DNA methylation is indispensable for leukemia inhibitory factor dependent embryonic stem cells reprogramming

Abstract

24 25 26 27 Highlights and novelty 1  LIF alone supports ESCs self-renew and pluripotency in chemically defined media. 2  L-ESCs re-establish the epigenetic memory in reprogramming. 3  DNA methylation is indispensable for L-ESCs reprogramming. 4  L-ESCs contribute to somatic tissues and germ cells in chimaeras. Abstract 7 Naï ve pluripotency can be maintained by the 2i/LIF supplements (CHIR99021, 8 PD0325901 and LIF), which primarily affect canonical WNT, FGF/ERK, and 9 JAK/STAT3 signaling. However, whether one of these tripartite supplements alone is 10 sufficient to maintain naï ve self-renewal remain unclear. Here we show that LIF alone is 11 sufficient to induce reprogramming of 2i/LIF cultured ESCs (2i/L-ESCs) to ESCs with 12 hypermethylated state (L-ESCs). In vitro, upon withdrawal of 2i, 2i/L-ESCs overcome 13 the epigenetic barrier and DNA hypermethylated, which accompanies transcriptional 14 changes and subsequent establishment of epigenetic memory. Global transcriptome 15 features also show that L-ESCs are close to 2i/L-ESCs and in a stable state between 16 naï ve and primed pluripotency. Notably, our results demonstrate that DNA methylation 17 is indispensable for LIF-dependent mouse ESCs reprogramming and self-renew. 18 LIF-dependent ESCs reprogramming efficiency is significantly increased in serum 19 treatment and reduced in Dnmt3a or Dnmt3l knockout ESCs. Importantly, unlike 20 epiblast and EpiSCs, L-ESCs contribute to somatic tissues and germ cells in chimaeras. 21 Such simple culture system of ESCs is more conducive to clarify the molecular 22 mechanism of ESCs in vitro culture.23 24 Key Words 25Embryonic stem cells; Leukemia inhibitory factor; Reprogramming; Epigenetic; Genomic 26 imprinting; DNA methylation 2 pre-implantation embryos (Evans & Kaufman, 1981; Martin, 1981). Since pluripotent mouse 3 embryonic stem cells were first established four decades ago, various culture systems of ESCs 4 have been developed including the initial feeder/serum/cytokines, then the 5 feeder/serum/Leukemia inhibitory factor (LIF) or Bone morphogenetic protein 4 (BMP4) 6 (Smith et al, 1988; Williams et al, 1988; Ying et al, 2003), and the recent 2i/LIF (two 7 inhibitors CHIR99021, PD0325901 and LIF) (Ying et al, 2008). It is generally believed that 8 the optimal culture condition for ground sate ESCs comprises three additive 2i/LIF 9 supplements which affect canonical WNT, FGF/ERK, and JAK/STAT3 signals. It has been 10 reported that combination of any two of these tripartite supplements was sufficient to maintain 11 naï ve self-renewal of ESCs (Hackett et al, 2017). However, whether any one of the tripartite 12 supplements plays a critical role with unique critical targets and signaling for ESCs 13 pluripotency and self-renew remain elusive. 14 15 LIF is the most pleiotropic member of the interleukin-6 family of cytokines, and utilizes a 16 receptor that consists of the LIF receptor B and gp130 (Ohtsuka et al, 2015). LIF is able to 17 activate three intracellular signaling pathway: the JAK/STAT pathway, the PI3...