Cloning & Transgenesis
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Multipotent Cell Types in Primary Fibroblast Cell Lines Used to Clone Pigs using Somatic Cell Nuclear Transfer

Sharon J. Harrison, Luke F.S. Beebe, Ivan Vassiliev, Stephen M. McIlfatrick and Mark B. Nottle

We have previously demonstrated that the use of porcine mesenchymal stem cells (MSCs) isolated from the bone marrow can increase the proportion of somatic cell nuclear transfer (SCNT) embryos that develop to the blastocyst stage compared with adult fibroblasts obtained from the same animal. The aim of the present study was to determine if MSCs are also present in primary cultures of adult fibroblasts which are commonly used for cloning live animals. To do this we chose a primary culture of adult fibroblasts that we had previously used to clone pigs. Single cell clones were isolated using low-density plating. After seven days of culture 63% of colonies displayed typical fibroblast morphology, while the remainder appeared cobblestone-like in appearance. Two of the 57 clones that displayed fibroblast morphology differentiated into adipocytes but not chondrocytes or osteocytes (uni-potent clones). Three of the 33 cobblestone-like clones differentiated into chondrocytes only, while 3 differentiated into adipocytes and chondrocytes but not osteocytes (bi-potent clones). One of the bi-potent cobblestone-like clones was then used for SCNT and in vitro development compared with a fibroblast-like clone which did not differentiate. Both cell types produced blastocysts at similar rates. In conclusion we have identified uni-potent and bi-potent cell types in primary cultures of adult fibroblasts used previously to clone live piglets.