Incomplete or defective nuclear reprogramming may be responsible for low cloning efficiencies. Less differentiated stem cells are thought to be more easily reprogrammed, resulting in improved survival of cloned mice (Rideout WM III et al., 2000 Nat. Genet. 24, 109–110). Our objective was to establish porcine mesenchymal stem cell (MSC) cultures and use these as donor cells in nuclear transfer (NT). A bone marrow (BM) aspirate was collected from an anesthetized gilt. BM mononuclear cells were isolated by centrifugation over a density gradient (Histopaque-1077; Sigma, St. Louis, MO, USA), resuspended in low glucose DMEM (Gibco) plus 10% FBS and plated on flasks; fibroblast-like MSCs were later passaged. Ear skin fibroblast (SF) cultures from the same BM donor gilt were established. Cultures of MSC and SF were exposed to lipogenic, osteogenic or chondrogenic differentiation media (Pittenger MF et al., 1999 Science 284, 143–147) for 14 days. Cells cultured in DMEM with 10% FBS served as controls. Differentiation was assessed by histochemical methods. Calcium deposits and alkaline phosphatase (AP) activity (Vector Red AP Substrate Kit, Vector Labs) were indicative of osteogenic differentiation. MSCs cultured under osteogenic conditions were positive for AP activity and developed a black color after von Kossa staining, indicative of calcium deposition. Oil red O stain identified cellular lipid accumulation. When exposed to adipogenic differentiation media, 10–15% of MSCs developed an adipocyte phenotype with lipid droplet accumulation and oil red O staining. Lipogenic differentiation was not observed in SF and control cultures. Presence of acidic mucopolysaccharides associated with cartilage formation was determined by alcian blue stain. MSCs exposed to chondrogenic conditions were alcian blue-positive, and SF and control cultures were alcian blue negative. For NT, confluent (passage 2) MSC and SF cultures were exposed to roscovitine (15μM; Sigma) for 24h. In vitro-matured oocytes were enucleated and a single cell (MSC or SF) was transferred into the periviteline space. Cell-oocyte couplets were fused in Zimmerman’s medium with a single electric pulse (250V/mm for 20μs) delivered through a needle-type electrode. NT units were electrically activated (2 pulses of 100V/mm for 60μs separated by 5s) in a chamber 1h after fusion and transferred to NCSU-23 medium. Embryos were examined for cleavage and blastocyst formation at 2 and 7 days after NT, respectively. Cleavage rates were 53.3% (40/75) for MSC and 59.7% (46/77) for SF NT embryos. Development to blastocyst stage was 6.6% (5/75) in the MSC group and 1.2% (1/77) in SF group. In conclusion, we established an adult MSC line from a live animal using a minimally invasive BM aspiration technique. Additionally, MSC donor-derived NTs developed to the blastocyst stage. Further experiments will determine nuclear reprogramming in MSC-derived NT embryos.
Nuclear reprogramming in cloned cows
