In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states (I, bulk RNA-Seq data)

Naive pluripotent cells in the implanting mouse blastocyst generate a rosette structure before undergoing lumenogenesis to form the egg cylinder. Simultaneously, they acquire primed pluripotency, the ability to differentiate into the primary germ layers. The existence of discrete intermediate pluripotent states during this transition has not been demonstrated. We identify here a distinct rosette pluripotent state, defined by co-expression of naive factors with transcription factor OTX2. Downregulation of WNT signals in the blastocyst drives transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells (RSCs), a self-renewing naive-primed intermediate. RSCs gain a unique epigenome that includes erasure of constitutive heterochromatin and bivalent marking of primed pluripotency genes. Notwithstanding this primed chromatin landscape, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate where control over pluripotency progression and morphogenesis pivots from WNT to MEK signals. Using Illumina HiSeq, mRNA profiles were generated from mouse embryonic stem cells cultured in various conditions or differentiated to the primed pluripotent state. As biological replicates, R1 and CGR8 embryonic stem cell lines were used. The cells were cultured in serumfree N2B27 medium supplemented with the cytokine LIF, and either the GSK3-inhibitor CHIR99021 and the MEK-inhibitor PD325901 (L2i), or a combination of PD325901 and the Wnt inhibitor IWP2 (LIM). Furthermore, mRNA profiles were also generated from cells differentiated towards the primed pluripotent state for 35 hrs and for 4 days by culture in the presence of LIF, FGF2, Activin A, and IWP2 (LFAI). Finally, mRNA profiles were generated from time courses of R1 embryonic stem cells switched from LIF+2i to LIF supplemented with either FGF2, Activin A and IWP2 (LFAI), or with IWP2 and PD325901 (LIM). The time courses lasted for 4 days, and mRNA profiles were generated from every day. No replicates were generated for the time courses.

着床期小鼠囊胚中的初始多能细胞（naive pluripotent cells）在发生腔发生（lumenogenesis）形成卵柱（egg cylinder）前，会先形成玫瑰花结结构。与此同时，这些细胞会获得始发态多能性（primed pluripotency），即分化为初级胚层的能力。目前尚未有研究证实该转变过程中存在离散的中间多能状态。
本研究在此鉴定出一种独特的玫瑰花结多能状态，其特征为初始多能因子与转录因子OTX2共表达。囊胚中WNT信号的下调会通过诱导OTX2的表达，推动细胞向玫瑰花结多能状态转变。随后，玫瑰花结结构会激活MEK信号通路，进而诱导腔发生并推动细胞进展至始发态多能状态。
因此，联合抑制WNT与MEK信号可支持玫瑰花结样干细胞（rosette-like stem cells, RSCs）的自我更新，这类细胞属于初始-始发态中间多能状态。RSCs具有独特的表观基因组（epigenome），其特征包括组成型异染色质（constitutive heterochromatin）的消除，以及始发态多能性相关基因的二价标记（bivalent marking）。尽管具备这类始发态染色质特征，WNT信号仍可诱导细胞逆转至初始多能状态。因此，玫瑰花结结构是一种可逆的多能中间状态，其多能性进展与形态发生的调控核心会从WNT信号切换至MEK信号。
本研究使用Illumina HiSeq测序平台，对多种培养条件下的小鼠胚胎干细胞（mouse embryonic stem cells）或分化至始发态多能状态的细胞进行了mRNA表达谱（mRNA expression profiles）分析。作为生物学重复，实验使用了R1与CGR8两种胚胎干细胞系。
细胞培养于无血清N2B27培养基（serum-free N2B27 medium），并添加细胞因子LIF（leukemia inhibitory factor, LIF），以及以下两种组合之一：① GSK3抑制剂CHIR99021与MEK抑制剂PD325901（即L2i培养体系）；② PD325901与Wnt抑制剂IWP2（即LIM培养体系）。
此外，本研究还对通过添加LIF、FGF2、激活素A（Activin A）与IWP2（即LFAI培养体系）培养35小时及4天、分化为始发态多能状态的细胞进行了mRNA表达谱分析。
最后，本研究对R1胚胎干细胞的时间梯度样本进行了mRNA表达谱分析：将细胞从LIF+2i培养体系切换至添加了以下物质的LIF培养基中：① FGF2、Activin A与IWP2（LFAI培养体系）；② IWP2与PD325901（LIM培养体系）。该时间梯度实验持续4天，每日采集样本进行表达谱分析，且未设置重复样本。