For induction of FGF-iPS cells, 200.000 MEFs were seeded onto gelatin-coated dishes in DMEM medium with 10% serum and transduced by pLIB-based retroviruses for mouse Oct4, Klf4, Sox2 and c-Myc. include blastocyst embryos, which give rise to embryonic stem cells (ES cells), and the post-implantation epiblast which gives rise to epiblast stem cells (EpiSCs)[1],[2]. ES cells and EpiSCs are both pluripotent as they are capable of generating derivatives of the three embryonic germ layers uponin vitroorin vivodifferentiation, yet important molecular and functional differences exist between these two pluripotent states. At the molecular level, the ES cell pluripotent state is maintained by a combination of LIF/JAK/STAT3 and BMP4 signaling, while EpiSCs require a combination of bFGF and TGF/Activin signaling for their continued self-renewal. The different culture conditions that maintain ES cells and EpiSCs are reflected in the morphological, molecular and functional properties of these cells. Murine ES cells form dome-shaped three dimensional colonies and are capable of generating chimeras with functional contribution to all somatic lineages as well as the germline. In contrast, EpiSCs form flatted colonies that are split by mechanical- or collagen-mediated passaging as small clusters of cells, since EpiSCs cannot be passaged as single cells by trypsin digest. 6-TAMRA EpiSCs are pluripotent and form derivatives of all three germ layers during in vitro differentiation and upon teratoma formation in vivo. Unlike ES cells, EpiSCs can even generate trophoectoderm derivativesin vitro. Yet, fail to integrate with the ICM upon morula aggragation and as a result, chimera forming potential of EpiSCs is very low or even absent. Thus, while 6-TAMRA EpiSCs are pluripotent, to-date theirin vivodevelopmental potential is limited to teratoma formation. Above results demonstrate that in the mouse, two functionally distinct pluripotent states exist, a nave LIF-dependent pluripotent state that is compatible with the pre-implantation ICM and a primed FGF-dependent state that is reminiscent of the post-implantation epiblast[3]. The ability to generate ES cell lines is restricted to only a SKP1A few inbred mouse strains whereas other, so-called non-permissive mouse strains fail to yield ES cells under standard culture conditions, but instead can give rise to to EpiSCs,Pluripotent stem cell lines from other species, including human and rat, share many of the defining characteristics of EpiSCs, suggesting that the EpiSC pluripotent state is the common stable pluripotent state for most strains of mice as well as other species. Interestingly, Hanna and colleagues recently demonstrated that the constitutive ectopic expression of either Klf4 or cMyc allows the derivation of LIF-dependent ES-like 6-TAMRA cells from blastocyst embryos of the non-permissive NOD mouse strain[4]. In addition, LIF/serum-dependent ES-like cell lines can be generated through somatic cell reprogramming of NOD fibroblasts with defined factors (Oct4, Sox2, Klf4, cMyc) that have recently been shown to allow the generation of induced pluripotent stem cells (iPS cells) from somatic cells[5],[6]. Yet, as with the blastocyst-derived NOD ES cell lines, the stable propagation of NOD iPS cells is dependent on the continued ectopic expression of Klf4 or cMyc. Small molecule inhibitors of glycogen synthase kinase beta 6-TAMRA (GSK3) and the mitogen-activated protein kinase (MAPK) signaling pathway can replace some of the reprogramming factors during iPS cell generation[7], and these inhibitors can similarly stabilize the LIF/serum-dependent pluripotent state in blastocyst-derived stem cells or iPS cells from the the non-permissive NOD mouse strain[4],[8],[9],[10]. Thus, it appears that the LIF-dependent pluripotent state is metastable in NOD mice, meaning it is dependent on either the constitutive expression of ectopic reprogramming factors or the presence of small molecule inhibitors of the GSK3 and/or the MEK/ERK signaling pathway. In the absence of these exogenous factors, NOD iPS cells assume a stable EpiSC-like state, even when LIF is present in the culture media. Genetic background appears to play an important part in stabilizing the LIF-dependent pluripotent state, yet its part in defining the FGF-dependent pluripotent stateis less obvious. We explored the possibility of generating EpiSCs by iPS reprogramming of murine embryonic fibroblasts from your permissive129 and/or BL6 mouse strains in EpiSC tradition conditions. Unexpectedly, we found that actually in the presence of EpiSC tradition 6-TAMRA conditions, iPS cells adopt a naive ICM/ES-like pluripotent state. Thus, it appears that strain-specific genetic elements dictate that in permissive mouse strains, the ES-like pluripotent state is.
