Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, 510095, China.
Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, State Key Laboratory of Respiratory Disease, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, 511436, China.
BMC Biol. 2021 Sep 9;19(1):201. doi: 10.1186/s12915-021-01138-6.
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.
此前，人们曾使用细胞表面标志物对小鼠造血干/祖细胞（HSPC）亚群进行研究，而最近则采用了单细胞技术。单细胞分析的最新革命正在极大地改变我们对造血的理解，证实了构成造血系统的细胞具有很大的异质性。尽管人们已经广泛研究了造血过程中 DNA/RNA 水平的动态分子变化，但对于造血信号转导程序和图谱的单细胞异质性的广泛理解，即在蛋白质水平上研究并反映转录后加工的信号转导程序和图谱的异质性，仍知之甚少。在这里，我们使用液质联用技术在单细胞水平上准确地定量了 9 种磷酸化蛋白和 2 种功能蛋白的细胞内水平，从而系统地捕获了包括 EGFR、Jak/Stat、NF-κB、MAPK/ERK1/2、MAPK/p38、PI3K/Akt、Wnt 和 mTOR 在内的 8 条信号通路在小鼠造血过程中的激活动态。
通过对 320 万个造血干/祖细胞（HSPC）和谱系细胞的精细分析，以及多参数细胞表型分析，我们绘制了 HSC 分化过程中信号转导程序的轨迹，并确定了循环 HSPC 和从干细胞到祖细胞和谱系细胞的多种分化途径的特定信号生物标志物。我们还研究了造血重建过程中造血细胞群体的恢复模式以及这些群体中的信号转导调节。总的来说，我们发现 HSPC 亚群中的信号通路激活具有很大的异质性，表现出不同的信号模式。
这些全面的单细胞数据为深入了解细胞内信号调控的造血提供了有力的依据，并为剖析 HSC 命运决定的本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，并进行功能验证，以验证 HSC 分化过程中 HSPC 亚群的异质性，并确定用于表型分析这些 HSPC 亚群的稳健标志物。
