Cultivation properties of human embryonic stem cells in the absence of serum and feeder layer


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Abstract

Cultivation conditions for five human embryonic stem cell lines (hESM01, hESM02, hESM03, hESM04, HES-9) were switched from serum replacement containing medium with mouse embryonic fibroblasts as a feeder to two commercialy available media (mTESR1 by StemCell Technologies, Canada and StemPro by lnvitrogen, USA). Cell lines were maintained in these media for more than 20 passages. Matrigel (BD Biosciences, USA) was used as a substrate. hESCs cultivated in defined media and without animal derived components retain their pluripotency and differentiation ability. Changes in morphology of feeder-free hESC colonies were observed. Feeder-free hESC cultivation gives possibility to get larger amount of cells due to increase of proliferative activity.

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About the authors

E. S. Philonenko

Vavilov lnstitute of general genetics

Author for correspondence.
Email: bozo.ilya@gmail.com
Russian Federation, Moscow

A. N. Kamnev

Vavilov lnstitute of general genetics

Email: bozo.ilya@gmail.com
Russian Federation, Moscow

V. G. Zautorov

Vavilov lnstitute of general genetics

Email: bozo.ilya@gmail.com
Russian Federation, Moscow

M. V. Shutova

Vavilov lnstitute of general genetics

Email: bozo.ilya@gmail.com
Russian Federation, Moscow

L. V. Tskhovrebova

Vavilov lnstitute of general genetics

Email: bozo.ilya@gmail.com
Russian Federation, Moscow

A. N. Bogomazova

Vavilov lnstitute of general genetics

Email: bozo.ilya@gmail.com
Russian Federation, Moscow

S. L. Kiselev

Vavilov lnstitute of general genetics

Email: bozo.ilya@gmail.com
Russian Federation, Moscow

M. A. Lagarkova

Vavilov lnstitute of general genetics

Email: bozo.ilya@gmail.com
Russian Federation, Moscow

References

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Supplementary files

Supplementary Files
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1. JATS XML
2. Fig. 1. Culture of hESC line hESM01: A- hESC colonies in bright field, 4th day. after passaging in mTESR-1 medium; B - triple immunohistochemical staining of hESC cells in the colony. Cells are stained with antibodies against oct-4 (red), b-tubulin (green). Nuclei are stained with OAPl (blue). Yellow arrows indicate cells in a state of mitosis, orange arrows indicate a dying cell with a fragmented nucleus

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3. Fig. 2. Expression of specific markers of hESCs during cultivation in feeder-free conditions: A - SSEA-4; B - TRA-1-60; B - CO-30. Nuclei were stained with OAPl. Scale - 100 mM

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4. Fig. 3. The result of RT-PCR analysis of the expression of specific transcription factors of the hESC line hESM01, when cultivated in the mTESR1 medium. m-molecular weight marker. Lanes (-) represent the negative control

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