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Effect of Proteinase 3 on the Proliferation and Differentiation of Hematopoietic Stem/Progenitor Cells

SUN Lu^1,2, LIU Huan^1,2, XIE Xuemei^1,2, WANG Lisha^1,2, ZHANG Shiyue^1,2, ZHAO Zihan^1,2, CHENG Tianran^1,2, JIANG Shan^1,2, WANG Xiaohan³, WANG Tong^1,2, XU Yuanfu^1,2*

(¹State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China; ²Tianjin Institutes of Health Science, Tianjin 301600, China; ³the Second School of Clinical Medicine, Guangdong Medical University, Dongguan 523808, China)

Abstract:

PRTN3 (proteinase 3) is a neutral serine protease, which is associated with pathogen elimination, tissue damage, and apoptosis. Recent studies have reported that Prtn3 is highly expressed in hematopoietic stem/progenitor cells, but the biological functions and annotations remain largely unknown. Here, this article aimed to investigate Prtn3 expression in the hematopoietic compartment of wild-type mice (WT) by single-cell transcriptomics and construct a Prtn3 knockout mouse model (Prtn3^–/–) to analyze the number and percentage of LT-HSC, ST-HSC, MPP, CMP, GMP, and MEP as well as the mature blood cells by flow cytometry and blood routine analysis. In vitro single-cell colony assay and CFC assay was used to investigate the proliferation and differentiation potential of LSKs. Competitive transplantation of isolated mouse bone marrow LSK cells was used to analyze the number and percentage of different lineages of in PB, SP, and BM, and compare the distribution of donor-derived cells in SP and BM. The results showed that Prtn3 was consistently high expression in mouse hematopoietic stem/progenitor cells, especially in myeloid progenitor cells (CMP and GMP). The proportion of LSK and LK cells in the bone marrow of Prtn3^–/– mice was significantly higher than that of WT mice, and the number and proportion of LT-HSC, STHSC, MPP, CMP and GMP in bone marrow cells significantly increase (P<0.05). Single-cell colony assays showed that the number of cells produced by Prtn3^–/– LSK cell division, especially the number of small clones, was significantly higher than that in the control group (P<0.05). In vitro colony, formation assays showed that the number of G, M and GM colony formation of Prtn3^–/– LSK cells increased significantly compared with the control group (P<0.05). Competitive transplantation assays showed that the progeny cells produced by Prtn3^–/– LSKs dropped significantly (P<0.05), and the proportion of neutrophils and monocytes produced by Prtn3^–/– LSKs increased (P<0.05), while the proportion of lymphocytes decreased (P<0.05). In conclusion, the Prtn3 gene is involved in the regulation of hematopoietic stem/progenitor cell proliferation and differentiation. This study provides a certain theoretical and experimental basis for the study of the regulation of proliferation and differentiation of hematopoietic stem/progenitor cells for the maintenance of hematopoietic homeostasis.

CSTR: 32200.14.cjcb.2023.05.0003