Efficient Derivation and Long Term Maintenance of Pluripotent Porcine Embryonic Stem-like Cells

Hye-Young Son,Eugene Lee,Hyun-Jong Kim,Hakhyun Ka,Hye-Sun Kim,Jin-Kyu Park,Jung-Eun Kim,Sang-Goo Lee,Chang-Kyu Lee

doi:10.5713/ajas.2009.80343

Abstract

Porcine embryonic stem (ES) cells have a great potential as tools for transgenic animal production and studies of regulation of differentiation genes. Although several studies showed successful derivation of porcine ES-like cells, these cells were not maintained long-term in culture. Therefore, this study was conducted to establish porcine pluripotent ES-like cells using in vivo fertilized embryos and to maintain these cells in long term culture. Porcine ES-like cells from in vivo embryos obtained by immunosurgery or whole explant culture were successfully cultured for over 56 passages. Morphology of porcine ES-like cells was flat-shaped with a monolayer type colony. These cells stained for alkaline phosphatase throughout the culture. Furthermore, porcine ES-like cells reacted with antibodies against Oct-4, SSEA-1, SSEA-4, Tra-1-60, and Tra-1-81, which are typical markers of undifferentiated stem cells. To characterize the ability of porcine ES-like cells to differentiate into three germ layers, embryoid body formation was induced. After plating of these cells, porcine ES-like cells were spontaneously differentiated into various cell types of all three germ layers. In addition, porcine ES-like cells were successfully derived from IVF blastocysts in media containing human recombinant basic fibroblast growth factor.

Highlights

Embryonic stem (ES) cells are undifferentiated and pluripotent cells established either from intact blastocysts (Evans et al, 1981) or isolated inner cell masses (ICMs) (Martin, 1981; Thomson et al, 1998) of preimplantation embryos
In vivo fertilized porcine embryos were cultured in DMEM/F10 (50% low glucose DMEM and 50% F10) or DMEM/F12 (50% high glucose DMEM and 50% F-12) with 15% FBS (Hyclone) or 20% Knockout serum replacement (KSR), 1% nonessential amino acids, 1.7 mM L-glutamine, 1% antibiotic-antimycotic, and 0.1 mM β-mercaptoethanol on a mytomycin-C (Roche, Mannheim, Germany) inactivated murine embryonic fibroblast (MEF) feeder layer
Six porcine ES-like cell lines out of 9 attached blastocysts were isolated from ICMs or whole blastocysts

Summary

INTRODUCTION

Embryonic stem (ES) cells are undifferentiated and pluripotent cells established either from intact blastocysts (Evans et al, 1981) or isolated inner cell masses (ICMs) (Martin, 1981; Thomson et al, 1998) of preimplantation embryos. The most common supplements used to establish and maintain ES-like cells in pigs include LIF (Shim et al, 1997; Saito et al, 2003; Li et al, 2004a), bFGF (Li et al, 2003; Li et al, 2004a), stem cell factor (SCF) (Saito et al, 2003), and various combination of these supplements (Piedrahita et al, 1998; Li et al, 2003; Tsung et al, 2003), or no supplemental growth factors (Chen et al, 1999; Mueller et al, 1999) None of these studies were demonstrated in long term culture of porcine ES-like cells while maintaining undifferentiated state. We tried to develop an efficient culture condition for the derivation of porcine ESlike cells from in vitro fertilized embryos using various media conditions

MATERIALS AND METHODS

Embryoid body formation and in vitro differentiation

Whole explant

RESULTS AND DISCUSSION

Final number of passages

CONCLUSION