i LIF ESCs serum LIF ESCs EPL cells and EpiSCs

2i/LIF ESCs, serum/LIF ESCs, EPL cells, and EpiSCs may represent different states of pluripotency, with 2i cells placed at the naive end of the hierarchy and EpiSCs at the primed end. Depending on the in vitro conditions for derivation, epiblast cells of a late atr inhibitor will settle on different positions along a descending gradient of pluripotency (conceptualized as a series of terraces on the slope) (Fig. 2 — permissive rodents). 2i/LIF conditions appear to have captured the cells at the state of pluripotency of the epiblast of the E3.75 to E4.5 blastocyst, as revealed by the similarity in gene expression profiles (Kolodziejczyk et al., 2015). However, it is unclear without interrogating the molecular properties of individual cells, if there is an admixture of heterogeneous cell types in the 2i/LIF colonies. A case in point is the presence of a minor population of apparently “totipotent” stem cells with the propensity of extraembryonic cell lineages in the LIF/Serum and the 2i/LIF ESC colonies (Macfarlan et al., 2012; Morgani et al., 2013). Whether these “totipotent” cells are generated specifically in conditions that promote a naive state of pluripotency is not known. LIF and serum might capture cells at another state of naive pluripotency, which may correspond to the pluripotency of cells in the diapause (delayed) blastocyst (Fig. 1A). EPL cells are positioned further along the gradient (the slope) than the LIF/Serum ESCs. FGF2 and Activin arrest the cells at an even further stage of pluripotency corresponding to that of the epiblast of gastrula-stage embryos (Kojima et al., 2013). In essence, the progression from a naive state to the primed state of pluripotency in the stem cells brings forth the commitment to, or the poising for, lineage differentiation. Other pluripotent stem cells with intermediate phenotypes have been generated in vitro. Pluripotent stem cells with dual responsiveness to LIF/STAT3 and Activin A/Smads and a mixed gene expression pattern, known as intermediate epiblast stem cells (IESCs), are comparable to a state in between ESCs and EpiSCs (Chang and Li, 2013). Activin or FGF2 could maintain ESCs derived from the mouse inner cell mass cells (Ozawa et al., 2014). ESCs derived with FGF2 could contribute to germline chimeras, attesting their naive-like characteristics. The ability to generate bona fide EpiSCs directly from the mouse blastocyst suggests that an intermediate 2i/LIF step for the transit through the naïve state may not be a prerequisite for acquiring primed pluripotency in vitro (Najm et al., 2011). Whether these atypical states of pluripotency reflect intermediate states that are native to the epiblast or are the results of response of the embryonic cells to the experimental conditions is not known.
Pluripotent stem cells from refractory/non-permissive mouse strains and rats The 129-related mouse strains are amenable for the derivation of germline-competent ESC lines from E4 blastocysts using LIF and selected batches of serum or BMP4, while many other mouse strains are refractory to the generation of ESCs under this condition. Epiblast cells of refractory rodents differentiate rapidly and die when grown in Serum/LIF condition. ESCs can be derived, albeit at a low efficiency, for some of the refractory strains, such as C57BL/6, DBA/1lacJ, CBA/Ca, and CD1, through the induction of implantation delay (that mimics embryonic diapause) to activate LIF signaling, and the isolation of an epiblast free of the primitive endoderm prior to in vitro culture in Serum/LIF on fibroblast feeders (Gardner and Brook, 1997; Brook and Gardner, 1997). Other mouse strains (C57BL/6 X CBA, CBA, MF1, and NOD) and several strains of rats are completely refractory to ESC derivation in Serum/LIF (or BMP4) conditions (Blair et al., 2011; Gardner and Brook, 1997) (Fig. 1B). The basis of the refractory nature of these mouse strains and the rats is not fully understood. However, the successful derivation of ESCs in 2i/LIF or 3i/LIF conditions (see below) suggests that autonomous FGF4-ERK signaling activity and/or strong activation of differentiation-promoting ERK signaling by LIF are probably the counter-productive factors. Using the serum-free 2i/LIF regime that eliminates most of the differentiation-induced signaling from ERK1/2, stable ESC lines can be generated with high efficiency from many strains of mouse, including the aforementioned refractory strains (Nichols et al., 2009; Ying et al., 2008). Germline-competent ESC lines have also been obtained from rat blastocysts (Buehr et al., 2008; Hirabayashi et al., 2010; Kawamata and Ochiya, 2010; Li et al., 2008). Rat ESCs have also been generated using the serum-free 3i condition (Buehr et al., 2008; Li et al., 2008), a condition based on the 2i/LIF medium supplemented additionally with a chemical inhibitor of the FGF4 receptor, SU5402. This synthetic medium was designed to block the inductive stimuli transduced by FGF/ERK signaling that drive cell differentiation. The 2i/LIF and 3i/LIF culture regime led to the notion of a ground state of pluripotency, a pristine state characterized by the self-renewing of rodent pluripotent stem cells in the absence of any known exogenous signaling activity (Ying et al., 2008) (Fig. 1B, Fig. 2 — non-permissive rodents). Provision of 2i/LIF or 3i/LIF condition, in particular the abolition of ERK activity, therefore enables capturing ESCs at the naïve pluripotency state for refractory rodent strains. However, culturing the epiblast cells from these strains in defined culture medium supplemented with KOSR, FGF2, and Activin A captures the stem cells at the primed state of pluripotency (Brons et al., 2007; Tesar et al., 2007). Cells from the refractory rodent species may be inherently permissive for the derivation of primed pluripotent stem cells.