Primed Pluripotent Cell Lines Derived from Various Embryonic Origins and Somatic Cells in Pig

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.

LIF signaling in stem cells and development

“…As such, and possibly as an evolutionary prevention of the development of this malignancy, JAK-STAT signaling is muted in pluripotent stem cells derived from many different species, including most strains of mice (non-permissive strains), and is unable to maintain their pluripotency (Brons et al, 2007;Park et al, 2013;Thomson et al, 1998Thomson et al, , 1995Wang et al, 2008). Indeed, the first mESC lines were derived from strains of mice (129) that spontaneously generate teratocarcinomas (Stevens and Little, 1954).…”

Porcine pluripotent stem cells: progress, challenges and prospects

“…We summarize below the methods used to evaluate the pluripotency of porcine stem cells in recent decades. (Tables 4-6), which is very similar to human ESCs or mouse epiblast stem cells (EpiSCs) [29,115,121,138,147,148] . They show a large, flat and round (polygonal in rare cases) shape with compact colonies and distinct borders, with relatively small diameters and a high nucleus-to-cytoplasm ratio, a single nucleus with multiple nucleoli, and are sensitive to tryptase.…”

“…Generally, the culture medium consists of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2-mercapthoethanol, L-glutamine, antibiotics, nucleosides, non-essential amino acids, fetal bovine serum (FBS) and different cytokines, and alternative components. For the basic medium, variations of DMEM from high glucose to low glucose [27,28] , α-MEM [29,30] , KO-DMEM [10,31] , and combinations of DMEM with Ham's-F10 [32,33] or F12 [10,31] have been examined. The results from different studies are not consistent, but pESC-like cell lines have predominantly been derived from DMEM medium without consideration of glucose concentrations.…”

“…Another type of porcine PSCs was shown to have a mouse ESCs morphology, i.e., small, not flat and with a compact, glistening, doom shaped appearance. These cells have a high nuclear-to-cytoplasmic ratio with short cell cycle interval, grow more vigorously and are capable of undergoing successful differentiation both in vivo and vitro, and are considered to have high developmental potential and show naive status of porcine PSCs [29,49] . In general, morphology of stem cell represents a basic indicator for pluripotency of Note: AP, alkaline phosphatase; EB, embryoid body; CP, chimeras piglets; CB, chimeric blastocysts; ND, not determined.…”

Use of pluripotent stem cells for reproductive medicine: are we there yet?

“…Also, day 6-7 blastocysts as well as ICMs from parthenogenetically activated porcine embryos appear to have an increased ESC derivation rate (Chen et al 1999;Brevini et al 2010). It is now possible to derive porcine ESCs from in vitro fertilized, in vivo derived, IVF aggregated and parthenogenetic embryos (Park et al 2013). However, in order to derive authentic porcine ESCs, the current culture conditions still require optimization (Hall et al 2013).…”