Abstract
Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1–60 and TRA 1–81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.
Highlights
Pluripotent stem cells are capable of differentiating into all three embryonic germ layers and maintain their pluripotency and selfrenewal capacity over a long period of time [1]
Our results suggest that pluripotent stem cells derived from embryos and iPSCs derived from embryonic fibroblasts in the porcine model possess a primed pluripotent state similar to that of mouse epiblast stem cells (mEpiSCs) or human ES cells (hESCs), rather than to that of mouse ES cells (mESCs)
Two to three days following the seeding of day 8 in vitro hatched blastocysts, giant cells originating from trophectoderm initially proliferated and gradually died out or disappeared
Summary
Pluripotent stem cells are capable of differentiating into all three embryonic germ layers and maintain their pluripotency and selfrenewal capacity over a long period of time [1]. Since pluripotent cells were first derived from the inner cell mass (ICM) of mouse blastocysts [2], attempts to establish embryonic stem (ES) cell lines have been tried from various mammal species, including pigs [3], cattle [4], rats [5], primates [6] and humans [1] Among these various ES cell lines, different characteristics concerning morphology, signaling pathways and cell surface marker antigens are observed across species [7,8,9]. A second ‘‘primed’’ state has been described and is possible in mouse epiblast stem cells (mEpiSCs) or human ES cells (hESCs) [10,11,12] These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and Activin/ Nodal signaling pathways, expressions of SSEA4, TRA 1–60 and TRA 1–81 surface marker antigens and X chromosome inactivation in female [10,12,13]_ENREF_13
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