Cloning competence of various somatic cell types
Many somatic cell types, including mammary epithelial cells, ovarian cumulus cells, fibroblast cells from skin and internal organs, various internal organ cells, Sertoli cells [38,56], macrophage [56] and blood leukocytes [34,35] have been successfully utilized for nuclear transfer. A clear consensus, however, has not yet been reached as to the superior somatic cell type for nuclear transfer. This is due in part to the fact that different laboratories employ diverse procedures; and cell culture, nuclear transfer, and micromanipulation all require critical technical skills. In order to make these comparisons valid, the procedures and techniques used, as well as the skill of lab personnel, must be identical for each donor animal and cell type. To compare the competence of different cell types for reprogramming by cloning, we avoided animal variation by looking at the cloning competence of three cell types: ovarian cumulus, mammary epithelial and skin fibroblast cells, all from the same donor animal, a 13-year-old elite diary cow.
The ability of donor cells to be reprogrammed was assessed by the development of cloned embryos in vitro and by the birth of cloned calves following embryo transfer. As shown in Tables 2 and 3, although no differences were detected in the cleavage rates of embryos from three different cell types, cumulus cells produced the highest rate of blastocyst development in this study and resulted in 6 full-term cloned calves. Furthermore, four out of the six calves derived from cumulus cells survived and were still healthy at nearly 4 years of age (Table 3). In contrast, the poorest in vitro development, and no full-term survival, was obtained with mammary epithelial cells. Skin fibroblast cells resulted in an intermediate rate of in vitro development and gave rise to 4 full-term cloned calves.
Table 2  Summary of in vitro development of cloned embryos from different cell types
Cell types  No. reconstructed embryos  Embryo development (%)
Cleavage  Blastocyst
Cumulus  92  65a  57a
Fibroblast  110  63a  34b
Epithelium  96  66a  23c
Numbers with different superscripts within columns are significantly different (P < 0.05).
Table 3  Summary of embryo transfer and calving of cloned embryos from different cell types
Cell type  No. embryo Transferred  No. recipients  No. (%) calves born  Alive to adulthood
Total  Pregnant*
Cumulus  109  58  10  6 (5.5)**  4
Fibroblast  57  29  8  4 (7.0)**  0
Epithelium  34  24  1  0  0
*: Pregnancy determined by ultrasound examination at 60 days of gestation. **: A set of twins included. Our results showed that the donor cell type can significantly affect embryo development in vitro as well as in vivo. Cumulus cells proved to be the most effective cell type for somatic cloning according to both the in vitro development test as well as full-term survival. These results suggest that DNA from cumulus cells is more effectively reprogrammed following nuclear transfer. Our results agreed with those obtained in mice [57] where they compared the nuclear transfer efficiency of neuronal, Sertoli and cumulus cells, and obtained the best live birth rate from cumulus cell-derived cloned embryos. Furthermore, it was reported that cumulus cell-derived cloned mice do not have widespread dysregulation of imprinting [23]. Kato et al. [15,36] compared cells from the liver, testis, skin, ear, along with cumulus and oviductal cells and concluded that cumulus and oviduct epithelial cells are the most suitable for nuclear donors. Evidence supporting the superiority of cumulus cells for nuclear transfer also comes from the study of Forsberg et al. [58] who conducted large numbers of embryo transfer in cattle. It was shown that cumulus cells gave an overall 15.2% calving rate, while fetal genital ridge cells, and fibroblast cells produced a 9% calving rate. Adult fibroblast cells, in this study, gave the lowest calving rate of only 5%.
In summary, among the somatic cell types tested, the consensus from numerous laboratories is that cumulus cells give the highest cloning efficiency and result in the least number of abnormalities in cloned animals.