The catalytic activity of TET1 is required for human germ cell fate choice [scRNA-seq]

Primordial germ cell (PGC) is the foundation of the germline which become gametes and transmit genetic and epigenetic information to the next generation. The segregation of germline and soma is critical yet well-characterized in human during embryonic development due to ethical consideration. Here we discover that human TET1 (Ten-eleven translocation 1) acts as a safety check of germline cell fate. Using CRISPR/Cas9 to knock-out TET1 catalytic domain, we demonstrate extremely low hPGCLC (hPGC-like cell) competency of undifferentiated pluripotent stem cell lines. We plot whole-genome 5'hydroxymethylation (5hmC) profiles to elucidate global as well as locus-specific 5hmC enrichment in hPGCLCs, indicating the DNA oxygenase activity of TET1 is active during hPGCLC differentiation. Overall design: We applied scRNAseq in hESCs, iMeLCs and D4 aggregate cells during hPGCLC differentiation in Control (CTRL, 3 replicates) and TET1 catalytic domain knockout (CDKO, 3 replicates) lines.

原始生殖细胞（primordial germ cell, PGC）是生殖系的奠基者，可分化为配子，并将遗传与表观遗传信息传递给子代。生殖系与体细胞的分离是胚胎发育的关键事件，但受伦理因素限制，人类胚胎发育阶段的该过程尚未得到充分解析。本研究发现，人类TET1（Ten-eleven translocation 1，十-十一易位蛋白1）可作为生殖细胞命运的安全检查点。通过CRISPR/Cas9技术敲除TET1催化结构域，我们证实未分化多能干细胞系的hPGCLC（人PGC样细胞，human PGC-like cell）生成能力极低。我们绘制了全基因组5-羟甲基化（5-hydroxymethylation, 5hmC）图谱，以解析hPGCLCs中的全局及位点特异性5hmC富集模式，结果表明TET1的DNA加氧酶活性在hPGCLC分化过程中处于激活状态。实验设计：在hPGCLC分化过程中，我们对对照组（CTRL，3次生物学重复）及TET1催化结构域敲除组（CDKO，3次生物学重复）的人类胚胎干细胞（human embryonic stem cells, hESCs）、诱导内中胚层样细胞（induced mesendoderm-like cells, iMeLCs）以及分化第4天的聚集细胞进行了单细胞RNA测序（single-cell RNA sequencing, scRNAseq）。