Hematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry. With fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling. These comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

中文翻译：

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蛋白质水平的单细胞分析描绘了造血过程中的细胞内信号传导动态

之前已经使用细胞表面标记物和最近的单细胞技术研究了小鼠的造血干细胞和祖细胞 (HSPC) 亚群。最近单细胞分析的革命极大地改变了我们对造血的理解，证实了构成造血系统的细胞的实质性异质性。虽然已经广泛探索了造血背后 DNA/RNA 水平的动态分子变化，但仍然缺乏对造血信号程序和景观中单细胞异质性的广泛理解，在蛋白质水平上研究并反映转录后加工。这里，我们在单细胞水平准确量化了 9 种磷酸化蛋白和 2 种功能蛋白的细胞内水平，以系统地捕捉 8 种信号通路的激活动态，包括 EGFR、Jak/Stat、NF-κB、MAPK/ERK1/2、MAPK/p38 、PI3K/Akt、Wnt 和 mTOR 通路，在小鼠造血过程中使用质谱流式细胞术。通过对 320 万个单一造血干细胞和祖细胞 (HSPC) 以及谱系细胞进行多参数细胞表型分析，我们绘制了 HSC 分化过程中信号传导程序的轨迹，并确定了循环 HSPC 和多种分化途径的特定信号传导生物特征从干细胞到祖细胞和谱系细胞。我们还研究了造血细胞群的恢复模式，以及在造血重建过程中这些人群的信号调节。总体而言，我们发现 HSPC 子集中的通路激活具有显着的异质性，其特征在于不同的信号传导模式。这些全面的单细胞数据提供了对细胞内信号调节的造血功能的有力洞察，并为剖析 HSC 命运决定的性质奠定了坚实的基础。未来需要整合转录组学和蛋白质组学数据以及功能验证，以验证 HSC 分化过程中 HSPC 子集的异质性，并确定可靠的标记来对这些 HSPC 子集进行表型分析。这些全面的单细胞数据提供了对细胞内信号调节的造血功能的有力洞察，并为剖析 HSC 命运决定的性质奠定了坚实的基础。未来需要整合转录组学和蛋白质组学数据以及功能验证，以验证 HSC 分化过程中 HSPC 子集的异质性，并确定可靠的标记来对这些 HSPC 子集进行表型分析。这些全面的单细胞数据提供了对细胞内信号调节的造血功能的有力洞察，并为剖析 HSC 命运决定的性质奠定了坚实的基础。未来需要整合转录组学和蛋白质组学数据以及功能验证，以验证 HSC 分化过程中 HSPC 子集的异质性，并确定可靠的标记来对这些 HSPC 子集进行表型分析。