Selective Advantage of Clonal Hematopoietic Mutant Stem Cells in Aging and Inflammatory Responses

Background and Research Motivation

Clonal Hematopoiesis (CH) is an aging-related phenomenon in the hematopoietic system, where hematopoietic stem cells (HSCs) acquire specific somatic mutations and undergo expansion, causing a particular mutated cell line to occupy a significant proportion in an individual’s blood. Recent research indicates that CH is associated with various adverse health outcomes, including cardiovascular diseases, myeloid malignancies, and other age-related diseases. However, how mutant cell clones gain competitive advantage and expand in the body remains unclear.

Current studies have identified that the most common gene mutations in CH involve the DNMT3A and TET2 genes. These mutations confer certain selective advantages to mutant cells, allowing them to gradually expand within the human hematopoietic system. Therefore, this study focuses on exploring the mechanisms of DNMT3A and TET2 gene mutations in CH, aiming to uncover their molecular regulatory mechanisms in inflammatory responses and aging environments, thereby providing new insights into the clonal selection and expansion of CH.

Research Source and Publication Information

This paper was completed in collaboration by scholars from internationally renowned research institutions including Oxford University, Cambridge University, and the University of Toronto in Canada, such as Jakobsen and Turkalj. It was published on August 1, 2024, in the journal “Cell Stem Cell.” The study used high-precision single-cell genotyping and transcriptome sequencing to analyze HSCs in human CH samples, offering new insights into the molecular mechanisms of CH.

Research Design and Experimental Process

Subjects and Sample Collection

The study selected 195 patients without a history of hematological malignancies who were undergoing hip replacement surgeries to collect bone marrow samples for analysis. High-depth sequencing identified CH in 57 of these patients, with 69% of CH patients carrying mutations in the DNMT3A or TET2 genes. To eliminate interference from malignancies, patients with inflammatory arthritis or those using systemic steroids long-term were excluded from the study.

Single-cell Multi-omics Analysis Method

The research adopted the optimized Target-seq+ single-cell sequencing method, combined with high-precision genotyping and transcriptome sequencing technologies, to analyze hematopoietic stem and progenitor cells (HSPCs) in CH and non-CH control samples. This technology has high sensitivity in detecting transcript levels of each cell, enabling the differentiation of mutated and non-mutated cells within the same tissue, thereby deeply analyzing the molecular characteristics of HSCs affected by CH mutations.

Analysis of Clone Expansion of Mutant Stem Cells

To quantify the clonal expansion patterns of CH mutant stem cells during hematopoietic differentiation, the study employed genotype-transcriptome-based MELD and scCODA analysis methods. Using UMAP visualization, mutant and wild-type (WT) cells were projected into transcriptomic space, observing the distribution differences of DNMT3A and TET2 mutant clones at different stages of hematopoietic differentiation.

Research Results

Clonal Expansion Patterns of DNMT3A and TET2 Mutations in HSCs

The study found that HSCs with DNMT3A and TET2 mutations exhibited significant differences in transcriptomic characteristics, each with distinct clonal expansion patterns: 1. DNMT3A Mutant Clones primarily expand in HSC and multipotent progenitors (MPPs) without showing a clear lineage bias. 2. TET2 Mutant Clones expand further into granulocyte-monocyte progenitors (GMPs) after expansion in HSCs and MPPs, demonstrating a more obvious myeloid differentiation bias.

Effects of Inflammation and Aging on Mutant and Non-mutant HSCs

By comparing the gene expression profiles of CH mutant and non-mutant HSCs, the study discovered that the inflammatory response and aging-related transcription programs of mutant HSCs were suppressed: - Compared to non-CH HSCs, mutant HSCs in CH samples exhibited a weaker response to TNF-α signaling pathways and NF-κB, indicating enhanced adaptability to inflammatory microenvironments. - In contrast, non-mutant HSCs in CH showed significantly enhanced expression of inflammation response-related genes, indicating that the inflammatory environment adversely affects these non-mutant HSCs.

The adaptability advantage of mutant HSCs in an inflammatory environment allows them to gain a competitive advantage over the long term and gradually expand, which may be a key mechanism for the clonal expansion of CH.

Non-cell Autonomous Effects of CH

The study also found that non-mutant HSCs in CH samples were significantly affected by the inflammatory microenvironment, exhibiting transcriptional characteristics similar to mutant HSCs. This suggests that the clonal expansion of CH is driven not only by the intrinsic properties of mutant cells but also by environmental factors. This “non-cell autonomous” effect may exacerbate the expansion of CH by spreading inflammatory factors to surrounding non-mutant cells.

Cell Type-specificity of Mutant HSCs in Clonal Selection

Through further subgroup analysis of HSCs and early progenitor cells, the study identified three HSC subgroups with different transcriptional characteristics, with one subgroup (HSC2) being more active in inflammation and aging signaling pathways, exhibiting higher TNF-α/NF-κB signaling activity and aging-related gene expression. However, TET2 mutant cells were less frequent in this subgroup, which may be a reason for the weakened inflammatory signaling response of mutant HSCs.

Conclusion and Significance

This study reveals the molecular mechanisms of DNMT3A and TET2 mutations in the clonal selection of CH, proposing a CH expansion model based on the inflammatory microenvironment: mutant HSCs gain competitive advantages in adverse environments by suppressing inflammation and aging signaling responses and gradually expanding. This mechanism not only explains the dynamics of CH clonal expansion but also provides new ideas for future CH therapy, suggesting that reducing inflammatory stress or decreasing the inflammation tolerance of mutant HSCs may help inhibit CH progression.

Research Highlights

1. High-precision Single-cell Sequencing Technology: The optimized Target-seq+ technology achieved high-precision genotyping and transcriptome sequencing of CH mutant and non-mutant HSCs.
2. Non-cell Autonomous Effects of Inflammation and Aging on CH: Revealed the selective advantage of mutant HSCs in inflammatory environments and the potential impact of CH on surrounding non-mutant cells.
3. Myeloid Differentiation Bias of TET2 Mutations: The expansion advantage of TET2 mutant HSCs in myeloid differentiation provides a new perspective for understanding the lineage selectivity of CH.
4. Adaptation of CH Mutations to Inflammatory Response: CH mutations confer competitive advantages to mutant HSCs in adverse microenvironments by weakening inflammation and aging-related transcriptional responses.

Scientific and Practical Value of the Research

This study provides a molecular basis for the selective expansion of CH mutant HSCs in inflammatory and aging environments, helping to explain the association of CH with various age-related diseases. In the future, by further studying the interaction mechanisms between CH and the inflammatory microenvironment, it is expected to develop intervention strategies for CH to mitigate the health risks it poses, such as reducing the incidence of cardiovascular diseases and myeloid malignancies. This finding offers new theoretical support and potential therapeutic directions for achieving healthy aging in an aging society.