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Human embryonic stem cell lines isolation, cultivation, and characterization

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Abstract

A large number of human embryonic stem cell (hESC) lines have been derived worldwide since the first hESC line establishment in 1998. Despite many common characteristics, most important of which is the pluripotency, hESC lines vary significantly in their transcriptional profiles, genetic, and epigenetic state. These differences may arise both from individual genetics of the cell lines and from variations in their handling such as isolation and cultivation. In order to minimize the latter differences, the standardized protocols of cultivation and inter-laboratory comprehensive studies should be performed. In this report, we summarized our experience of derivation and characterization of hESC lines as well as of adaptation of hESCs to novel cultivation protocols. We have successfully derived five hESC lines and characterized them by previously established criteria, including expression of specific markers and the capacity to differentiate both in vitro and in vivo. Four of these lines, namely hESM01–04, were initially derived using mouse fibroblasts as a feeder and currently are maintained under feeder-free, serum-free conditions using mTeSR1 and Matrigel. The fifth line, hESMK05 was derived in feeder-free, serum-free conditions using mTeSR1 and Matrigel. Cell lines retain their pluripotent status and normal karyotype for more than 70 passages and are available to the scientific community.

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Acknowledgments

This work was supported by Russian Foundation for Basic Research, Russian Academy of Sciences, and LKT Ltd. We thank A. Bogomasova for excellent work regarding karyotype analysis, A. Zhelezova for teratoma experiments, and T. Karamisheva for FISH analysis. We specially thank D. Kuprash for helpful comments during manuscript preparation.

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Authors and Affiliations

  1. Vavilov Institute of General Genetics RAS, Gubkina str., 3, 119991, Moscow, Russia

    Maria A. Lagarkova & Sergei L. Kiselev

  2. Center for Reproductive Medicine, Krasnoyarsk, Russia

    Artem V. Eremeev & Anatoly V. Svetlakov

  3. Institute of Cytology and Genetics RAS, Novosibirsk, Russia

    Nikolay B. Rubtsov

Authors
  1. Maria A. Lagarkova
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  2. Artem V. Eremeev
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  3. Anatoly V. Svetlakov
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  4. Nikolay B. Rubtsov
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  5. Sergei L. Kiselev
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Correspondence to Sergei L. Kiselev.

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Editor: J. Denry Sato

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Lagarkova, M.A., Eremeev, A.V., Svetlakov, A.V. et al. Human embryonic stem cell lines isolation, cultivation, and characterization. In Vitro Cell.Dev.Biol.-Animal 46, 284–293 (2010). https://doi.org/10.1007/s11626-010-9282-6

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  • DOI: https://doi.org/10.1007/s11626-010-9282-6

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