Genomic profiling of human spermatogonial stem cells [scRNA-Seq]. Homo sapiens

To better understand human spermatogonial stem cells (SSCs), we profiled their transciptome and epigenome, which revealed the mechanism how human SSCs regulates their self-renewal versus differentiation dermination, as well as how latent pluripotency is established in human SSCs. Remarkly, we discovered signaling pathways (e.g. LIF, BMP, WNT) that differentially regulated self-renewal vesus differentiation in SSCs. We also discovered that SSCs repress core pluripotent factors (Sox2, Pou5f1 and Nanog) yet activate ancillary factors (e.g. Klf4, Mbd3, Tcf3, Sall4) transcriptionally and epigenetically. Overall design: Using SSEA4 as self-renewal marker and Kit as differentiating marker, we isolated self-renewal and differentiation SSCs by magnetic antibody cell sorting (MACS). SSEA4+ or Kit+ cells were loaded into 5-10 μm integrated fluidic circuits (IFCs) using Fluidigm C1 instrument. Single cells in IFCs were lysed and total RNA was harvested for polyadenylation selection, reverse transcription and PCR amplification. Library constructions were performed according to Fluidigm Library preparation with Nextera XT protocol and sequenced on a 50-cycle single end run.

为更深入阐释人类精原干细胞（spermatogonial stem cells, SSCs）的生物学机制，我们对其转录组（transcriptome）与表观基因组（epigenome）开展了全景式谱学分析，由此揭示了人类精原干细胞调控自我更新与分化命运决定的内在机制，以及人类精原干细胞中潜伏多能性的建立过程。值得注意的是，我们发现了若干可差异性调控精原干细胞自我更新与分化的信号通路，例如白血病抑制因子（LIF）、骨形态发生蛋白（BMP）及WNT信号通路。此外我们还发现，精原干细胞可在转录与表观遗传层面同时抑制核心多能因子（Sox2、Pou5f1及Nanog）的表达，并激活辅助多能因子（如Klf4、Mbd3、Tcf3、Sall4）的转录。整体实验设计：以阶段特异性胚胎抗原4（SSEA4）作为自我更新标志物，以Kit作为分化标志物，我们通过磁珠激活细胞分选（magnetic antibody cell sorting, MACS）分离得到处于自我更新状态与分化状态的精原干细胞。将SSEA4阳性或Kit阳性细胞通过Fluidigm C1仪器加载至5-10 μm集成流体回路（integrated fluidic circuits, IFCs）中。随后对集成流体回路内的单细胞进行裂解，提取总RNA并进行多聚腺苷酸尾筛选、逆转录及PCR扩增。按照Fluidigm文库制备流程结合Nextera XT试剂盒标准操作完成文库构建，并采用50个循环的单端测序模式完成测序。