Dysfunctional Clusterin-Positive Hematopoietic Stem Cells Expand with Aging

Abstract

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes dysregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old), and aged (20-month-old) mice to gain insight into the dynamics of HSC aging. scRNA-seq data revealed that transcriptomic profiles of HSCs change with aging, although middle-aged and aged HSCs showed very similar profiles to each other. scRNA-seq data divided young and aged HSCs into 2 and 3 clusters, respectively. While both young and aged HSCs had clusters with myeloid or lymphoid gene signature, the cluster with myeloid signature dominated in aged HSCs, in which aged gene signature (Nupr1, Mt1, Mt2, Selp) was highly enriched. Clusterin (Clu) was also significantly up-regulated in this cluster. Clu encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis (Itakura et al., JCB 2020). We hypothesized that Clu would be useful as a marker of aged HSCs, and conducted further analysis using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory elements of the Clu gene. Bone marrow (BM) analysis of Clu-reporter mice revealed that Clu expression is confined to a part of HSCs in BM and significantly up-regulated within the HSC compartment with aging (10 and 70 % of HSCs are Clu-positive in young and middle-aged mice, respectively). In competitive repopulation assays, Clu-positive HSCs showed severely impaired repopulation capacity and preferentially differentiated into myeloid cells while Clu-negative HSCs largely retained repopulation capacity and showed balanced differentiation. We analyzed the transcriptomic profile of Clu-positive HSCs with aging. RNA-sequence analysis revealed that young and middle-aged Clu-positive HSCs were significantly enriched for aged HSC gene signature and myeloid-biased HSC signature compared to Clu-negative HSCs. We evaluated the transcriptional regulation mechanism of Clu by Assay for Transposase-Accessible Chromatin sequence (ATAC-seq) analysis. Chromatin accessibility at the Clu promoter was already open in young HSCs, and largely enhanced in Clu-positive HSCs but not in Clu-negative HSCs among middle-aged HSCs. The open chromatin region at the Clu promoter had binding motifs of AP-1 and STAT family transcription factors, suggesting that external signals or stresses activate Clu expression. These results suggest that Clu-positive cells represent myeloid-biased HSCs which expand with aging and that Clu serves as a faithful marker to trace alterations in HSC heterogeneity with aging.