To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.

The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission.

Cultured primary cells exhibit a finite proliferative lifespan, termed the Hayflick limit. Cloning by nuclear transfer can reverse this cellular ageing process and can be accomplished with cultured cells nearing senescence. Here we describe nuclear transfer experiments in which donor cell lines at different ages and with different proliferative capacities were used to clone foetuses and animals from which new primary cell lines were generated. The rederived lines had the same proliferative capacity and rate of telomere shortening as the donor cell lines, suggesting that these are innate, genetically determined, properties that are conserved by nuclear transfer.

In the present study, a zona-free nuclear transfer (NT) technique, which had been originally developed in cattle, was modified for the mouse. Steps involved in this approach include removing the zona pellucida and enucleating without a holding pipette; sticking donor cells to the cytoplast before electric pulses are applied to fuse them and culturing reconstructed embryos individually in single droplets, to prevent aggregation. Control zona-free and zona-intact embryos from mated donors showed no significant difference in development to blastocyst, but did show reduced development to term. Removal of the zona pellucida affected the response to activation by strontium in the absence of calcium as a significant proportion of zona-free control oocytes and embryos reconstructed by NT lysed during this treatment. A comparison between cumulus and ES cells as donor cells revealed significant differences in fusion efficiency (58.1 +/- 4.0%, n = 573 vs. 42.9 +/- 2.2%, n = 2064, respectively, p < 0.001), cleavage (77.2 +/- 3.4%, n = 334 vs. 40.8 +/- 2.7%, n = 903, respectively, p < 0.001) but not for development to morula/blastocyst (8.7 +/- 2.1%, n = 334 vs. 13.9 +/- 1.8%, n = 903, respectively, p < 0.1). The stage at which embryo development arrested was also affected by donor cell type. A majority of embryos reconstructed from cumulus cells arrested at two-cell stage, usually with two nuclei, whereas those reconstructed from ES cells arrested at one-cell stage, usually with two pseudo-pronuclei. After transfer of ES cell-derived NT embryos, a viable cloned mouse was produced (3.0% of transferred embryos developed to term). These observations establish that a zona-free cloning approach is possible in the mouse, although further research is required to increase the efficiency.

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