Alessandro Donada

Hematopoietic aging is marked by declining regenerative capacity and a shift from lymphoid to myeloid output. Previous studies have focused primarily on hematopoietic stem cells (HSCs), yet whether aging uniformly remodels the hematopoietic stem and progenitor cell (HSPC) compartment, or selectively affects distinct populations, remains largely unknown. To address this, we profile HSCs and multipotent progenitors MPP1-6 from young and aged mice across transcriptomic, epigenomic, metabolomic and lipidomic layers, together with single-cell division and in vivo serial transplantation assays. We identify young MPP6 (negative for CD150, CD34, CD48 and CD135) as an HSC-like, lymphoid-biased population with long-term reconstitution capacity, positioned within the top-tier of the differentiation hierarchy and marked by low global chromatin accessibility and metabolic activity. Upon aging, MPP6 preserved long-term lymphoid reconstitution, underwent limited transcriptional and regulatory remodeling, and did not accumulate polyunsaturated phospholipids or show mitochondrial oxidation. By contrast, most HSPCs, and particularly HSCs, underwent pronounced age-associated changes, including myeloid-biased expansion, metabolic rewiring, extensive distal regulatory alteration and accumulation of long-chain polyunsaturated phospholipids linked to increased oxidative stress. Pseudotime analysis further resolved the aged MPP6 compartment into two subsets, including a dormant, lymphoid-biased Dlk1-expressing population characterized by reduced mitochondrial activity, which was distinctly affected by inflammatory challenge in vivo. Together, these findings establish MPP6 as a comparatively resilient population within the aged HSPC compartment and suggest that it may represent a potential target for restoring hematopoietic balance.

MYC overexpression is a crucial element of acute myeloid leukaemia (AML) physiopathology. By advanced cellular modelling and molecular analysis, Stokes and colleagues showed that MYC overexpression induces a myeloid block. Sustained expression of this oncogene is required and sufficient to maintain the block, a feature shared by multiple AML subtypes.

Summary: Clonal memory, a cellular property inherited across at least two divisions, has emerged as a key driver of cell heterogeneity. To uncover its roles in human haematopoiesis, we developed high-resolution ex vivo tools that track both division and fate commitment of individual primary human haematopoietic stem and progenitor cells (HSPCs). We show that human HSPCs display a clonal memory of division, as cells descending from the same ancestor cell divide synchronously over multiple generations. In parallel, HSPCs inherit a clonal memory of fate commitment, independently of lineage identity. Both forms of clonal memory persist over at least two divisions, across different HSPC commitment stages and cell culture conditions. In contrast, malignant haematopoiesis exhibits lower synchronicity, revealing a disruption of clonal memory in leukemic cells. Epigenetic remodelling using a bromodomain inhibitor partially restores the clonal memory in division in leukemic HSPCs, highlighting the plasticity of this trait and its potential for therapeutic modulation. Our findings position clonal memory as a key regulator of human haematopoietic stem cell behaviour. Demonstrating that clonal memory can be modulated opens new avenues for tuning cell heterogeneity in healthy and pathological tissues.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes Thrombocytopenia 2 (THC2), an inherited platelet disorder associated with leukemia predisposition. Some of those patients present also erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patient cells and patient-derived iPSCs) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly impacts the proliferation/differentiation balance for these three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization, which leads to increased signaling and cytokine hypersensitivity. Altogether these findings show that ANKRD26 overexpression or the absence of its silencing during differentiation are responsible for myeloid blood cell abnormalities in THC2 patients.

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Filamin A (FLNa) links the cell membrane with the cytoskeleton and is central in several cellular processes. Heterozygous mutations in the X-linked FLNA gene are associated with a large spectrum of conditions, including macrothrombocytopenia, called filaminopathies. Using an isogenic pluripotent stem cell model derived from patients, we show that the absence of the FLNa protein in megakaryocytes (MKs) leads to their incomplete maturation, particularly the inability to produce proplatelets. Reduction in proplatelet formation potential is associated with a defect in actomyosin contractility, which results from inappropriate RhoA activation. This dysregulated RhoA activation was observed when MKs were plated on fibrinogen but not on other matrices (fibronectin, vitronectin, collagen 1, and von Willebrand factor), strongly suggesting a role for FLNa/α_IIbβ₃ interaction in the downregulation of RhoA activity. This was confirmed by experiments based on the overexpression of FLNa mutants deleted in the α_IIbβ₃-binding domain and the RhoA-interacting domain, respectively. Finally, pharmacological inhibition of the RhoA-associated kinase ROCK1/2 restored a normal phenotype and proplatelet formation. Overall, this work suggests a new etiology for macrothrombocytopenia, in which increased RhoA activity is associated with disrupted FLNa/α_IIbβ₃ interaction. Publisher9s Note: There is a Blood Commentary on this article in this issue. Key Points FLNA mutations lead to the absence of FLNa in patient MKs and a defect in proplatelet formation via RhoA overactivation. The increased RhoA activity in FLNA-mutated MKs is driven by the disruption of FLNa/α_IIbβ₃ interaction. Visual Abstract