Mitochondria-Enriched Hematopoietic Stem Cells Exhibit Elevated Self-Renewal Capabilities Within the Context of Aged Bone Marrow

Geriatrics Hematology Immunology Basic Medical Sciences Cell Biology Biochemistry Genetics Life Sciences Medicine
hematopoietic stem cells mitochondria self-renewal aging bone marrow single-cell analysis cell differentiation

Mitochondria-Enriched Hematopoietic Stem Cells Reveal Enhanced Self-Renewal Activity in Aged Bone Marrow: In-Depth Analysis of the Latest Study in Nature Aging

I. Academic Background and Research Significance

Hematopoietic stem cells (HSCs) are the essential foundation underlying life-long production of blood and immune cells. However, with aging, the functional characteristics of these HSCs undergo significant changes, resulting in decreased regenerative capacity, imbalanced differentiation tendency of blood cells, and increased risk of hematological disorders. Studies have shown that HSC aging is not only manifested as functional decline but also involves profound alterations in metabolism, gene expression, and organelle (especially mitochondrial) dynamics. Nevertheless, the specific role of mitochondria in the aging process of HSCs and how mitochondrial quality and quantity affect the stemness and self-renewal capacity of aged HSCs remain inconclusive in academia.

In recent years, mitochondria, as the core of cellular metabolism and energy production, have been recognized as a key hallmark of HSC aging when their function is impaired. Related studies have hypothesized that the regulation of mitochondrial quality affects HSC fate decisions and the balance between stemness and differentiation. However, the current consensus has largely focused on the adverse impact of mitochondrial damage on HSC function, while the true biological characteristics of “high mitochondrial quality/quantity” HSCs in the aged bone marrow environment have not been deeply explored. There is concern that HSCs with high mitochondrial content may simply be a subpopulation with accumulated damage and imminent exhaustion, lacking self-renewal capability and true regenerative potential. Therefore, this study focuses on the impact of mitochondrial quality on the functional state of aging HSCs, aiming to clarify the characteristics and significance of high mitochondrial quality HSCs in aged bone marrow, and to reveal if they possess unique molecular markers and regulatory mechanisms.

II. Source of the Paper and Author Information

The paper, entitled “mitochondria-enriched hematopoietic stem cells exhibit elevated self-renewal capabilities, thriving within the context of aged bone marrow”, was completed by Haruhito Totani, Takayoshi Matsumura (corresponding author), Rui Yokomori, Terumasa Umemoto, Yuji Takihara, and other scholars, with participating institutions including the Cancer Science Institute of Singapore, National University of Singapore, Jichi Medical University (Japan), Kumamoto University (Japan), Institute of Hematology, Chinese Academy of Medical Sciences, Nagoya City University Graduate School of Medical Sciences (Japan), etc. The paper was published in May 2025 in “Nature Aging”, an internationally renowned journal in the field of gerontology.

III. Detailed Research Workflow

1. Overall Study Design

This is a systematic original experimental study, employing transgenic mouse models, single-cell multiomics, cell transplantation, cell sorting, and multiple functional assays to decipher the biological characteristics and functions of “mitochondria-enriched HSCs” in the context of aging.

1.1 Mouse Model and Mitochondrial Labeling System

To accurately and dynamically measure mitochondrial quality in HSCs, the authors used “mito-dendra2 mice” that globally express a mitochondria-targeted Dendra2 fluorescent protein. This system overcomes interference from dye efflux pumps in HSCs in traditional labeling methods, providing precise and stable quantitative analysis of mitochondrial quality.

1.2 HSC Subtype Grouping and Cell Sorting

Young (8-12 weeks) and old (50-140 weeks) mice were used for systematic bone marrow collection, with stringent selection of hematopoietic stem cells via labeling and flow cytometry techniques. HSC subtypes included:

  • LSK (Lin^-Sca1^+c-Kit^+) cells
  • SLAM-HSC (CD150^+CD48^-LSK)
  • ESLAM-HSC (Endothelial protein C receptor^+, or EPCR^+SLAM-HSC)

Based on fluorescence intensity, HSCs from the same batch were further divided into “mito-dendra2 high” (top 20% of fluorescence intensity) and “mito-dendra2 low” (bottom 20%) subgroups.

1.3 Mitochondrial Quality, Function, and Marker Detection

  • Comprehensive assessment of mitochondrial-related parameters, including mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential (MMP), autophagy/mitophagy levels, expression of genes related to oxidative phosphorylation (OXPHOS), etc.
  • Combined analysis of molecular markers (such as EPCR, CD150, GPR183, etc.) to reveal the association between function and molecular phenotype.

1.4 Stem Cell Functional Assays and Transplantation Model

  • Bone marrow transplantation (BMT): High and low mitochondrial quality HSCs, after sorting, were transplanted into lethally irradiated syngeneic recipient mice (with competitor cells); peripheral blood and bone marrow chimerism were analyzed 16 weeks later.
  • In vitro expansion and differentiation: Serum-free culture system containing thrombopoietin (TPO), SCF, and Soluplus polymer was used to assess self-renewal and differentiation tendency of different HSC subgroups.
  • Colony forming unit (CFU-C) assay: In vitro evaluation of the multilineage differentiation potential of HSCs.

1.5 Single-Cell and Multiomics (Multiome) Analysis

  • Using the 10x Genomics platform, SLAM-HSCs from young/old and high/low mitochondrial quality groups were subjected to joint single-cell RNA-seq and ATAC-seq, constructing a panoramic map of cell heterogeneity and molecular regulation.
  • Data analyses were conducted with Seurat, Signac and other R packages, identifying key genes and regulatory networks through nearest neighbor clustering, GSVA, GSEA, differential expression, and multiomics integration algorithms.

1.6 Screening and Functional Validation of the Novel Marker GPR183

  • By cross-validating multiomics data, the specifically stemness-related marker GPR183 for aged HSCs was identified from multiple aged HSC data sources, and its expression specificity and association with HSC stemness were verified through qPCR, flow cytometry, and other methods.

2. Major Experimental Steps and Technical Highlights

  • Innovative use of mito-dendra2 mice avoids interference of traditional mitochondrial dyes by efflux pumps in HSCs, improving the accuracy of distinguishing high and low mitochondrial quality.
  • Integration of multiomics sequencing methods, combining cell phenotype, gene expression, and chromatin accessibility data, enables fine classification and molecular network analysis of HSC subpopulations.
  • Multiple functional experiments coupled with molecular detection directly delineate the connections among “stemness–differentiation–aging,” and establish powerful operational evaluation workflows.

IV. Main Results and Scientific Discoveries

1. High Mitochondrial Quality HSCs in Aged Bone Marrow Retain High Stemness

  • SLAM-HSCs from aged mice exhibited significantly higher mitochondrial quality than those from young mice, partially attributable to decreased autophagy/mitophagy levels and impaired mitochondrial clearance.
  • High mitochondrial quality (mito-dendra2^high) HSCs showed increased mtDNA copy number and enhanced expression of stemness markers such as EPCR and CD150.
  • Bone marrow transplantation experiments revealed that mice receiving high mitochondrial quality HSCs had significantly higher bone marrow long-term HSC (LT-HSC) chimerism, with this subgroup maintaining original stemness for a prolonged post-transplant period.
  • In vitro expansion experiments showed that the high mitochondrial quality HSCs possessed outstanding self-renewal ability, expanding up to 30-fold in two weeks, and remained enriched among stem cell subgroups; in contrast, the low mitochondrial quality group tended to differentiate more readily.

2. High Mitochondrial Quality HSCs Maintain Elevated OXPHOS Activity and ATP Production, Without Obvious ROS Increase

  • Single-cell omics analysis found that high mitochondrial quality HSCs are enriched for both classical and recently identified stemness genes (such as Sca1, EPCR, vwf, alcam, CD63, PDZK1IP1, etc.).
  • GSVA and KEGG enrichment analyses revealed significant enrichment of oxidative phosphorylation (OXPHOS) and iron homeostasis pathways in this HSC group.
  • While mitochondrial aggregation and fusion (with upregulation of MFN2) were observed, there was no marked increase in mitochondrial biogenesis, indicating functional accumulation rather than damage-associated accumulation.
  • ATP production was significantly elevated, primarily relying on OXPHOS; treatment with 2-DG had a greater effect on low mitochondrial quality HSCs, whereas oligomycin A predominantly impacted the high mitochondrial quality subgroup. However, neither cellular nor mitochondrial ROS significantly increased in the high mitochondrial quality group, supporting the notion of being “energized but not damaged.”

3. GPR183 (Epstein–Barr virus-induced gene 2, EBI2) Defines a Self-Renewal-Advantaged Subgroup of Mitochondria-Rich Aged HSCs

  • Through cross-validation of multiomics data, GPR183 emerged as a novel, specific marker for high mitochondrial quality aged HSCs. Its expression is concentrated within aged SLAM-HSC subgroups and is absent in young and differentiated cells.
  • The GPR183^high HSC subgroup also demonstrates high mitochondrial quality, high EPCR, high CD150, and elevated autophagic/mitophagic activity, indicating greater adaptability to the aged bone marrow microenvironment and self-renewal capacity.
  • Both bone marrow transplantation and in vitro expansion experiments confirmed that GPR183^high HSCs possess stronger engraftment, long-term maintenance of stemness, and expansion advantages.
  • As a G protein-coupled receptor, GPR183 expression in animal models and pharmacological manipulations is regulated upstream by mitochondrial biogenesis (PGC1a), but direct activation (e.g., with its ligand 7α,25-OHC) does not increase mitochondrial accumulation, indicating correlation rather than direct causation between the two.

4. Proposal of a Novel Molecular Profile and Functional Subtype of Aged HSCs

  • Besides GPR183, the study identified 17 positive regulatory genes highly relevant to mitochondria-rich aged HSCs (e.g., alcam, jam2, sult1a1, nupr1, etc.) through integration of multiomics and database cross-validation. Most of these genes are closely related to stemness maintenance, iron homeostasis, and autophagy regulation.
  • Contrary to the previous notion that high mitochondrial accumulation simply represents exhausted, low-function states associated with aging, this study presents a new subtype concept of “mitochondria-rich–stemness/autophagy-maintaining” and substantiates it with functional data.

V. Conclusions and Academic/Application Value

1. Scientific Significance

  • This study systematically reveals that, within the aged bone marrow environment, high mitochondrial quality is not a sign of HSC functional exhaustion but instead designates a subgroup with strong self-renewal ability, capacity to adapt to microenvironmental stress, and persistent hematopoietic potential.
  • The identification of HSC subgroups with new molecular markers such as GPR183 extends the biological understanding of stem cell heterogeneity in aging and provides a new paradigm for investigating stemness maintenance and microenvironmental adaptation mechanisms.

2. Application Value

  • Provides new molecular targets and HSC selection criteria for precision medicine in elderly bone marrow transplantation and stem cell therapy, which may help enhance the efficacy and safety of transplantation in elderly patients.
  • Offers important theoretical and translational significance for developing methods to support repair of aged HSCs through mitochondrial regulation and GPR183-related pathways.

3. Research Highlights

  • The first use of mito-dendra2 transgenic mice enables high-throughput, accurate in vivo sorting of HSCs based on mitochondrial quality, supplying a powerful tool for HSC subtyping and functional testing.
  • Innovatively proposes GPR183 as a novel molecular marker for mitochondria-rich HSC subgroups in the aged population and systematically validates its association with functional stemness advantage.
  • Through integration of multiomics data and functional experiments, the study demonstrates that, contrary to traditional views, mitochondrial accumulation can accompany stemness maintenance and does not invariably indicate aging-related damage.

VI. Other Valuable Information

  • The discussion section systematically reviews iron metabolism, ferritin, iron homeostasis, HSC autophagy, and oxidative stress adaptation, supplying theoretical support for follow-up mechanistic studies.
  • The materials and methods section details parameter settings for analysis platforms, algorithms, antibodies, and transgenic mice models, ensuring experimental reproducibility and the authority of results.
  • The study suggests that the phenotype and function of aged HSCs can be dynamically modulated by the microenvironment, providing new insights for theories of dynamic stemness maintenance.

VII. Summary

This study, published in Nature Aging, systematically delves into the self-renewal advantages and molecular basis of mitochondria-rich aged HSCs in the bone marrow, from model establishment, molecular markers, functional assays, to the integration of multiomics big data. The discovery of new markers such as GPR183 not only expands the understanding of heterogeneity in aging hematopoietic stem cells but also provides a theoretical and practical basis for stem cell transplantation and the treatment of hematopoietic diseases in elderly populations. This research supplies important scientific materials and ideas for future exploration of HSC aging mechanisms and related clinical translation.