43 INFLUENCE OF DONOR CELL DIFFERENTIATION ON CLONING EFFICIENCY IN PIGS

Abstract

We recently reported the discovery of a novel type of stem cells which could be derived from primary cultures of fibroblasts by high density culture (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). The goal of the present study was to analyze the suitability of this specific stem cell population (fetal somatic stem cells, FSSCs) in NT and to test their ability to produce normal offspring upon transfer of cloned embryos. In the first of 4 experiments, FSSCs from isolated attached colonies were compared with fetal fibroblasts in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups [FSSCs: 75.3 ± 10.5% (mean ± SD) vs. 83.7 ± 9.2%; fetal fibroblasts: 64.8 ± 17.3% vs. 82.5 ± 5.6%, respectively]. Blastocyst rate differed significantly between the two groups (6.4 ± 3.5% vs. 24.9 ± 8.6%). In the second experiment, FSSCs of 3 different sizes (<14 µm, 15–20 µm, >21 µm), obtained from dissociation of spheroids, were compared in their ability to form blastocysts upon use in NT. No differences were found among the 3 groups (fusion rates: 93.0 ± 3.1 vs. 91.3 ± 10.1 vs. 92.3 ± 5.1; cleavage rates: 83.5 ± 7.9 vs. 83.1 ± 1.6 vs. 83.2 ± 5.8; blastocyst rates: 15.3 ± 7.9 vs. 17.6 ± 6.8 vs. 10.4 ± 2.7, respectively). In the third experiment, FSSCs 15–20 µm in size, derived from spheroids, were compared with fetal fibroblasts. No differences were detected between groups (fusion rates: 83.3± 7.3% vs. 86.8 ± 5.3%; cleavage rates: 86.1 ± 6.7% vs. 80.7 ± 5.9%; blastocyst rates: 21.4 ± 5.6% vs. 18.4 ± 5.6%, respectively). In the final experiment, 70–100 nuclear transfer complexes cloned from FSSCs were transferred immediately after activation to prepubertal gilts to evaluate their in vivo developmental potential. Pregnancies were established in 3 of 7 recipients, which delivered 7 piglets, of which 3 piglets were vital and showed normal development. Four piglets were lost due to dystocia. These results show that FSSCs are able to generate cloned embryos, and pregnancies can be established and vital piglets can be produced.