In Vitro and In Vivo Characterization of Putative Porcine Embryonic Stem Cells

Abstract

We have developed a new method for the isolation of porcine embryonic stem cells (ESCs) from in vivo-derived and in vitro-produced embryos. Here we describe the isolation and characterization of several ESC lines established using this method. Cells from these lines were passaged up to 14 times, during which they were repeatedly cryopreserved. During this time, ESCs maintained their morphology and continued to express Oct 4, Nanog, and SSEA1. These cells formed embryoid bodies in suspension culture, and could be directed to differentiate into various lineages representative of all three germ layers in vitro. When injected into blastocysts these cells localized in the inner cell mass of blastocysts. To examine their pluripotency further, cells were injected into host blastocysts and transferred to recipient animals. Of the six transfers undertaken, one recipient became pregnant and gave birth to a litter of one male and three female piglets. Microsatellite analysis of DNA extracted from the tail tissue of these piglets indicated that two female piglets were chimaeric.

The current state of reproductive biology research in Australia and New Zealand: core themes from the Society for Reproductive Biology Annual Meeting, 2016

“…This question is confounded by the fact that the loss of pluripotency and the onset of cell differentiation occur at different stages for different species (at least for mice, humans, and pigs); suggesting caution is required when drawing conclusions from experimental models. For example, mouse embryonic stem cells (ESC) are more pluripotent than human embryonic stem cells at equivalent stages of embryo development (Vassiliev et al 2010;Davidson et al 2015). Furthermore, there are limited naïve human ESC lines used for research, restricting the full potential of future ESC therapies (Ware 2014).…”

Effect of cytochalasin types on the production of heterozygous parthenogenetic porcine embryos and the isolation of putative parthenogenetic embryonic stem cells

“…pESC were isolated from parthenotes as described previously [11][12][13]. Day-7 blastocysts with zona pellucidae removed were gently pressed onto mitotically inactivated MEF feeder layers using a 30-gauge syringe and cultured in porcine ESC isolation medium for 6-12 days to obtain embryonal outgrowths.…”

“…Medium was changed every 4-6 days. Putative porcine pESCs were identified according to morphological criteria described previously [11][12][13] including polygonal shape, 10-15 μm in diameter, a small cytoplasmic/nuclear ratio, multiple lipid inclusions in the cytoplasm and a single nucleus with multiple nucleoli. Homogenous embryonal outgrowths, which had reached 3-5 mm in diameter, were cut into 200 μm square pieces and re-plated onto fresh feeder layer to passage the cells.…”

“…pESCs were vitrified and warmed as previously described [11]. Briefly, pESC colonies were cut into pieces and vitrified using the CryoLogic Vitrification Method (CVM; CryoLogic Pty.…”

“…The aim of the present study therefore was to determine the efficiency with which CB and CD could be used to generate heterozygous parthenotes in pigs and whether pESCs could be isolated from these using a method developed previously by us for the isolation of porcine ESCs from in vitro produced and somatic cell nuclear transfer (SCNT) embryos [11][12][13].…”