Derivation of stable ESC lines has been reported for the mouse 
, hamster 
, rhesus monkey 
, rat 
, and human 
, but validated ESC lines have yet to be established in farm animal species. There are several reasons for the reported difficulties in deriving ESCs from large animals including difficulty in isolating cells at the appropriate stage of embryonic development, suboptimal culture conditions and improper cell passaging methods. Although production of sheep ESC-like cells from early embryos has been reported, these sheep ESC-like cells have not been able to be maintained beyond two passages 
. Induction of pluripotency in somatic cells by defined factors has been reported in large animals such as pig and dog 
. Therefore, iPSC technology offers hope for a practical method by which pluripotent stem cells may be generated from other domestic animals. Here, we generated pluripotent stem cells using drug-inducible expression of defined factors in sheep fetal fibroblasts.
The Doxycycline (Dox) controlled tet-on-inducible system has successfully been applied to generate iPSCs from mouse, human and pig somatic cells 
. Use of this system is a predictable and highly reproducible platform for iPSC generation and should facilitate the study of early molecular events leading to epigenetic reprogramming of somatic cells 
. In the present study, we used the Dox controlled tet-on-inducible system to generate and maintain stable sheep ESC-like colonies from fetal fibroblasts. As addition of Dox to culture media was necessary for the induction and maintenance of sheep ESC-like colonies, it is likely that the culture conditions used in this paper are not sufficient to maintain pluripotency in sheep iPSCs alone. Withdrawal of Dox in cultures of stably reprogrammed iPSCs resulted in the silencing of exogenous genes, and differentiation of cells as evidenced by a switch from positive to negative AP staining. To culture sheep iPSCs without use of a drug inducible system, certain growth factors or chemical inhibitors may be required to prevent differentiation. The challenge of optimizing culture conditions for maintenance of pluripotency is the primary reason stable ESC lines have yet to be established in large animals 
. Cytokines and growth factors that inhibit spontaneous differentiation in mouse and primate ES cell lines such as LIF and bFGF do not inhibit differentiation of pluripotent cells for many large animal ICM and epiblast primary cultures 
. In this study, sheep iPSCs were not able to be maintained in an undifferentiated state following withdrawal of Dox even when hLIF and hFGF was added to the medium. Although cells were characterized by a cobble stone patterning with large nucleoli, sheep iPSC colonies generated from the drug-inducible system did not possess a defined, highly refractive edge characteristic of human or mouse ESC and iPSCs. The differences in cell morphology between sheep iPSCs and pluripotent stem cells derived from other animals may be due to imperfect culturing conditions. The maintenance of sheep iPSCs under this drug-inducible system provides a useful platform for studying the role of different cytokines and growth factors in sheep iPSCs self-renewal. This in turn should facilitate the optimization of culture conditions for iPSCs maintenance, and eventual establishment of true ESCs from sheep blastocysts.
While FBS has been used for the establishment of mouse ESC and iPSC lines in a majority of laboratories 
, knockout serum replacement (KSR), which is a defined, serum-free formulation, has been widely used to support the growth of human and primate ESCs and iPSCs in culture 
. Although detailed information concerning its composition is restricted, KSR does not contain any undefined growth factors or factors that promote differentiation 
. Our results showed that the replacement of KSR with FBS in culture medium improved the reprogramming efficiency and integrity of sheep iPSCs. These results suggest that serum substrates in FBS may play a vital role in generation of sheep iPSCs, and that these putative factors are not included in KSR.
Cell surface markers provide a powerful tool for characterization and isolation of pluripotent stem cells. Therefore, identification of surface markers expressed on iPSCs is important for pluripotent stem cells. SSEAs including SSEA-1 SSEA-3 SSEA-4, have widely used as cell surface marker to monitor pluripotency of ESCs. Dattena et al. reported that sheep ESCs express SSEA-1, SSEA-3, and SSEA-4 
. In this study, sheep iPSCs showed expression of SSEA-4, but not SSEA-1 and SSEA-3. Moreover, our results indicate that sheep iPSCs also lack Tra-1-60 and Tra-1-81, which are characteristic of human ESCs and iPSCs 
. For future experiments it will be important to compare global gene expression and epigenetic status of pluripotent cell-specific genes between sheep ESCs, iPSCs and somatic cells targeted for reprogramming such as fibroblasts to understand the molecular processes behind induction of pluripotency in sheep cells. Unfortunately, these assays will be difficult to conduct in a sheep model without the release of more comprehensive sheep genomes and commercial sheep gene-chips.
An important proof for the establishment of ungulate pluripotent stem cell lines is demonstration of pluripotency either by differentiation into defined cell types in vitro
or by teratoma formation in vivo
. These proofs have been common practice with both mouse and primate ESC and iPSC lines 
. In the present study, pluripotency of sheep iPSCs was demonstrated by derivation of cells of all three germ layers in EB and teratoma formation assays. However, future work such as chimera formation though injection of sheep iPSCs into developing blastocysts must be performed to truly confirm that these cells are ESC-like.