Aging alters the epigenetic asymmetry of HSC division [scRNA-Seq]

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain homeostasis. With aging, the frequency of polar HSCs decreases. Cell polarity in HSCs is controlled by the activity of the small RhoGTPase Cdc42. Here we demonstrate, using a comprehensive set of paired daughter cell analyses that include single cell 3D-confocal imaging, single cell transplants, single cell RNA-seq as well as single cell ATAC-seq, that the outcome of HSC divisions is strongly linked to the polarity status before mitosis, which is in turn determined by the level of the activity Cdc42 in stem cells. Aged apolar HSCs undergo preferentially self-renewing symmetric divisions, resulting in daughter stem cells with reduced regenerative capacity and lymphoid potential, while young polar HSCs undergo preferentially asymmetric divisions. Mathematical modeling in combination with experimental data implies a mechanistic role of the asymmetric sorting of Cdc42 in determining the potential of daughter cells via epigenetic mechanisms. Therefore, molecules that control HSC polarity might serve as modulators of the mode of stem cell division regulating the potential of daughter cells. Overall design: Sorted single cells were cultured with and without treatment in the presence of cytokines until first cell division (40-44hrs). The daughter cells were manually separated, washed with PBS and collected for RNA sequencing.造血干细胞（Hematopoietic stem cells, HSCs）通过平衡自我更新与分化进程维持机体稳态。随着机体衰老，极化型造血干细胞的占比逐渐降低。造血干细胞的细胞极性由小RhoGTP酶Cdc42的活性水平调控。本研究借助一系列配套的配对子代细胞分析实验——涵盖单细胞三维共聚焦成像、单细胞移植、单细胞RNA测序（RNA-seq）以及单细胞ATAC测序（ATAC-seq）——证实，造血干细胞的分裂结局与有丝分裂前的细胞极性状态密切相关，而该极性状态又由干细胞内Cdc42的活性水平决定。 衰老的非极化造血干细胞更倾向于发生自我更新型对称分裂，所产生的子代干细胞再生能力与淋巴系分化潜能均有所下降；而年轻的极化型造血干细胞则更偏好进行不对称分裂。结合实验数据构建的数学模型提示，Cdc42的不对称分配可通过表观遗传机制决定子代细胞的功能潜能。因此，调控造血干细胞极性的分子或可作为干细胞分裂模式的调节剂，进而调控子代细胞的分化潜能。 实验整体设计如下：将分选得到的单个细胞置于添加细胞因子的培养体系中，设置给药与未给药两组对照，培养至首次细胞分裂（耗时40~44小时）。随后手动分离子代细胞，经PBS洗涤后收集样本用于RNA测序。