig. 2B). These results reveal that TrypLE may be used as an alternative to accutase in the culture of human ES cell H9 after single cell dissociation.


Chromosomal Modification in Human Embryonic Stem Cells Cultured in a Feeder-Free Condition after Single Cell Dissociation using Accutase.

Kim YE, Park JA, Ha YW, Park SK, Kim HS, Oh SK, Lee Y - (2012)

Comparison of stem cell marker expression in human ES cells passaged after single cell-dissociation with accutase and TrypLE. The H9 human ES cells were dissociated to single cells with accutase (A) or TrypLE (B) and then cultured in a feeder-free condition using MEF conditioned medium. Expression of stem cells markers was determined by FACS analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4282239&req=5

Figure 2: Comparison of stem cell marker expression in human ES cells passaged after single cell-dissociation with accutase and TrypLE. The H9 human ES cells were dissociated to single cells with accutase (A) or TrypLE (B) and then cultured in a feeder-free condition using MEF conditioned medium. Expression of stem cells markers was determined by FACS analysis.
Mentions: As we confirmed that human ES cells can be maintained by culture of single cells dissociated with accutase, we next investigated whether other enzymes such as TrypLE can be used in the same way to culture human ES cells as single cells. As shown in Fig. 2, expression of stem cell markers in TrypLE-passaged human ES cells was analyzed by FACS. Expression levels of stem cell markers such as Tra-1-60, SSEA-4, Nanog, Sox-2, and Klf-4 were similar in accutase-passaged cells (Fig. 2A) and TrypLE-passaged cells (Fig. 2B). These results reveal that TrypLE may be used as an alternative to accutase in the culture of human ES cell H9 after single cell dissociation.

Bottom Line: Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery.Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology.Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Korea ; Biotechnology Research Institute, Chungbuk National University, Cheongju 361-763, Korea.

ABSTRACT
Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.

No MeSH data available.