Why is it so Difficult to Derive Pluripotent Stem Cells in Domestic Ungulates? F Gandolfi, G Pennarossa, S Maffei and TAL Brevini Department of Health, Animal Science and Food Safety, Universita ` degli Studi di Milano, Milan, Italy Content Pluripotent stem cells are the focus of an extremely active field of investigation that is bringing new light on our understand- ing of the mechanisms that control pluripotency and differen- tiation. Rodent and primates are the only species where true, or bona fide, pluripotent stem cells have been derived. The attempts to derive pluripotent stem cells from domestic ungulates have been going on for more than 20 years with little progress. Cell lines from these species present a series of limitations that have precluded their use for both basic and clinically oriented studies. However, in the last 3 years, some substantial progress have been made making the currently available ungulate pluripotent stem cells closest than ever before to their human and mouse counterpart. This result has been achieved through both conceptual and technical progress that will be illustrated and discussed in this review. Introduction Organs counteract the physiological wear and tear thanks to a small population of cells known as organ- specific or adult stem cells. They are classified on the basis of their potency that can span from unipotency when only a single cell type can be generated to multipotency when a stem cell can originate to all or many cells of a single germ layer. When a stem cell can originate, all cells from all three germ layers are defined as pluripotent. Pluripotency is limited to the epiblast, a transient tissue that exists only for brief stage of embryonic development; therefore, stable pluripotent cells are not a physiological component of the body but exist only in vitro (Smith 2001). The translation of the knowledge on stem cell biology into clinical applications is taking place at an increas- ingly fast pace. The ease of such translation depends on a delicate balance that includes the degree of plasticity of the stem cell of interest, its physiological rate of proliferation and its accessibility. As a result, clinical applications of stem cells of say hematopoietic (Shepp- ard et al. 2012) or epithelial (De Luca et al. 2006; Pellegrini et al. 2009) origin are well advanced and in many case have already entered the level of accepted procedures, whereas the use of stem cells such as those of muscle (Tedesco et al. 2010) or neuronal origin (Lindvall and Kokaia 2010) are still far from a clinical application both because their proliferation potential is more limited and because they are not easy to access especially those located in the central nervous system. These limitations could be overcome by the use of pluripotent stem cells that are attractive because easy to propagate in vitro and therefore readily available. An important step for the translation of basic research into clinical applications is the use of animal models that are intermediate between laboratory rodents and humans. Domestic ungulates like ruminants and pig have often been used for pre-clinical research and their use in regenerative medicine could be benefi- cial as well. However, whereas the derivation of organ-specific stem cells has been successful (Spencer et al. 2011), pluripotent stem cells have so far been difficult to obtain in these species. True Embryonic Stem Cells It is widely acknowledged that true, so-called bona fide, embryonic stem cells (ESC) can only be derived from mouse (Evans and Kaufman 1981), human (Thomson et al. 1998) and non-human primate embryos (Thomson et al. 1995). Cell lines derived from these species share some major properties such as unlimited replication in vitro (self- renewal), capacity to differentiate into any of the different tissues that make the body, expression of core pluripotency factors such as OCT4, SOX2 and NANOG, formation of teratomas if injected into immunodeficient mice. However, in the years following the derivation of primate ESC, it became progressively clear that they present substantial differences when compared to mouse ESC (mESC), even if they share the same definition of ESC. Differences begin in the culture dish where the presence of LIF and BMP4 in the culture medium is required to maintain the undifferentiated state of pro- liferating mouse ESC through the activation of the JAK ⁄ STAT3 pathway (Ying et al. 2003); however, the stimulation of the same pathways in primate ESC leads to a rapid differentiation (Xu et al. 2005). On the contrary, primate ESC require the presence in the culture medium of activin A and ⁄ or basic fibroblast growth factor (bFGF or FGF2) and depend on the activation of the activin ⁄ Nodal pathway (Xu et al. 2008). Mouse ESC can be derived only from a small number of ‘permissive’ strains, whereas primate ESC, and human ESC in particular, show no limitations related to a specific genetic background. Mouse and primate ESC differ also in their morphology. Small, compact and domed colonies are typically formed by mESC as opposed to primate ESC, which grow in larger, flat colonies. mESC colonies are propagated after dissociation to single cells, but the same treatment would rapidly kill primate ESC whose colonies need to be detached from the feeder layer and fragmented mechanically. In general, mESC grow more vigorously and can easily adapt to clonal culture conditions enabling the derivation of lines from single cells. This Reprod Dom Anim 47 (Suppl. 5), 11–17 (2012); doi: 10.1111/j.1439-0531.2012.02106.x ISSN 0936-6768 Ó 2012 Blackwell Verlag GmbH