State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin, 300020, China.
School of Medicine, Nankai University, Tianjin, China.
Stem Cell Res Ther. 2022 Jun 11;13(1):245. doi: 10.1186/s13287-022-02926-9.
The ubiquitin-proteasome system plays important roles in maintaining the self-renewal and differentiation of stem and progenitor cells through highly ordered degradation of cellular proteins. Fbxw11, an E3 ligase, participates in many important biological processes by targeting a broad range of proteins. However, its roles in hematopoietic stem/progenitor cells (HSPCs) have not been established.
In this study, the effects of Fbxw11 on HSPCs were studied in vitro and in vivo by an overexpression strategy. Real-time PCR was performed to detect the expression of Fbxw11 in hematopoietic subpopulations. Colony-forming assays were performed to evaluate the in vitro function of Fbxw11 on HSPCs. Hoechst 33342 and Ki67 staining was performed to determine the cell-cycle distribution of HSPCs. Competitive transplantation experiments were used to evaluate the effect of Fbxw11 on the reconstitution potential of HSPCs. Single-cell RNA sequencing (scRNA-seq) was employed to reveal the transcriptomic alterations in HSPCs.
The expression of Fbxw11 was higher in Linc-KitSca-1 (LSK) cells and myeloid progenitors than in lymphoid progenitors. Fbxw11 played negative roles in colony-forming and quiescence maintenance of HSPCs in vitro. Furthermore, serial competitive transplantation experiments revealed that Fbxw11 impaired the repopulation capacity of HSPCs. The proportion of granulocytes (Gr-1CD11b) in the differentiated mature cells was significantly higher than that in the control group, T cells and B cells were lower. Moreover, scRNA-seq revealed seven cell clusters in HSPCs. In addition, Fbxw11 downregulated the expression of Cebpa, Myc and Arid5b, which are significant regulators of HSPC activity, in most cell clusters.
Our data demonstrate that Fbxw11 plays a negative role in the maintenance of HSPCs in vitro and repopulation capacity in vivo. Our data also provide valuable transcriptome references for HSPCs in homeostasis.
泛素-蛋白酶体系统通过对细胞蛋白的高度有序降解，在维持干细胞和祖细胞的自我更新和分化方面发挥着重要作用。Fbxw11 是一种 E3 连接酶，通过靶向广泛的蛋白质参与许多重要的生物学过程。然而，其在造血干细胞/祖细胞（HSPCs）中的作用尚未确定。
在这项研究中，通过过表达策略在体外和体内研究了 Fbxw11 对 HSPCs 的影响。实时 PCR 用于检测造血亚群中 Fbxw11 的表达。集落形成测定用于评估 Fbxw11 对 HSPCs 体外功能的影响。Hoechst 33342 和 Ki67 染色用于确定 HSPCs 的细胞周期分布。竞争移植实验用于评估 Fbxw11 对 HSPCs 重建潜能的影响。单细胞 RNA 测序（scRNA-seq）用于揭示 HSPCs 中的转录组改变。
Fbxw11 在 Linc-KitSca-1（LSK）细胞和髓系祖细胞中的表达高于淋巴系祖细胞。Fbxw11 在体外对 HSPCs 的集落形成和静止维持发挥负作用。此外，连续的竞争移植实验表明，Fbxw11 损害了 HSPCs 的重殖能力。分化成熟细胞中粒细胞（Gr-1CD11b）的比例明显高于对照组，T 细胞和 B 细胞的比例较低。此外，scRNA-seq 揭示了 HSPCs 中的七个细胞簇。此外，Fbxw11 下调了大多数细胞簇中 HSPC 活性的重要调节因子 Cebpa、Myc 和 Arid5b 的表达。
我们的数据表明，Fbxw11 在体外维持 HSPCs 和体内重建能力方面发挥负作用。我们的数据还为 HSPCs 在体内平衡中提供了有价值的转录组参考。
