Stem Cells

Mesenchymal stromal cells (MSCs) are promising cell therapeutics, but their development as off-the-shelf products requires cryopreservation, which impairs cell function after thaw. Inflammatory licensing enhances MSC therapeutic attributes, yet protocols vary considerably and little is known about how these variables affect MSC metabolism or post-thaw recovery. We characterized mitochondrial and glycolytic flux in umbilical cord (UC) and bone marrow (BM) MSCs licensed with interferon-{gamma} (IFN-{gamma}), tumor necrosis factor- (TNF-), or both. Brief exposure to either cytokine shifted MSC metabolism toward glycolysis and reduced mitochondrial respiration, whereas prolonged exposure led to heterogeneous responses. Conversely, combined IFN-{gamma} and TNF- increased glycolytic flux and respiratory capacity in most donors, with responses detectable after 2 hours and at concentrations below those conventionally used. Cryopreservation impaired aerobic metabolism, followed by donor-dependent recovery. Preconditioning with combined IFN-{gamma} and TNF- partially improved post-thaw metabolism, particularly in BM MSCs, and enhanced early responsiveness to inflammatory challenge, particularly in UC MSCs, as detected by IDO and candidate biomarkers CTSS and C1S. However, donors with improved post-thaw metabolism did not consistently show increased biomarker responses. These findings demonstrate that metabolic fitness and inducible biomarkers capture complementary aspects of post-thaw MSC quality and may inform preconditioning strategies for cryopreserved MSC products.

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

Background: The ability to repopulate all lineages of blood cells from a single hematopoietic stem cell (HSC) requires a constant balancing between maintenance of quiescence/self-renewal and the need for differentiation and adoption of specific lineage phenotypes. A classical interpretation suggests that during homeostasis, long- and then short-term HSCs differentiate into multipotent progenitors (MPPs), which then lack self-renewal capacity but can still differentiate into all downstream lineages. While much is known about the intrinsic and extrinsic regulators that promote HSC homeostasis, less is known about factors which influence HSC behavior following significant bone marrow (BM) injury, such as sublethal irradiation (SLI). Methods: Conditional knock-out of follistatin-like 1 (FSTL-1) was achieved by breeding mice with floxed Fstl1 sites to mice expressing CreERT2 under control of the ROSA locus, followed by systemic tamoxifen administration. ELISA and RT-PCR quantitated protein and RNA level loss of FSTL-1, respectively. HSC populations were assessed by flow cytometry under multiple conditions. Sublethal irradiation induced BM injury and blocking polyclonal FSTL-1 antibodies were used to recapitulate results seen with genetic ablation. Finally, single cell RNA sequencing defined both cell-specific expression of Fstl1 within the BM compartment, but also identified transcripts and pathways upregulated in the absence of FSTL-1. Results: These studies delineate a novel role for FSTL-1 in regulating HSC biology. In addition to selective expression of Fstl1 within HSCs, genetic deletion of Fstl1 increases Lineage negative;Sca-1+c-Kit+ (LSK) cell number and alters HSC homeostasis. These findings are further amplified in the BM in the context of recovery from sublethal irradiation. Importantly, the post-irradiation increase in BM cellularity seen with Fstl1 deletion is replicated by treatment with anti-FSTL-1 polyclonal antibodies. Finally, single cell RNA sequencing reveals distinct Fstl1 expression in specific LSK subpopulations, including HSCs and proliferating cells, and identifies a novel relationship between decreased FSTL-1 levels and enrichment of canonical metabolic pathways including oxidative phosphorylation, fatty acid metabolism, and glycolysis. Conclusions: Taken together, these results highlight a novel role for FSTL-1 in promoting HSC homeostasis. They also unlock an innovative way to promote HSPC recovery following BM injury, knowledge predicted to be of significant therapeutic value.

Human pluripotent stem cells (hPSCs) are increasingly expanded in 3D suspension culture to support scalable cell culturing, yet how the transition from 2D to 3D cultures affects hPSCs remains poorly understood. Here, we compare hPSCs in 2D and 3D cultures using transcriptomics, proteomics, spent medium analysis, and stable-isotope tracing. hPSCs maintained pluripotency in both formats, although suspension culture showed altered growth dynamics and increased initial cell loss. Multi-omics analysis identified culture format as the dominant driver of molecular variation and revealed changes in adhesion, cytoskeletal organization, glycolysis, and oxidative phosphorylation. Functional analyses showed increased lactate production from glucose and stable-isotope tracing revealed increased glucose-derived pyruvate formation and altered partitioning of glucose-derived carbon within the TCA cycle in 3D suspension cultures. Glutamine contribution to the TCA cycle was reduced, whereas relative reductive carboxylation was increased in 3D suspension cultures. Together, these findings show that transition from 2D to 3D suspension culture induces metabolic rewiring in hPSCs.

Microglia, the immune cells of the brain parenchyma, play a pivotal role in neurodevelopment and neuroprotection. Human stem cell-derived microglia-like cells (MGL) offer a valuable, human-relevant system to study microglia in vitro. However, their cell states and intercellular interactions within complex 3-dimensional (3D) tissue contexts in vitro remain underexplored. Here, we analyze the gene expression profiles of MGL generated by two differentiation protocols in conventional 2D cultures. We show that co-culture with neural organoids promotes a more mature, in vivo-like MGL phenotype but this signature is lost during long-term co-culture. By varying media compositions, we modulate 2D MGL states toward more active or more homeostatic identities, which may enhance the similarity to primary microglia identities upon subsequent co-culture. Moreover, forced expression of the microglial lineage regulator SALL1 shifts MGL away from a general myeloid state toward a more defined pre-microglial fate. SALL1 overexpression within a 3D neural organoid environment further amplifies the shift toward a homeostatic developmental microglia population. Collectively, our transcriptomic analysis delineates human MGL states in 2D and 3D contexts, refines existing differentiation protocols toward more authentic MGL, and establishes a framework to investigate the intricate microglia-neural tissue interplay.

Acutely induced cellular senescence is increasingly recognized as vital for tissue repair but the precise roles of senescent cells in skeletal muscle regeneration remain paradoxical. Here, using single-cell RNA sequencing and a Unified Senescence Score (USS) methodology, we identify a distinct USSHigh (USSHi) muscle stem cell (MuSC) subpopulation emerging in early phase of acute injury induced muscle regeneration. Isolation via the surface marker MHCII enables characterization of these cells as exhibiting canonical senescent-like features. Further characterization through integrated transcriptomics, proteomics, and secretomics reveals their highly pro-inflammatory secretory capacity. Moreover, lipidomic profiling uncovers that the USSHi MuSCs undergo profound sphingolipid metabolic remodeling to generate upregulated bioactive lipid Sphingosine-1-phosphate (S1P). In vivo specific ablation of this subpopulation using a Pax7-CreERT; Ccl2-LSL-DTA genetic strategy significantly impaired muscle regeneration. Furthermore, spatial transcriptomics and cell-cell communication analyses revealed that USSHi MuSCs act as critical niche modulators, directing crosstalk with multiple cell types. Specifically, S1P is essential for mediating direct USSHi interaction with endothelial cells and USSLo cells to promote endothelial angiogenesis and MuSC expansion in the regenerating muscle. Crucially, exogenous S1P rescues the regenerative deficits caused by USSHi MuSC ablation. Altogether our findings define a senescent-like MuSC population emerging in acute injury induced muscle regeneration and provide definitive evidence to demonstrate the positive role of these cells in enhancing muscle repair via their robust secretory phenotypes and inter-cellular communicative capacity.

Homozygosity for the HBB codon 8/9 (+G) frameshift (c.27dup (p.Ser10ValfsTer14)) causes transfusion-dependent {beta}-thalassaemia and is common in South Asia. Prime editing can reverse this insertion without double-strand breaks or donor DNA, but its efficiency depends on pegRNA design. We derived Sendai-reprogrammed iPSCs from a homozygous patient, optimised PEmax editing (spacer, pegRNA extension, secondary nick, MLH1dn) and compared patient, PEmax-treated and control iPSC-derived erythroid cells by flow cytometry, colony assays, RT-qPCR, western blotting and cation-exchange HPLC. Patient iPSCs had a normal 46,XY karyotype, expressed pluripotency markers, formed all three germ layers and were Sendai-free by passage 15. A PAM-disrupting spacer with a +68 nicking sgRNA gave the highest intended-edit frequency 8.4%; MLH1dn added little. PEmax-treated cultures matured and formed colonies more like control than patient cultures, restored HBB transcript (about 4-fold control iPSC-derived cells) and detectable {beta}-globin protein, and contained an HPLC fraction consistent with HbA ( undetectable in patient cells). Fetal haemoglobin (about 80%) and embryonic globin remained predominant. Prime editing corrects HBB c.27dupG in patient iPSCs and restores {beta}-globin expression, with partial restoration of adult haemoglobin in a fetal/embryonic-type erythroid background. Validation in additional donors, haematopoietic stem cells and in vivo is required.

Lack of robustness and high variability in cardiomyocyte (CM) differentiation from human induced pluripotent stem cells (hiPSCs) remain critical challenges for cardiac regenerative medicine and biological research. Guided by developmental principles of lineage specification, we investigated whether early WNT inhibition could expand the process window of CM differentiation and thereby improve robustness. To test this, we compared early WNT inhibition from days 1--5 with standard inhibition from days 3--5 following a 1-day CHIR99021 (CHIR) treatment for WNT activation. Focusing on CHIR concentration as a critical parameter, we found that early WNT inhibition substantially broadened the concentration window yielding [≥]80% differentiation efficiency across distinct hiPSC lines. Extending the analysis to a two-dimensional parameter space incorporating cell density revealed a markedly expanded [≥]80% process window. Temporal analysis of lineage-specific transcription factor expression showed that early WNT inhibition suppressed paraxial mesoderm (PM) specification, likely through MSGN1 suppression, in the range of high initial WNT activation that otherwise favored PM fate, thereby extending the process window. It also suppressed definitive endoderm (DE) specification within the expanded window, promoting a more favorable lateral plate mesoderm/cardiac fate. We further demonstrated substantially reduced variability under parameter perturbations and across wells and batches, accompanied by higher differentiation efficiency, without compromising CM phenotypes. These findings demonstrate early WNT inhibition as an effective strategy for expanding the process window of CM differentiation and improving robustness, while exemplifying the process engineering principle that a broader window can support more robust differentiation, with potential relevance to stem cell--based bioprocesses.

Metabolism and mitochondrial remodelling are increasingly recognised as key regulators of stem cell identity, governing the balance between self-renewal and differentiation. Here we compared mitochondrial and metabolic properties of stem and progenitor cells from the male germline using transmission electron microscopy, quantitative proteomics and extracellular flux analysis. While the stem cell-to-progenitor transition was accompanied by increased mitochondrial abundance and cristae density, our analyses unveiled a remarkable and unexpected capacity for spermatogonial stem cells (SSCs) to engage oxidative phosphorylation. When oxygen availability was unrestricted, SSCs displayed markedly greater maximal and spare respiratory capacity than progenitors, despite comparable glycolytic flux. SSCs were also enriched for glutathione-dependent antioxidant enzymes and maintained lower basal mitochondrial superoxide levels than progenitors. Collectively, these findings challenge the view that SSCs are predominantly glycolytic and instead identify metabolic plasticity, marked by latent oxidative capacity and enhanced redox control, as a hallmark of the stem cell state.

Hematopoietic stem and progenitor cells (HSPC) depend on interactions with nearby stromal cells to support their homeostatic functions and mount responses to perturbations. The murine bone marrow (BM) niche can be profiled using single-cell RNA sequencing (scRNA-seq), but there is considerable inconsistency in cell type identification, and uncertainty over how populations can be isolated prospectively by flow cytometry. Here, we produced new scRNA-seq reference datasets using purified populations of murine BM stromal cells, which we used to create a bespoke classifier for stromal cells. Our NicheScribe approach is consistent, accurate, and generalizable to query datasets irrespective of methods used for cell isolation. Moreover, consistent annotation permits comparative analyses of the same stromal cells across different bone types and perturbations like inflammation, neoplasia, and aging. Our work provides new tools for consistent cell type annotation in the murine BM niche and new insights for combined molecular and functional analyses.

During embryogenesis, the cortical and medullary compartments of the adrenal gland emerge from two distinct sources: intermediate mesoderm-derived steroidogenic progenitors that comprise the cortex, and migratory neural crest-derived catecholaminergic progenitors that give rise to the medulla. The distinct cellular origins and paracrine signaling environments that foster induction, differentiation, and/or migration of cortical and medullary layers present challenges to recapitulating their co-emergence and incorporation in a human pluripotent stem cell (hPSC) based system. To address this, we developed an approach for modeling the adreno-gonadal primordium (AGP) in compound organoids that are organized along a gradient of NR5A1 expression and which unexpectedly incorporate SOX10-expressing Schwann cell precursors that ultimately generate chromaffin cells. Deconstruction of paracrine signaling within AGP-like organoids (AGPLOs) identified a fibroblast growth factor 9 (FGF9)-driven mechanism that expands bridge cell-like chromaffin progenitors and correlates to paracrine signaling cues encountered by physiological correlates along their migration corridor to the cortex in vivo. Combination of NR5A1-expressing cells with chromaffin cell clusters generated corticomedullar-like assembloids (CMLAs) that generated steroids and catecholamines and were responsive to adrenocorticotropic hormone. This work establishes a platform for modeling the reciprocal signaling relationships that drive adrenal gland development and function in health and disease and provides an approach for generating hPSC-derived adrenal cells/tissues that can be applied therapeutically.

Aims: Mature, contractile vascular smooth muscle cells (vSMC) in muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Given the complicated embryonic origins and conflicting views on vSMC heterogeneity, our aim was to establish how closely hPSC-vSMC of a known germ layer lineage and defined phenotype are representative of vSMC obtained from aorta (AoSMC). Methods and Results: We generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines and performed phenotypic and mechanical comparisons with AoSMC. Synthetic hPSC-vSMC and cultured AoSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMC are non-proliferative, capable of phenotype switching and have elevated levels of contractile proteins (CNN1, TAGLN, ACTA2). These contractile proteins also displayed density-dependent, cell-to-cell heterogeneity in both AoSMC and hPSC-vSMC. Single-cell traction force measurements on micropost array detectors (mPADs) yielded comparable forces per micropost in the nN range across cell types. Quantitative 3D force measurements on humanized smooth muscle cell microtissues (SMTs) developed similar forces per cell. Larger forces were generated by MEKi-treated hPSC-vSMC and AoSMC versus controls. Conclusions: We have systematically studied the properties of hPSC-vSMC and performed comprehensive phenotypic and mechanical comparisons with AoSMC. These results demonstrate that synthetic hPSC-vSMC generated in vitro and cultured AoSMC derived from tissues are comparable and mechanically similar, but results with hPSC-vSMC are more reproducible.

Aging reduces skeletal muscle regenerative capacity, but how the aged environment impairs muscle stem cells (MuSCs) remains unclear. Skeletal muscle acts as a secretory organ, yet age-related changes in its secretome and impact on stem cells are poorly understood. We performed proteomic and lipidomic analyses of conditioned media from primary muscle cells of young (15-25 years) and aged (60-86 years) donors. We identified 127 age-dependent secreted proteins, revealing major remodeling. The lysosomal enzyme acid sphingomyelinase (ASM), which converts sphingomyelin into ceramide, was the most upregulated factor. Recombinant ASM reduced PAX7+ MuSCs in young cultures, showing extracellular ASM impairs stem cell maintenance. Lipidomics revealed disruption of the sphingomyelin-ceramide-S1P axis, with decreased sphingomyelin and S1P, a lipid promoting MuSC proliferation. Exogenous S1P restored the stem cell pool in aged cultures. These findings link secretome remodeling and sphingolipid metabolism to MuSC decline and identify ASM and S1P as targets

Achieving near-single-cell precision in the prospective identification of long-term engrafting hematopoietic stem cells (LT-HSCs) remains challenging, likely because current static, point-in-time single-cell phenotyping technologies do not capture the dynamic nature of cell fate decisions. Here, we overcome this barrier by longitudinally tracking individual hematopoietic stem and progenitor cells (HSPCs) in ex vivo niche cultures using live imaging, coupled with downstream immunophenotyping, single-cell transcriptomics, and transplant assays. Deep learning analysis of live imaging data achieved 98.5% average precision in predicting serially-engrafting LT-HSC (serial-LT-HSC) identity from the behaviorome (the totality of observed behaviors) of each cell, outperforming currently available technologies. Serial-LT-HSCs exhibited lower motility among HSPC populations, which was associated with reduced Activin signaling activity and elevated expression of cell adhesion genes. Correspondingly, antagonizing Activin signaling by Inhibin-A treatment in ex vivo niche cultures suppressed HSPC motility while increasing the frequency of post-expansion serial-LT-HSCs, revealing a novel role for Activin signaling in HSPC behavior and fate determination. Collectively, this study establishes a broadly applicable framework that connects behavioromic analysis and single-cell transcriptomics to enable near-single-cell precision in predicting stem cell fate and uncover molecular mechanisms governing behavior-fate relationships.

Stem cell factor (SCF) from Leptin Receptor-expressing (LepR+) mesenchymal stromal cells and endothelial cells is required for hematopoietic stem and progenitor cell maintenance in bone marrow. Consistent with this, Scf is highly expressed by LepR+ cells and at lower levels by endothelial cells in bone marrow. However, endothelial cells outside the bone marrow produce circulating SCF, raising the question of whether the SCF that promotes stem and progenitor cell maintenance is produced inside or outside bone marrow. We found that Scf deletion in spleen and liver endothelial cells reduces SCF levels in the blood without affecting stem or progenitor numbers in the bone marrow. Moreover, deletion of Scf from mesenchymal cells in the long bones, but not vertebrae, of Prx1Cre; Scf fl/fl mice depletes stem and progenitor cells in long bones but not vertebrae. SCF thus acts locally within the bones in which it is made to promote stem and progenitor cell maintenance.

Mitochondrial transplantation has recently emerged as an alternative treatment for cardiovascular disease (CVD), aimed at increasing mitochondrial number and improving mitochondrial function. Though mitochondrial dysfunction is a key factor in the development of right ventricular hypertrophy/failure, the effects of mitochondrial transplantation on disease mitigation have been largely unexplored. Therefore, this in vitro study aimed to determine whether the transplantation of exogenous L6 myotube mitochondria mitigates negative outcomes in H9C2 cardiomyocytes that were stimulated to hypertrophy with phenylephrine. Control (CTL), control with mitochondrial transplantation (MitoTx), Phenylephrine only (Phe-only), and phenylephrine with mitochondrial transplantation (Phe+MitoTx) treatments were evaluated. Pilot experiments indicated mitochondrial transplantation into healthy cardiomyocytes acutely increased Complex I-linked oxidative phosphorylation (OXPHOS) capacity (p=0.005) and maximal respiratory capacity (p=0.022) within 24 hours, and these data alongside microscopic evidence of fluorescently labeled L6 mitochondria in H9C2 cardiomyocytes suggested successful transplantation. Regarding treatment comparisons, Phe-only showed a significant increase in cell area (p<0.05), while Phe+MitoTx blunted the hypertrophic cardiomyocyte response. Consistent with these results, proteomic analysis of 4,806 proteins showed that transplantation enriched the mitochondrial proteome, impacting pathways including OXPHOS, respiration, fatty acid and amino acid metabolism, while suppressing extracellular matrix remodeling and de-differentiation signatures. This response was observed in healthy and phenylephrine-stressed cardiomyocytes. In conclusion, our in vitro data indicates that transplantation of L6 skeletal muscle mitochondria mitigates negative effects induced by phenylephrine in H9C2 cardiomyocytes. However, more rigorous in vivo studies are needed to determine if this is a suitable approach for disease mitigation.

The pace of fracture repair declines with age, increasing the risk of delayed union and nonunion. Delayed union and nonunion are associated with significant morbidity and even mortality with few effective non-surgical interventions. Here, we identify cathelicidin antimicrobial peptide (CAMP) as an age-dependent regulator of bone repair. Single-cell RNA sequencing and immunohistochemistry demonstrated higher CAMP expression during fracture healing in young compared with aged mice. Global or myeloid lineage-depletion of Camp delayed fracture repair in young mice, whereas Camp depletion in aged mice increased callus bone formation. Heterochronic bone marrow transplantation using the conditional genetic knockout further implicated hematopoietic-lineage derived cells in these effects and demonstrated that myeloid-specific Camp expression is important in the rejuvenation of the pace of fracture healing. CAMP promotes osteogenic differentiation through P2X7 receptor (P2X7R)-dependent signaling, influencing the downstream regulation of NLRP3 expression. NLRP3 expression is also correlated with osteogenesis following Camp modulation in both young and old mice, supporting CAMP-P2X7R-NLRP3 signaling as a regulator of osteogenesis. Together, these findings identify CAMP as a hematopoietic-derived regulator of bone repair whose function is age-dependent, promoting fracture healing in young animals while constraining it in aged animals.

A central question in developmental biology is how spatiotemporal embryo morphological events and cell differentiation are precisely coordinated to eventually develop into a mature organism. Previous research has shown cell positions and asymmetric division stages are related to cell lineage specification since morula stage. However, the detailed spatiotemporal dynamics and histories of embryonic cells and their relations with cell lineage specification remain incompletely understood from the onset of embryo development onward. My study on in-vitro embryos showed that continuous live cell tracking mapped community-like patterns in cell origin and differentiation. Cell positions, cell temporal factors (such as cell division order and cell cycle) and their histories intricately interacted over pre- and peri-implantation stages. These interactions of spatiotemporal cellular activities directed embryo morphological events and asymmetric cell lineage origin and differentiation throughout 2-cell to around 100-cell stage. In conclusion, this study provides comprehensive insights into the cellular spatiotemporal dynamics of cell inheritance and differentiation during pre- and peri-implantation morphological events and cell lineage specification. By longitudinally integrating spatial and temporal parameters relating to early embryogenesis, the findings not only bridge divergent explanations stemmed from different studies in the field but also offers a refined foundation for evaluating embryo potential and improving outcomes in assisted reproductive technologies, stem cell research, and regenerative medicine.

Fibro-adipogenic progenitors (FAPs) have emerged as central regulators of the skeletal muscle homeostasis and the muscle microenvironment. However, the role of FAPs in the context of exercise-induced muscle hypertrophy remains largely unexplored. Here, we utilized six weeks of blood flow restricted resistance exercise (BFRRE) to study cellular adaptations within the muscle microenvironment accompanying muscle hypertrophy in healthy older individuals. Using flow cytometry, we characterized global changes of key cell populations within the skeletal muscle microenvironment, including FAPs, muscle stem cells (MuSCs), and immune cells. BFRRE induced significant enlargement of both FAPs and MuSCs, consistent with cellular adaptation to exercise. Notably, exercise shifted the FAP pool toward an increased predominance of the CD90high FAP phenotype, without altering total FAP abundance. In addition to matrix and collagen-related genes, transcriptional analysis of genes associated with secretory proteins revealed enrichment of promyogenic factors in CD90high versus CD90low FAPs. Conditioned media experiments of freshly isolated FAPs demonstrated that CD90high FAPs promote myotube growth in vitro compared to CD90low counterparts, suggesting that this phenotypic shift may facilitate muscle hypertrophy. In parallel, BFRRE increased the proportion of pro-inflammatory (CD11c+) macrophages within the skeletal muscle niche, highlighting a dynamic immune response during adaptation. Finally, we show identified possible link between pro-inflammatory macrophages and FAPs, as TNF markedly reduced the proliferation of human primary FAPs ex vivo, suggesting that macrophage-derived signals may attenuate excessive FAP expansion during tissue remodelling. Together, these findings provide new insight into how the remodelling of the cellular niche may support muscle hypertrophy in response to exercise. The coordinated expansion and phenotypic remodeling of FAP and immune cell populations may represent an important mechanism through which exercise supports hypertrophy in older individuals.

Somatic mutations in the de novo DNA methyltransferase DNMT3A are frequent in clonal hematopoiesis, but how they alter the function of differentiated myeloid cells remains unclear. Here, we used isogenic human embryonic stem cell-derived macrophage models to define the consequences of DNMT3A dysfunction during myeloid differentiation. DNMT3A-mutant macrophages selectively upregulated bivalent Polycomb target genes, accompanied by DNA hypomethylation, reduced H3K27me3 and increased promoter accessibility. Despite broad epigenetic changes across bivalent promoters, transcriptional derepression occurred at a subset of loci characterized by lower baseline DNA methylation and higher H3K27me3, identifying pre-existing chromatin state as a determinant of transcriptional response. This Polycomb-associated program was conserved in murine macrophages and enriched for genes involved in cell migration and wound repair. DNMT3A-mutant macrophages exhibited enhanced migration and preferential early recruitment to injured tissue. These findings link clonal hematopoiesis-associated epigenetic alterations to selective transcriptional reprogramming and altered function of differentiated myeloid cells.