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. 2021 Aug 9;12(1):444.
doi: 10.1186/s13287-021-02455-x.

Using a new HSPC senescence model in vitro to explore the mechanism of cellular memory in aging HSPCs

Yongpin Dong #  1   2 , Chunni Guo #  3 , Wuxiong Zhou  1 , Wenfang Li #  4 , Lina Zhang #  5
Affiliations

Using a new HSPC senescence model in vitro to explore the mechanism of cellular memory in aging HSPCs

Yongpin Dong et al. Stem Cell Res Ther. .

Retraction in

Abstract

Background: Age-associated changes attenuate human blood system functionality through the aging of hematopoietic stem and progenitor cells (HSPCs), manifested in human populations an increase in myeloproliferative disease and even leukemia; therefore, study on HSPC senescence bears great significance to treat hematopoietic-associated disease. Furthermore, the mechanism of HSPC aging is lacking, especially the cellular memory mechanism. Here, we not only reported a new HSPC senescence model in vitro, but also propose and verify the cellular memory mechanism of HSPC aging of the Polycomb/Trithorax system.

Methods: HSPCs (Lin-c-kit+ cells) were isolated and purified by magnetic cell sorting (MACS). The proportions and cell cycle distribution of cells were determined by flow cytometry; senescence-related β-galactosidase assay, transmission electron microscope (TEM), and colony-forming unit (CFU)-mix assay were detected for identification of the old HSPC model. Proteomic tests and RNA-seq were applied to analyze differential pathways and genes in the model cells. qPCR, Western blot (WB), and chromatin immunoprecipitation PCR (CHIP-PCR) were used to detect the gene expression of cell memory-related proteins. Knockdown of cell memory-related key genes was performed with shRNA interference.

Results: In the model old HSPCs, β-gal activity, cell cycle, colony-forming ability, aging-related cell morphology, and metabolic pathway were significantly changed compared to the young HSPCs. Furthermore, we found the model HSPCs have more obvious aging manifestations than those of natural mice, and IL3 is the major factor contributing to HSPC aging in the model. We also observed dramatic changes in the expression level of PRC/TrxG complexes. After further exploring the downstream molecules of PRC/TrxG complexes, we found that Uhrf1 and TopII played critical roles in HSPC aging based on the HSPC senescence model.

Conclusions: These findings proposed a new HSPC senescence model in vitro which we forecasted could be used to preliminary screen the drugs of the HSPC aging-related hemopathy and suggested cellular memory mechanism of HSPC aging.

Keywords: Cellular memory; HSPC; Senescence model; TOPOIIα; UHRF1.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
HSPCs were effectively isolated and purified by MACS. a Schematic illustrates the isolation of mouse bone marrow mononuclear cells (MNCs). b For lineage depletion, cells were magnetically labeled with a cocktail of biotinylated antibodies that are against a panel of so-called lineage antigens (CD5, CD45R (B220), CD11b, anti-Gr-1, and Ter-119 antibodies) and anti-Biotin MicroBeads. This labeling procedure leaves the lineage-negative cells undisturbed, thus allowing further separation of lineage− cells. Then, the lineage− cells (lin− cells) were purified by c-Kit MACS, namely lin-c-Kit+ cells. c HSPCs were cultured with a specific modeling medium (stem cell culture medium + 10 ng/mL IL3 + 10 ng/mL IL6 + 30 ng/mL SCF) and incubated for 8 days, its the model group. d Flow cytometric analysis of the purity of lin–c-Kit+ cells at different phases. The results were expressed as mean ± S.D., and the p values (*P < 0.05, **P < 0.01, ***P < 0.001) were determined by the ANOVA test
Fig. 2
Fig. 2
Model HSPCs showed significant manifestations of aging such as increased β-galactosidase activity, G0/G1 phase arrest, decreased colony-forming capacity, and changes in cell morphology. a Photomicrographs of SA-β-gal staining(× 200). The percentage of SA-β-gal stain-positive cells was significantly increased in the model group compared with the young group, n = 10. b Flow cytometric analysis of cell cycle distribution. The model HSPCs were arrested in G0/G1 phase compared with the young group, n = 10. c Photomicrographs of CFU-Mix. The size and number of CFU-Mix significantly decreased in the model group compared with the young group. d. Photomicrographs of TEM. The nuclear membranes of young HSPCs were smooth and flat, chromatin evenly distributed; there were few to no inclusion bodies found in the cytoplasm. But the perinuclear cisternae in the model HSPCs widened, and chromatin edge aggregated. A large number of inclusion bodies appeared in the model HSPCs
Fig. 3
Fig. 3
Integrated proteomic and transcriptomic analysis showed significant changes of metabolic processes associated with aging in Model HSPCs. a The volcano plot of proteomic, which plots significance versus fold change on the y- and x-axes, respectively, found upregulated and downregulated proteins using t-test. The red color represents the upregulated proteins, and the blue color represents downregulated proteins. b The heat map of proteomics showed protein expression levels of the young group and the model group. Color intensity indicates the level of expression, where green signifies low expression and red signifies high expression. c Gene Ontology analysis of proteomics, p < 0.05. d KEGG analysis of proteomics. Go enrichment (c) and KEGG pathway (d) analysis showed that glycolysis, lysosomal metabolism, ribosomal synthesis, and mRNA splicing were significantly changed in the model. e STRING analysis of proteomics showed that the common target proteins of PcG and TrxG are UHRF1 and TOPOIIa. f The scatter plot of transcriptomic: red dots were upregulated, and blue dots were downregulated. g Go enrichment analysis of transcriptomics showed genes associated with the stress response, inflammation, lysosomal metabolism, and protein aggregation dominated the upregulated expression profile, while the downregulated profile was marked by genes involved in the preservation of genomic integrity and chromatin remodeling
Fig. 4
Fig. 4
The factors driving the model HSPC aging. a–h Photomicrographs of SA-β-gal staining (× 100). The cells cultured with IL3 alone or together with IL3 were mainly SA-β-gal-positive. There was no significant change in the SA-β-gal activity at the IL6,SCF alone group or together with the IL6,SCF group. i, j Photomicrographs of CFU-Mix. The cells cultured with IL3 alone or together with IL3 showed a significant decrease in colony-forming ability. There was no significant change in the colony-forming ability at the IL6,SCF alone group or together with the IL6,SCF group
Fig. 5
Fig. 5
Polycomb/Trithorax system disturbance in HSPC aging process. RT-PCR was applied to determine the transcription levels of PcG/TrxG genes (a). Western blot was applied to determine the levels of PcG/TrxG proteins and TOP2a,URHF1 (c) and H3K4,H3K27 (b)
Fig. 6
Fig. 6
CHIP-PCR showed H3K4me3 of TOPOIIα and UHRF1 promoter decreased in senescent HSPCs. a, b The level of H3K4me3 in TOPOIIα/UHRF1 promoter was decreased in aging HSPCs. c, d There was no significant change in the level of H3K27me3 in TOPOIIα/UHRF1 promoter in aging HSPCs
Fig. 7
Fig. 7
Bmi-1 and Trx were important members of PCG/TrxG protein and knocking down Bmi-1/Trx downregulated TOPOIIα/UHRF1 expression. a, b HSPCs from 4-week-old mice were transfected with Bmi-1-siRNA and Trx-siRNA at 48 h. Bmi-1 gene expression (b, c) and Trx gene expression (a, d) were validated by real-time PCR and WB analysis. mRNA expression of TOPOIIα (e) and UHRF1 (f) decreased significantly in the Bmi-1 or the Trx knocked down group. SA-β-gal-stained cells increased in the Bmi-1 or the Trx knocked down group, but there was no significant difference (h). The colony-forming ability of HSPCs significantly decreased in the Bmi-1 (j) or the Trx (i) knocked down group compared with the control group
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