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Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition

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Abstract

Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3′-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 μM BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers.

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Acknowledgment

We thank Professor Hao Qi in Shaanxi Normal University for histological characterization of teratomas. This study was supported by National Natural Science Foundation of China (31201865).

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Authors and Affiliations

  1. College of Animal Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China

    Xiaokun Liu, Qiang Wei, Junhong Zhang, Wanli Yang, Xiaoe Zhao & Baohua Ma

  2. Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Shanxi, 712100, China

    Xiaokun Liu, Qiang Wei, Junhong Zhang, Wanli Yang, Xiaoe Zhao & Baohua Ma

Authors
  1. Xiaokun Liu
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  2. Qiang Wei
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  3. Junhong Zhang
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  4. Wanli Yang
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  5. Xiaoe Zhao
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  6. Baohua Ma
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Corresponding author

Correspondence to Baohua Ma.

Additional information

Editor: T. Okamoto

Xiaokun Liu and Qiang Wei are equal contributors.

Electronic supplementary material

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Supplemental Table 1

Primers sequences used for reverse transcription PCR (DOC 37 kb)

Supplemental Fig. 1

Filtration of appropriate BIO concentration (JPEG 96 kb)

Supplemental Fig. 2

Expression of pluripotency-related transcript factor genes (JPEG 39 kb)

Supplemental Fig. 3

Expression of pluripotency-related transcript factor genes and differentiation marker genes (JPEG 15 kb)

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Liu, X., Wei, Q., Zhang, J. et al. Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition. In Vitro Cell.Dev.Biol.-Animal 51, 541–545 (2015). https://doi.org/10.1007/s11626-014-9863-x

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  • DOI: https://doi.org/10.1007/s11626-014-9863-x

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