Endomucin marks quiescent long-term multi-lineage repopulating hematopoietic stem cells and is essential for their transendothelial migration [RNA-seq: wildtype_EMCN]

Endomucin (EMCN) currently represents the only hematopoietic stem cell (HSC) marker expressed by both murine and human HSCs. Here, we report that EMCN+ long-term repopulating HSCs (LT-HSCs; CD150+CD48-LSK) have a higher long-term multi-lineage repopulating capacity compared to EMCN- LT-HSCs. Cell cycle analyses and transcriptional profiling demonstrated that EMCN+ LT-HSCs were more quiescent compared to EMCN- LT-HSCs. Emcn-/- and Emcn+/+ mice displayed comparable steady-state hematopoiesis, as well as frequencies, transcriptional programs, and long-term multi-lineage repopulating capacity of their LT-HSCs. Complementary functional analyses further revealed increased cell cycle entry upon treatment with 5-fluorouracil and reduced granulocyte colony-stimulating factor (GCSF) mobilization of Emcn-/- LT-HSCs, demonstrating that EMCN expression by LT-HSCs associates with quiescence in response to hematopoietic stress and is indispensable for effective LT-HSC mobilization. Transplantation of wild-type bone marrow cells into Emcn-/- or Emcn+/+ recipients demonstrated that EMCN is essential for endothelial cell-dependent maintenance/self-renewal of the LT-HSC pool and sustained blood cell production post-transplant. Overall design: RNA was isolated from 3000-5000 FACS sorted EMCN+CD150+CD48-LSK (EMCN+ LT-HSCs) and EMCN-CD150+CD48-LSK (EMCN- LT-HSCs) for RNA sequencing. Granulocyte-Monocyte progenitors (GMPs) were additionally sorted as well characterised population to serve as more differentiated population for comparison. BM cells were isolated from C57Bl6 mice purchased at Taconic (Denmark). 4 biological replicates for each population, one EMCN-LT-HSC replicate was removed from analysis due to poor quality of the sequenced library.

内皮粘蛋白（Endomucin, EMCN）是目前唯一一种同时在小鼠和人类造血干细胞（hematopoietic stem cell, HSC）中表达的造血干细胞标志物。本研究报道，相较于EMCN阴性的长期重建造血干细胞（long-term repopulating HSCs, LT-HSCs; CD150+CD48-LSK），EMCN阳性的长期重建造血干细胞具有更强的长期多系重建造血能力。细胞周期分析与转录组谱分析结果显示，EMCN阳性LT-HSCs的静息状态较EMCN阴性LT-HSCs更为显著。Emcn基因敲除（Emcn-/-）与野生型（Emcn+/+）小鼠在稳态造血状态下的造血功能相当，其LT-HSCs的频率、转录程序以及长期多系重建造血能力均无明显差异。补充功能分析进一步揭示，经5-氟尿嘧啶（5-fluorouracil）处理后，Emcn-/- LT-HSCs的细胞周期进入过程增强，而粒细胞集落刺激因子（granulocyte colony-stimulating factor, GCSF）介导的动员能力则降低，这表明LT-HSCs表达EMCN与造血应激状态下的静息状态相关，且对于有效的LT-HSC动员不可或缺。将野生型骨髓细胞移植至Emcn-/-或Emcn+/+受体小鼠体内的实验证实，EMCN对于内皮细胞依赖性的LT-HSC池维持/自我更新以及移植后持续的血细胞生成均至关重要。
实验整体设计：从3000~5000个经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）得到的EMCN+CD150+CD48-LSK（EMCN阳性LT-HSCs）与EMCN-CD150+CD48-LSK（EMCN阴性LT-HSCs）中分离RNA，用于RNA测序。此外，我们还分选了粒细胞-单核细胞祖细胞（granulocyte-monocyte progenitors, GMPs）这一特征明确的细胞群，作为更分化的细胞群用于对照比较。骨髓（bone marrow, BM）细胞从购自丹麦Taconic公司的C57Bl6小鼠中分离得到。每个细胞群设置4个生物学重复，由于其中1个EMCN阴性LT-HSCs样本的测序文库质量较差，故将该重复样本从分析中剔除。