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  • br Introduction Pluripotent stem cells PSCs harbor core prop

    2018-10-24


    Introduction Pluripotent stem EZ Cap Reagent AG Supplier (PSCs) harbor core properties of self-renewal and pluripotency (Hackett and Surani, 2014; Martello and Smith, 2014). Mouse embryonic stem cells (mESCs), established from the inner cell mass (ICM) of preimplantation blastocysts, are classically derived from a permissive 129-strain and kept in serum-containing medium supplemented with leukemia inhibitory factor (LIF). They are considered to possess “naive” pluripotency. Bone morphogenetic proteins (BMPs) have been shown to substitute for serum and sustain self-renewal of naive mESCs together with LIF (Ying et al., 2003). On the other hand, human ESCs, which have characteristics similar to those of mouse epiblast stem cells (mEpiSCs) derived from postimplantation embryos, are more mature and are referred to as being “primed” for differentiation. These cells are LIF independent, and are usually maintained in the presence of fibroblast growth factor 2 (FGF2) and activin A, which stabilize the primed pluripotent state. Although the serum-free medium supplemented with LIF and BMP-4 is well established, PSCs maintained with BMP-4 are rather exceptional. To derive mESCs from non-permissive strains, suppression of the differentiation-inducing mitogen-activated protein kinase kinase 1/2 (MEK1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway is required (i.e. LIF+BMP-4+MEK1/2i) (Batlle-Morera et al., 2008). A cocktail of two inhibitors, i.e. for the MEK1/2 and GSK3 pathways (2i), has been proved to be sufficient to maintain mESCs with full pluripotency, or a “ground state” (Ying et al., 2008), and LIF supplementation (2i+LIF, or 2iL) strengthens the robustness. In the ground state, the BMP-SMAD pathway has been shown to be less active (Boroviak et al., 2014). Furthermore, BMP-4 induces differentiation of primed pluripotent cells. Thus, clarifying the role of BMP in mESCs from permissive strains will provide important clues for the molecular basis of naive pluripotency. BMPs are members of the transforming growth factor β (TGF-β) family, and transduce their signals through distinct sets of type I and type II receptors (Derynck and Miyazono, 2008; Miyazono et al., 2010). Activated type I receptors phosphorylate receptor-regulated SMAD proteins (R-SMADs; SMAD1, SMAD5, and SMAD8 in the BMP signaling pathway, and SMAD2 and SMAD3 in the TGF-β/activin pathway), which form heterotrimeric complexes with SMAD4 and translocate into the nucleus. SMAD complexes are recruited to cell-type-specific binding sites through interaction with master transcription factors, controlling the expression of specific target genes (Morikawa et al., 2013). In naive mESCs, SMAD1 has been reported to co-localize with OCT4, SOX2, and NANOG (abbreviated as OSN) and STAT3 on ESC-specific enhancers (Chen et al., 2008). Consistently, the BMP-SMAD target genes Id1 and Dusp9 play critical roles in naive mESCs (Li et al., 2012; Ying et al., 2003). However, the existence of Smad4 homozygous mutant mESCs (Sirard et al., 1998) suggests that the SMAD pathway is not required for maintenance of naive pluripotency, leading us to revisit the roles of the BMP-SMAD pathway in PSCs. In the present study, we have performed both RNA-sequencing and SMAD1/5 genome-wide chromatin immunoprecipitation and sequencing (ChIP-seq) analyses of mESCs in the naive or primed states, and have employed a genome editing method. We show that the BMP-SMAD pathway is dispensable for maintaining naive pluripotency. Instead, BMP utilizes the MEK5-ERK5 pathway, which induces Klf2, an essential factor for the ground state (Yeo et al., 2014). Our study thus sheds important light on the molecular mechanisms underlying naive pluripotency.
    Results
    Discussion BMP ligands induce the outgrowth of ICM during the derivation of mESC lines (Di-Gregorio et al., 2007; Ying et al., 2003). Inactivation of either Bmp4 or Bmpr1a (which encodes ALK3) in mice results in early embryonic lethality due to impaired growth of the epiblast as early as embryonic day 6.5 (E6.5). However, Smad1- and/or Smad5-deficient mice exhibit abnormalities in extra-embryonic tissue development and angiogenesis from E7.5 or later (Derynck and Miyazono, 2008). In addition, Bmpr1a KO mESCs show self-renewal defects, while BMP-7 can rescue the defects through ALK2 activation (Di-Gregorio et al., 2007). Interestingly, activin/ALK4 can partially rescue the self-renewal defects of Bmpr1a KO mESCs (Di-Gregorio et al., 2007), suggesting that BMP and activin commonly activate non-SMAD pathway(s) and regulate the proliferative activity of epiblast at an early stage of development. Consistently, Smad1/5 dKO mESCs and Smad4 KO mESCs exhibit characteristics of naive pluripotency when maintained in LIF+serum (Figures 3 and S2) (Sirard et al., 1998).