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Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts

Published online by Cambridge University Press:  01 February 2007

H-S. Kim ,
H-Y. Son ,
S. Kim ,
G-S. Lee ,
C-H. Park ,
S-K. Kang ,
B-C. Lee ,
W-S. Hwang  and
C-K. Lee
H-S. Kim
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul, Korea.
H-Y. Son
Affiliation:
Department of Food and Animal Biotechnology, Seoul National University, Seoul 151–742, Korea.
S. Kim
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul, Korea.
G-S. Lee
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul, Korea.
C-H. Park
Affiliation:
Department of Food and Animal Biotechnology, Seoul National University, Seoul 151–742, Korea.
S-K. Kang
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul, Korea.
B-C. Lee
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul, Korea.
W-S. Hwang
Affiliation:
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul, Korea.
C-K. Lee*
Affiliation:
Department of Food and Animal Biotechnology, Seoul National University, Seoul 151–742, Korea.
*
All correspondence to: C-K. Lee, Department of Food and Animal Biotechnology, Seoul National University, Seoul 151–742, Korea. Tel: +82 2 880 4805. Fax: +82 2 873 4805. e-mail: [email protected]
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Summary

The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.

Keywords

BlastocystsIn vitro fertilizationInner cell massPorcineSomatic cell nuclear transfer
Type
Research Article
Information
Zygote , Volume 15 , Issue 1 , February 2007 , pp. 55 - 63
Copyright
Copyright © Cambridge University Press 2007

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