Introduction Pluripotency is transiently induced during the

Introduction Pluripotency is transiently induced during the early stages of mammalian embryo development. Blastocyst stem baicalein progress from a “naive/ground” state to a “primed” state of pluripotency before lineage commitment (Martinez Arias et al., 2013). Two distinct pluripotent stem cells, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), are considered as their in vitro counterparts. ESCs and EpiSCs differ with respect to their morphology, metabolism, DNA methylation levels, transcription profiles, and growth factors requirement (Weinberger et al., 2016). Pluripotency states are unstable and thus difficult to stabilize in vitro. Indeed, ESC cultures consist of heterogeneous cells dynamically fluctuating between different pluripotent states (Hayashi et al., 2008; Toyooka et al., 2008). EpiSCs frequently lose the primed state, acquiring features of late pre-gastrula embryos (Wu and Izpisua Belmonte, 2015). Known molecular determinants of such plasticity are mainly transcription factors, while the role of metabolism has been largely unexplored until recently. Indeed, it has now become evident that metabolites, including amino acids, act as key regulators of pluripotent stem cell plasticity and behavior. For instance, it has been shown that ESC self-renewal depends on l-threonine (Wang et al., 2009), while ESC identity is regulated by l-proline (l-Pro) availability (Casalino et al., 2011; Comes et al., 2013; D\'Aniello et al., 2015; Washington et al., 2010). Moreover, several metabolites act as epigenetic signals (Blaschke et al., 2013; Comes et al., 2013; Shyh-Chang et al., 2012), thus defining a regulatory network among metabolism, epigenetic modification, and pluripotency, knowledge of which is still limited (Harvey et al., 2016). Here we provide evidence that pluripotency is finely controlled by the mutual availability of two physiological metabolites, vitamin C (VitC) and l-Pro, and propose that naive and early primed pluripotency states can be captured in vitro by exploiting the epigenetic activity of these metabolites.
Results
Discussion
Experimental Procedures Full details of the Experimental Procedure are included in Supplemental Experimental Procedures.
Author Contributions
Acknowledgments We are grateful to members of the Integrated Microscopy and FACS Facilities of IGB-ABT, CNR. We thank Gennaro Andolfi for excellent technical assistance. This study was supported by Epigenomics Flagship Project (EPIGEN) MIUR-CNR to G.M. and C.A., and AIRC (grant 11599), Italian Ministry of Education-University-Research (grant CTN01_00177 Cluster ALISEI_IRMI and PRIN) and CARIPLO to G.M.
Introduction Somatic cells can be reprogrammed to pluripotency through the ectopic expression of defined factors such as Oct4, Sox2, Klf4, and c-Myc (Takahashi et al., 2007; Takahashi and Yamanaka, 2006). The resultant induced pluripotent stem cells (iPSCs) provide unlimited cell sources in studying disease and in regenerative medicine (Yamanaka, 2012). Pigs show many similarities to humans in organ anatomy, physiology, and metabolism (Vodicka et al., 2005) and could be a suitable source of xenotransplantation and a model for the study of human diseases (Giraud et al., 2011). Derivation of pig iPSCs can complement research on human iPSCs (Montserrat et al., 2010). With fewer ethical concerns, robust transplantation experiments using pigs as a model could test the safety and effectiveness of iPSCs in pre-clinical translational medicine, such as retinal (Zhou et al., 2011) and myocardial therapy (Li et al., 2013). Currently, the main obstacle in achieving fully reprogrammed porcine iPSCs (piPSCs) is inadequate silencing of exogenous genes (Esteban et al., 2009; Ezashi et al., 2009; Fujishiro et al., 2013; Montserrat et al., 2012; West et al., 2010), even after using non-integrating episomal plasmids (Du et al., 2015) or Sendai virus (Congras et al., 2016).