Optimization of porcine embryonic germ cell culture system
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Optimization of porcine embryonic germ cell culture system Yang Li 1 & Yu Qiao 1 & Fei Li 1 & Hongjun Wang 2 & Xiao Dong 1 Received: 3 June 2020 / Accepted: 4 August 2020 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2020
Abstract Homologous feeder culture system can efficiently promote the proliferation of embryonic germ (EG) cells or embryonic stem (ES) cells while avoiding contamination by exogenous proteins and pathogens. In this study, we compared the potency of using homologous porcine embryonic fibroblasts (PEFs), gonadal stromal cells (GSCs), porcine adipose–derived stem cells (PASCs), or porcine amniotic fluid stem (PAFS) cells as feeder cells for porcine EG growth, with the commonly used mouse embryonic fibroblasts (MEFs). We compared the feeder cell growth rates; secretion of growth factors including stem cell factor (SCF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF); the effects of growth factors on porcine PGC growth; and EG growth rates when individual cells were used as feeders. Our results showed that feeder cells secreted limited amounts of growth factors, and supplementation of growth factors can significantly improve the formation of EG colonies and number of passages (P < 0.05). GSC and PEF were more suitable for EG growth because of their faster growth rate and their support on EG growth. In conclusion, this study identified novel homologous cells that can be used for EG production. Keywords EG . Homologous feeders . Porcine . Pluripotency . Differentiation
Introduction Embryonic stem (ES) and embryonic germ (EG) cells are widely used to study development, disease prevention, and treatment. Feeder cells are often used in ES/EG cell culture to provide stem cell matrix for cell attachment and growth. Feeder cells also secrete proliferation-promoting and differentiation-inhibiting factors, such as leukemia inhibitory factor (LIF), stem cell growth factor (SCF), and basic fibroblast growth factor (bFGF), and others (Matsui et al. 1992). Currently, SIM mouse embryo–derived thioguanine- and ouabain-resistant fibroblast cell line (STO) and mouse embryonic fibroblasts (MEFs) are the major cell types used as feeder cells (Evans and Kaufman 1981; Shiue et al. 2016). Although many groups including us have obtained pluripotent porcine EG on STO or MEF feeders (Shim et al. 1997; Piedrahita et al.
Yang Li and Yu Qiao contributed equally to this work. * Xiao Dong [email protected] 1
College of Life Science, Qingdao Agricultural University, 700 Changcheng Road, Qingdao 266109, Shandong, People’s Republic of China
2
Department of Surgery,, Medical University of South Carolina, Charleston, SC 29425, USA
1998; Mueller et al. 1999; Petkov et al. 2011; Dong et al. 2014, 2016), porcine EG germline chimeras have not been produced. In addition, many porcine EG generated so far proliferate slowly and differentiate easily, which suggest that the culture system need to be optimized. Compared with STO/MEF, homologous feeders can provide a microenvironment that resemble
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