Here we describe a procedure for cloning pigs by the use of in vitro culture systems. Four healthy male piglets from two litters were born following nuclear transfer of cultured somatic cells and subsequent embryo transfer. The initiation of five additional pregnancies demonstrates the reproducibility of this procedure. Its important features include extended in vitro culture of fetal cells preceding nuclear transfer, as well as in vitro maturation and activation of oocytes and in vitro embryo culture. The cell culture and nuclear transfer techniques described here should allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes, ID1, BCL2L1 and HM13, expressed in human ES cells, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

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Human embryonic stem (ES) cells are known to derive from the inner cell mass of blastocyst. Although the embryos of other developmental stages have also been used as a source for ES cells in animal models, the feasibility of obtaining ES cell lines from human morula is not known, despite being an obvious source available through assisted reproduction and preimplantation genetic diagnosis programmes. This study describes an original technique for derivation of ES cells from human morula, which enabled the establishment of eight morula-derived ES cell lines. These ES cell lines were shown to have no morphological differences from the ES cells derived from blastocysts, and expressed the same ES cell specific markers, including Oct-4, tumour-resistance antigens TRA-2-39, stage-specific embryonic antigens SSEA-3 and SSEA-4, and high molecular weight glycoproteins TRA-1-60 and TRA-1-81, detected in the same colony of morula-derived ES cells showing specific alkaline phosphatase expression. No differences were observed in these marker expressions in the morula-derived ES cells cultured in the feeder layer free medium. Similar to ES cell originating from blastocyst, the morula-derived ES cells were shown to spontaneously differentiate in vitro into a variety of cell types, including the neuron-like and contracting primitive cardiocyte-like cells.

Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.

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The pregnancy initiation and maintenance rates of nuclear transfer embryos produced from several bovine cell types were measured to determine which cell types produced healthy calves and had growth characteristics that would allow for genetic manipulation. Considerable variability between cell types from one animal and the same cell type from different animals was observed. In general, cultured fetal cells performed better with respect to pregnancy initiation and calving than adult cells with the exception of cumulous cells, which produced the highest overall pregnancy and calving rates. The cell type that combined relatively high pregnancy initiation and calving rates with growth characteristics that allowed for extended proliferation in culture were fetal genital ridge (GR) cells. Cultured GR cells used in nuclear transfer and embryo transfer initiated pregnancies in 40% of recipient heifers (197), and of all recipients that received nuclear transfer embryos, 9% produced live calves. Cultured GR cells doubled as many as 85 times overall and up to 75 times after dilution to single-cell culture. A comparison between transfected and nontransfected cells showed that transfected cells had lower pregnancy initiation (22% versus 32%) and calving (3.4% versus 8.9%) rates.

A total of 14 microsurgically produced zona pellucida-free bovine demi-blastocysts were cultured for 3 days in tissue culture medium (TCM) 199 supplemented with 10% heat-inactivated newborn calf serum (NBCS). Developing embryos were continuously cultured in TCM 199 plus 10% NBCS on a feeder-layer of murine embryonic fibroblasts, that had been incubated with mitomycin C (10 μg/ml) for 3 h prior to the onset of embryo cultivation to block mitotic activity of the fibroblasts. After 2 days, 3 expanded blastocysts were attached to the feeder-layer and both trophoblastic cells and inner cell mass (ICM) cells became apparent on the 9th day of culture in 2 out of the 3 expanded blastocysts. Five days later, the ICM cells were disaggregated by a short-term trypsin treatment. The resulting dissociated clumps were seeded on a new murine embryonic fibroblast feeder-layer and covered with modified minimum essential medium (MEM)-Alpha with 10% fetal calf serum (FCS), 0.1 mm mercaptoethanol, 4.5 g/l glucose and 20 mM HEPES-buffer (=passage 0). To prevent differentiation of the cells, approximately 1/3 of the MEM-Alpha was replaced by MEM previously incubated on cell line 5637 containing leucaemia inhibitory factor (LIF) for 3 days. Colonies of embryonic stem cell (ES)-like cells were observed 5 days after the 1st passage. These colonies were repeatedly passaged at approximately 2-week intervals. Two bovine ES-like cell lines were established, which grew considerably slower than murine ES cells, but were lost after the 4th passage, possibly because of toxic effects of a new FCS batch. After cytogenetic analysis, 16 out of 18 metaphase plates contained an euploid number of chromosomes with 2 X-chromosomes and 58 autosomes. Distribution of G-banding on the chromosomes of ES-like cells was in accordance with the diploid set of the bovine genome. ES-like cells were fused to in vitro matured bovine oocytes and, upon successful fusion, cultured in vitro over 5 days. Successful fusion was observed in 79.8% (67/84), 31.3% initiated cleavege and 10.4% reached the 8-16 cell stage at termination of culture.