Analysis of multi-lineage gene expression dynamics during primordial germ cell induction from human induced pluripotent stem cells
Analysis of multi-lineage gene expression dynamics during primordial germ cell induction from human induced pluripotent stem cells
复制标题
人诱导多能干细胞原始生殖细胞诱导过程中多谱系基因表达动态分析
DOI：
10.1186/s13287-020-01620-y
复制
发表时间：
2020-03-04
影响因子：
7.5
通讯作者：
Ni, Ke
中科院分区：
文献类型：
--
作者：
Fang, Fang;Li, Zili;Ni, Ke
哺乳动物原始生殖细胞（PGCs）的特化是在植入后早期胚胎中建立的。骨形态发生蛋白（BMP）-SMAD和WNT 3-β-连环蛋白信号传导启动PGC特化的基因调控网络。SOX 17-BLIMP 1轴的激活对于人类PGC程序是至关重要的。此外，EpCAM和INTEGRIN α 6最近被鉴定为人PGC-like细胞（PGCLC）的表面标志物。然而，非啮齿类哺乳动物中PGC特化的信号机制仍有待阐明。方法体外诱导人多能干细胞（hiPSCs）向PGCLC分化，诱导剂为激活素A和BMP 4。通过流式细胞术分析EpCAM/INTEGRIN alpha 6双阳性细胞（PGCLC）的百分比。通过qRT-PCR和免疫荧光评价PGC基因的表达。通过qRT-PCR评估分化过程中多谱系基因的表达动态。结果在PGCLC诱导的刺激下，hiPSCs来源的胚状体中早期PGC基因（BLIMP 1、TFAP 2C和NANOS 3）显著上调，而DPPA 3和晚期PGC基因（DAZL和DDX 4）维持低水平或不表达。EpCAM/INTEGRIN α 6双阳性PGCLC的百分比在诱导第6天达到最高。在预诱导后，初始的中胚层样细胞（iMeLC）上调大部分中胚层基因（EOMES、T、MSXI、RUNX 2和MIXL 1）。分化的胚状体显示出高水平的关键多能性基因OCT 4和NANOG，但对SOX 2呈阴性。与iMeLCs相比，分化的胚状体下调中胚层基因RUNX 2和EOMES以及外胚层基因PAX 6，但增加内胚层基因SOX 17的表达。结论在PGCLC体外诱导过程中，分化的胚状体不仅激活了PGCLC相关基因，而且表现出多能性基因和多谱系基因的复杂调控。这些结果将对未来研究人类早期生殖细胞发育的调控有意义。
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-beta-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 INTEGRIN alpha 6 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/INTEGRIN alpha 6 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/INTEGRIN alpha 6 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.