Induction of the mouse germ cell fate by transcription factors in vitro [exp2]

The germ cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have established a culture system that recapitulates the mouse germ-cell specification pathway: Using cytokines, embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) are induced into epiblast-like cells (EpiLCs) and then into primordial germ cell-like cells (PGCLCs) with capacity both for spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous over-expression of three transcription factors (TFs), Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not ESCs, swiftly and highly efficiently into a PGC state with endogenous transcription circuitry. The induction of the PGC state on EpiLCs minimally requires Prdm14 but not Blimp1 or Tfap2c. The TF-induced PGC state reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC specification in vivo and in vitro by cytokines including BMP4. Importantly, the TF-induced PGC-like cells robustly contribute to spermatogenesis and fertile offspring. Our findings provide not only a novel insight into the transcriptional logic that creates a germ cell state, but also a foundation for the TF-based reconstitution and regulation of mammalian gametogenesis. Aim of this analysis is identification of genes whose expression was altered by each of key transcription factor for transcription factor-induced primordial germ cells (TF-PGCLCs) induction (Blimp1 (B), Prdm14 (P14), and Tfap2c (A)). Both of epiblast-like cells (EpiLCs) (Hayashi et al., 2011, Cell) and aggregates of EpiLCs cultured with doxycycline on day 1 were harvested for 5 cell lines, including BP14A (Clone #3-3), B (#2-4), P14 (#7-109), A (#8-2), and the parental clone (BVSCR26rtTA embryonic stem cells). Total RNA was isolated and analyzed. Two biological duplicates for each cell type were included.

生殖细胞谱系（germ cell lineage）通过产生雄性配子与雌性配子以维持生命延续，二者结合后可形成全能合子（totipotent zygote）。我们构建了一套可重现小鼠生殖细胞特化通路的培养体系：借助细胞因子，可将胚胎干细胞（ESCs）/诱导多能干细胞（iPSCs）诱导为上胚层样细胞（epiblast-like cells, EpiLCs），随后进一步诱导为同时具备精子发生（spermatogenesis）与卵子发生（oogenesis）能力的原始生殖细胞样细胞（primordial germ cell-like cells, PGCLCs），这为在体外研究并调控两性哺乳动物的生殖细胞发育提供了重要途径。

本研究发现，在无需细胞因子的条件下，同时过表达三个转录因子（transcription factors, TFs）——Blimp1（又名Prdm1）、Prdm14与Tfap2c（又名AP2γ）——可快速且高效地将EpiLCs（而非ESCs）诱导为具备内源转录调控环路的原始生殖细胞（PGC）状态。在EpiLCs上诱导PGC状态仅需Prdm14即可，Blimp1与Tfap2c并非必需。经转录因子诱导获得的PGC状态可重现PGC中关键的转录组与表观遗传重编程过程，但可绕过由包括骨形态发生蛋白4（BMP4）在内的细胞因子在体内及体外伴随PGC特化所激活的中胚层程序。尤为重要的是，经转录因子诱导获得的PGC样细胞可高效促成精子发生并产生可育后代。

本研究结果不仅为解析生殖细胞状态形成的转录调控逻辑提供了全新视角，同时也为基于转录因子的哺乳动物配子发生（gametogenesis）重建与调控奠定了研究基础。

本分析的核心目标是鉴定在转录因子诱导的原始生殖细胞样细胞（TF-PGCLCs）诱导过程中，各关键转录因子（Blimp1(B)、Prdm14(P14)及Tfap2c(A)）所调控的差异表达基因。我们收集了上胚层样细胞（EpiLCs，Hayashi等，2011，《Cell》）以及第1天经多西环素（doxycycline）处理的EpiLCs聚集体，共涉及5个细胞系，分别为BP14A（克隆#3-3）、B（#2-4）、P14（#7-109）、A（#8-2）以及亲本克隆（BVSCR26rtTA胚胎干细胞）。

提取所有样本的总RNA并进行转录组分析，每个细胞类型均设置两个生物学重复样本。