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.

Mesenchymal stem cells (MSCs) isolated from bone marrow were used to examine the hypothesis that a less differentiated cell type could increase adult somatic cell nuclear transfer (SCNT) efficiencies in the pig. SCNT embryos were produced using a fusion before activation protocol described previously and the rate at which these developed to the blastocyst stage compared with that using fibroblasts obtained from ear tissue from the same animal. The use of bone marrow MSCs did not increase cleavage rates compared with adult fibroblasts. However, the percentage of embryos that developed to the blastocyst stage was almost doubled, providing support for the hypothesis that a less differentiated cell can increase cloning efficiencies. As MSCs are relatively difficult to isolate from the bone marrow of live animals, a second experiment was undertaken to determine whether MSCs could be isolated from the peripheral circulation and used for SCNT. Blood MSCs were successfully isolated from four of the five pigs sampled. These cells had a similar differentiation capacity and marker profile to those isolated from bone marrow but did not result in increased rates of development. This is the first study to our knowledge, to report that MSCs can be derived from peripheral blood and used for SCNT for any species. These cells can be readily obtained under relatively sterile conditions compared with adult fibroblasts and as such, may provide an alternative cell type for cloning live animals.

We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White x Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half-sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half-sib and subsequently a full-sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.

We report here the establishment and characterization of putative porcine embryonic stem cell (ESC) lines derived from somatic cell nuclear transfer embryos (NT-ESCs). These cells had a similar morphology to that described previously by us for ESCs derived from in vitro produced embryos, namely, a polygonal shape, a relatively small (10-15 μm) diameter, a small cytoplasmic/nuclear ratio, a single nucleus with multiple nucleoli and multiple lipid inclusions in the cytoplasm. NT-ESCs could be passaged at least 15 times and vitrified repeatedly without changes in their morphology, karyotype, or Oct-4 and Nanog expression. These cells formed embryoid bodies and could be directed to differentiate in vitro to cell types representative of all three germ layers. Following their injection into blastocysts, these cells preferentially localized in the inner cell mass. In conclusion, we have isolated putative porcine ESCs from cloned embryos that have the potential to be used for a variety of applications including as a model for human therapeutic cloning.

Abstract. The present study examined whether delipated porcine oocytes and embryos at various stages of development can be cryopreserved by conventional slow cooling or vitrification. Most (93%) of the 27 delipated morulae developed to blastocysts after freezing with 1.5 M propanediol + 0.1 M sucrose. Late morulae and early blastocysts delipated at 2-4 cell stage and cultured in vitro survived freezing either with 1.5 M glycerol + 0.25 M sucrose (10/18, 56%) or 1.8 M ethylene glycol + 0.25 M sucrose (14/19, 74%). Delipated 2-4 cell stage embryos and oocytes could be cryopreserved by vitrification with 40% ethylene glycol, 1 M sucrose and 20% fetal calf serum. Half (7/14) of the vitrified, delipated embryos developed to blastocysts after thawing. Of 48 delipated oocytes, 27 (56%) maintained an intact outline of the ooplasm after vitrification and underwent subzonal sperm injection. Fertilization was confirmed in 12 (25%) of these oocytes and 3 (6%) developed to morula stage. This study also aimed at developing a non-invasive method for cryopreserving porcine embryos after reducing their cytoplasmic lipid content without micromanipulation. Morulae and early blastocysts were centrifuged in the presence of cytochalasin B and cryoprotectants and then frozen immediately. More than half (14/24, 58%) of the centrifuged morulae developed to blastocycts after freezing with 1.5 M propanediol + 0.1 M sucrose. Greater than 70% of centrifuged early blastocysts survived freezing either with 1.5 M propanediol (30/31, 97%), 1.5 M glycerol (22/29, 76%) or 1.8 M ethylene glycol (21/29, 72%). These results demonstrated that delipation (lipid removal) from porcine oocytes and embryos at various stages enables their cryopreservation. A new insight into the development of a non-invasive method for cryopreserving porcine embryos was also provided.