Effects of Culture Dimensions on Maintenance of Porcine Inner Cell Mass-Derived Cell Self-Renewal
Song Baek1, Na Rae Han1, Jung Im Yun2, Jae Yeon Hwang3, Minseok Kim4, Choon Keun Park1,5, Eunsong Lee6, and Seung Tae Lee1,5,*
1Department of Animal Life Science, 2Division of Animal Resource Science, Kangwon National University, Chuncheon 24341, Korea, 3Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven 06510, USA, 4Animal Nutrition and Physiology Team, National Institute of Animal Science, RDA, Wanju 55365, Korea, 5Division of Applied Animal Science, 6College of Veterinary Medicine, Kangwon National University, Chuncheon 24341, Korea
*Correspondence: stlee76@kangwon.ac.kr
Received September 19, 2016; Revised January 4, 2017; Accepted January 6, 2017.; Published online February 15, 2017.
© Korean Society for Molecular and Cellular Biology. All rights reserved.

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ABSTRACT
Despite the fact that porcine embryonic stem cells (ESCs) are a practical study tool, in vitro long-term maintenance of these cells is difficult in a two-dimensional (2D) microenvironment using cellular niche or extracellular matrix proteins. However, a three-dimensional (3D) microenvironment, similar to that enclosing the inner cell mass of the blastocyst, may improve in vitro maintenance of self-renewal. Accordingly, as a first step toward constructing a 3D microenvironment optimized to maintain porcine ESC self-renewal, we investigated different culture dimensions for porcine ICM-derived cells to enhance the maintenance of self-renewal. Porcine ICM-derived cells were cultured in agarose-based 3D hydrogel with self-renewal-friendly mechanics and in 2D culture plates with or without feeder cells. Subsequently, the effects of the 3D microenvironment on maintenance of self-renewal were identified by analyzing colony formation and morphology, alkaline phosphatase (AP) activity, and transcriptional and translational regulation of self-renewal-related genes. The 3D microenvironment using a 1.5% (w/v) agarose-based 3D hydrogel resulted in significantly more colonies with stereoscopic morphology, significantly improved AP activity, and increased protein expression of self-renewal-related genes compared to those in the 2D microenvironment. These results demonstrate that self-renewal of porcine ICM-derived cells can be maintained more effectively in a 3D microenvironment than in a 2D microenvironment. These results will help develop novel culture systems for ICM-derived cells derived from diverse species, which will contribute to stimulating basic and applicable studies related to ESCs.
Keywords: agarose, culture dimension, embryonic stem cells, pig, self-renewal


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