Isolation and in vitro characterization of putative porcine embryonic stem cells from cloned embryos treated with trichostatin A.

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.

In vitro and in vivo characterization of putative porcine embryonic stem cells.

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.

Development of culture conditions for the isolation of pluripotent porcine embryonal outgrowths from in vitro produced and in vivo derived embryos.

In the present study we examined the effect of culture media and protein source on the formation of pluripotent primary outgrowths from in vitro produced and in vivo derived porcine embryos as the first step towards the isolation of embryonic stem cells (ESCs). To do this we compared high glucose Dulbeccos Modified Eagles Medium (DMEM) with Minimal Essential Alpha Medium (αMEM) both supplemented with fetal bovine serum (FBS) or serum replacement (SR) in a 2 × 2 factorial design. Culture in DMEM or αMEM supplemented with 10% SR resulted in the establishment of homogenous populations of cells which expressed Oct 4 and Nanog. In contrast culture in either media with FBS resulted in the formation of embryonal outgrowths composed entirely of differentiated cells or a mixture of differentiated cells and putative ESCs which grew poorly and could not be passaged. Using αMEM medium containing 10% SR and culturing in 5% oxygen, putative ESC lines were isolated from in vitro and in vivo derived embryos at efficiencies of 2 and 10% respectively.

Cytochalasin B and trichostatin a treatment postactivation improves in vitro development of porcine somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) is a useful technique for the production of transgenic pigs that can be used for biomedical research. However, the efficiency of SCNT in pigs is low. In this study, we examined the effect of two postactivation treatments, cytochalasin B (CB) and trichostatin A (TSA), on the in vitro development of porcine SCNT embryos. Treating porcine parthenotes with 7.5 microg/mL CB for 3 h after electrical activation was effective in preventing the extrusion of the second polar body in 65% of the oocytes compared to 17% in the control group. Treating SCNT embryos with CB for 3 h after electrical activation significantly increased the average blastocyst cell number compared to the control group (CB treatment 51, Control 34, p < 0.05). Treatment of porcine SCNT embryos with CB for 3 h and 50 nM TSA for 24 h after electrical activation resulted in a threefold increase in blastocyst rate (CB + TSA 64%, CB 20%, p < 0.05) and an increase in the average blastocyst cell number (CB + TSA 63, CB 46, p < 0.05), compared to CB treatment alone. These results show that treatment with TSA and CB significantly improves the in vitro morphological development and quality of porcine SCNT embryos.

Adding essential amino acids at a low concentration improves the development of in vitro fertilized porcine embryos.

The aims of this study were to investigate improvements to the pig preimplantation embryo culture system using in vitro produced embryos. For experiment 1, the optimum time to change the medium from NCSU23 containing 0.6 mM glucose, 0.2 mM pyruvate, 5.7 mM lactate and nonessential amino acids to NCSU23 containing 5.6 mM glucose and both essential and nonessential amino acids was examined. There were no statistically significant differences in blastocyst rates or cell number when the medium was changed at 48, 72 or 96 h, although there was a consistent trend for the 96 h treatment to produce fewer blastocysts with fewer cells. For experiment 2, the addition of essential amino acids at either a 1:50 or a 1:100 dilution of the purchased stock solution for day 1 to 6 or for days 3 to 6 only was investigated. Adding essential amino acids at a 1:50 dilution for day 3 to 6 significantly reduced the blastocyst rate and adding them at a 1:50 dilution from day 1 to 6 significantly reduced both the blastocyst rate and blastocyst cell number compared to when it was added at a 1:100 dilution. Embryos were produced by IVF, cultured for 6 days and good quality blastocysts were transferred into 6 synchronized pseudopregnant recipients (24 to 35 blastocysts per recipient) resulting in 4 pregnancies and 21 live birth piglets. These results show that adding essential amino acids at a 1:100 dilution provided the best culture conditions and the blastocysts produced were able to attain full term development after transfer.

Production of homozygous alpha-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer.

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.

Use of adult mesenchymal stem cells isolated from bone marrow and blood for somatic cell nuclear transfer in pigs.

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.