BACKGROUND In mammals, specification of primordial germ cells (PGCs) is established in the early post-implantation embryo. The bone morphogenetic protein (BMP)-SMAD and WNT3-β-catenin signaling initiate the gene regulatory network for PGC specification. The activation of SOX17-BLIMP1 axis is critical for human PGC program. Moreover, EpCAM and INTEGRINα6 were identified as surface markers of human PGC-like cells (PGCLCs) recently. However, the signaling mechanism for PGC specification in non-rodent mammals remains to be clarified. METHODS We differentiated human induced pluripotent stem cells (hiPSCs) into PGCLCs in vitro in response to Activin A and BMP4. The percentage of EpCAM/INTEGRINα6 double-positive cells (PGCLCs) was analyzed by flow cytometry. The expression of PGC genes was evaluated by qRT-PCR and immunofluorescence. The expression dynamic of multi-lineage genes during the differentiation process was evaluated by qRT-PCR. RESULTS Under the stimulation for PGCLC induction, the embryoids derived from hiPSCs initiated significant upregulation of the early PGC genes (BLIMP1, TFAP2C, and NANOS3), but maintained low or no levels of DPPA3 and late PGC genes (DAZL and DDX4). The percentage of EpCAM/INTEGRINα6 double-positive PGCLCs reached the highest at day 6 of induction. After pre-induction, the incipient mesoderm-like cells (iMeLCs) upregulated most of the mesoderm genes (EOMES, T, MSXI, RUNX2, and MIXL1). The differentiating embryoids showed high levels of key pluripotency genes, OCT4 and NANOG, but became negative for SOX2. In contrast to iMeLCs, the differentiating embryoids downregulated mesoderm genes RUNX2 and EOMES, and ectoderm gene PAX6, but increased the expression of endoderm gene SOX17. CONCLUSIONS During PGCLC induction process in vitro, the differentiating embryoids not only activated the PGC-related genes, but also displayed complex regulation of pluripotency genes and multi-lineage genes. These results would be meaningful for future research investigating the regulation of human early germ line development.

中文翻译：

---

人类诱导的多能干细胞诱导原始生殖细胞过程中多谱系基因表达动态的分析。

背景技术在哺乳动物中，在植入后的早期胚胎中建立了原始生殖细胞（PGC）的规格。骨形态发生蛋白（BMP）-SMAD和WNT3-β-catenin信号启动了PGC规范的基因调控网络。SOX17-BLIMP1轴的激活对于人类PGC程序至关重要。此外，最近将EpCAM和INTEGRINα6鉴定为人PGC样细胞（PGCLC）的表面标记。然而，非啮齿类哺乳动物中PGC规格的信号传导机制仍有待阐明。方法我们将人诱导的多能干细胞（hiPSC）在体外对激活素A和BMP4的分化为PGCLC。通过流式细胞术分析EpCAM /INTEGRINα6双阳性细胞（PGCLC）的百分比。通过qRT-PCR和免疫荧光评估PGC基因的表达。通过qRT-PCR评估了多谱系基因在分化过程中的表达动态。结果在刺激PGCLC的刺激下，来自hiPSC的胚状体开始显着上调早期PGC基因（BLIMP1，TFAP2C和NANOS3），但保持低水平或无水平的DPPA3和晚期PGC基因（DAZL和DDX4）。EpCAM /INTEGRINα6双阳性PGCLC的百分率在诱导第6天达到最高。预诱导后，初期的中胚层样细胞（iMeLC）上调了大多数中胚层基因（EOMES，T，MSXI，RUNX2和MIXL1）。分化的胚状体显示出高水平的关键多能性基因OCT4和NANOG，但对SOX2呈阴性。与iMeLC相比，分化的胚状体下调了中胚层基因RUNX2和EOMES，以及外胚层基因PAX6，但增加了内胚层基因SOX17的表达。结论在体外PGCLC诱导过程中，分化的胚状体不仅激活了PGC相关基因，而且还显示了多能性基因和多谱系基因的复杂调控。这些结果对于未来研究人类早期种系发育调控的研究将是有意义的。