Human Primordial Germ Cells are Specified from Lineage Primed Progenitors. Human Primordial Germ Cells are Specified from Lineage Primed Progenitors

In vitro gametogenesis is the process of making germline cells from human pluripotent stem cells. The foundation of this model is the quality of the first progenitors called primordial germ cells (PGCs), which in vivo are specified during the peri-implantation window of human development. Here, we show using human embryo attachment culture that hPGC specification begins at day 12 post fertilization. Using single cell RNA-sequencing of hPGC-like cells (hPGCLCs) differentiated from pluripotent stem cells we discovered that hPGCLC specification involves resetting pluripotency towards a transitional state with shared characteristics between naïve and primed pluripotency followed by differentiation into lineage primed TFAP2A+ progenitors. Applying the germline trajectory to TFAP2C mutants reveals that TFAP2C functions in the TFAP2A+ progenitors upstream of PRDM1 to regulate the expression of SOX17. This serves to protect hPGCLCs from crossing the Weismann’s barrier to adopt somatic cell fates and therefore is an essential mechanism for successfully initiating in vitro gametogenesis. Overall design: We applied single cell RNA-sequencing (scRNA-seq) with 10x Genomics to hESCs, iMeLCs and aggregate cells at D1-4 with two biological replicates in UCLA1 and UCLA2 cell lines, respectively. In total, we sequenced 85,309 cells with high quality to uncover the germline trajectory and explored the progenitors involved in establishing germline cell fate. Moreover, we performed scRNA-seq of TFAP2C -/- cells at 6 time points (hESCs, iMeLCs, aggregates at D1, D2-D4, 19,808 cells sequenced) and compared this to the single cells from UCLA1 wild type cells which is the genetic background used to make TFAP2C mutant. We applied ChIP-seq to hESCs, iMeLCs, and day 4 aggregates using anti-TFAP2C antibodies and to ITGA6/EPCAM-isolated day 4 hPGCLCs using anti-H3K27ac antibodies. Two biological replicates were included for each sample.体外配子发生（in vitro gametogenesis）是指利用人类多能干细胞制备生殖系细胞的过程。该模型的核心基础是首批被称为原始生殖细胞（primordial germ cells，PGCs）的前体细胞质量，而在体内，原始生殖细胞的特化发生于人类发育的围植入窗口期。本研究通过人类胚胎贴附培养实验证实，人类原始生殖细胞（hPGCs）的特化始于受精后第12天。通过对由多能干细胞分化获得的人类原始生殖细胞样细胞（human primordial germ cell-like cells，hPGCLCs）开展单细胞RNA测序（single cell RNA-sequencing，scRNA-seq），本研究发现hPGCLC的特化过程包含两个阶段：首先将多能性重编程至介于初始态与激活态多能性之间的过渡状态，随后分化为谱系预定向的TFAP2A+前体细胞。将生殖系发育轨迹应用于TFAP2C突变体分析后可知，TFAP2C在TFAP2A+前体细胞中作用于PRDM1的上游，调控SOX17的表达。这一机制可保护hPGCLCs避免跨越魏斯曼屏障（Weismann’s barrier），防止其向体细胞命运转化，因此是成功启动体外配子发生的核心必需机制。整体实验设计：本研究依托10x Genomics平台开展单细胞RNA测序（scRNA-seq），分别对UCLA1与UCLA2细胞系中的人类胚胎干细胞（hESCs）、诱导中内胚层样细胞（iMeLCs）以及第1至4天的聚集细胞进行检测，每组设置2次生物学重复。最终共获取85309个高质量细胞，以解析生殖系发育轨迹并探究参与确立生殖细胞命运的前体细胞。此外，本研究对6个时间点的TFAP2C敲除细胞（TFAP2C -/-）进行scRNA-seq检测，涵盖hESCs、iMeLCs、第1天聚集细胞、第2至4天聚集细胞，共测序19808个细胞，并将其与用于构建TFAP2C突变体的遗传背景对照——UCLA1野生型细胞的单细胞数据进行比对。本研究还使用抗TFAP2C抗体对hESCs、iMeLCs以及第4天的聚集细胞进行染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing，ChIP-seq），并使用抗H3K27ac抗体对经整合素α6（ITGA6）与上皮细胞黏附分子（EPCAM）分离得到的第4天hPGCLCs开展ChIP-seq检测，每个样本均设置2次生物学重复。