Bone regeneration is impaired in type 1 diabetes mellitus (T1D), but the underlying mechanisms remain incompletely understood. Neutrophil extracellular traps (NETs) are implicated in diabetic complications, yet their effects on bone marrow mesenchymal stem cell (BMSCs) osteogenesis are unclear. Single-cell RNA sequencing was used to characterize BMSCs alterations in healthy and T1D mice. NETs accumulation and its contribution to extraction-socket healing were evaluated using Pad4−/− mice. In vitro, healthy-derived NETs (H-NETs) and T1D-derived NETs (D-NETs) were compared for their effects on BMSCs osteogenesis, proliferation, apoptosis, oxidative stress, and signaling. TSPAN11 function was examined by knockdown and overexpression. T1D was associated with fewer osteogenic-prone and more adipogenic-prone BMSCs subpopulations and reduced osteogenic gene expression. Diabetic extraction sockets showed excessive NETs accumulation, whereas PAD4 deficiency reduced NETs formation and partially improved bone regeneration. Compared with H-NETs, D-NETs more strongly impaired osteogenesis and proliferative activity, increased apoptosis and oxidative stress, and induced greater ERK1/2 activation. D-NETs also downregulated TSPAN11; TSPAN11 depletion impaired osteogenesis, whereas its overexpression alleviated D-NETs-induced inhibition. D-NETs contribute to impaired bone regeneration under T1D conditions by exerting enhanced detrimental effects on BMSCs function. Persistent ERK1/2 activation and TSPAN11 downregulation both contribute to D-NETs-induced osteogenic impairment. Targeting excessive NETs formation and its downstream effects on BMSCs may provide a potential strategy for improving bone healing in T1D.
Development of human motor neurons (MNs) is fundamental both for understanding human neurodevelopment and for advancing regenerative strategies targeting MN disorders. However, how mechanical microenvironmental cues, particularly gravity, shape chromatin organization and regulate human MN differentiation remains largely unclear. Human pluripotent stem cells (hPSCs) were chemically induced to generate motor neuron progenitors (MNPs), which were subsequently exposed to time-averaged gravity vector conditions for five days using a clinostat. We investigated the effects of time-averaged gravity vector conditions on the proliferation and differentiation of MNPs. RNA-seq was used to profile transcriptional changes. High-resolution Hi-C was performed to assess clinostat-induced alterations in 3D genome architecture. Clinostat conditions markedly enhanced the differentiation of MNPs into MNs, increasing the proportions of NEUN+, MNX1+, ISL1+, and FOXP1+ cells while reducing OLIG2+ and Ki67+ populations, indicating accelerated neurogenic commitment and diminished progenitor proliferation. RNA-seq revealed upregulation of neuronal differentiation pathways and suppression of gliogenic programs under clinostat conditions. Hi-C analysis demonstrated widespread clinostat-induced remodeling of 3D genome organization, including compartments, TADs and loops. Notably, new chromatin loops emerged at key neurogenic and mechanosensitive loci FOXP1, NRP1, and PIEZO2. Clinostat conditions accelerate MNP-to-MN differentiation by reshaping 3D genome architecture and reprogramming lineage-specific transcriptional networks. These findings provide new mechanistic insight into how physical stimuli modulate neurodevelopment and may inform strategies for neural regeneration.
Distraction osteogenesis (DO) is a powerful reconstructive technique for bone lengthening and reconstruction, but its utility is limited by prolonged consolidation and complications. Mesenchymal stem cells (MSCs) show promise as adjunctive therapies, yet their functions are highly dependent on the dynamic mechanical and biological microenvironment. This review summarizes the phase-specific roles of MSCs during DO, focusing on mechanotransduction pathways (e.g., integrin-FAK, Wnt/β-catenin, Hippo-YAP/TAZ, Piezo1), paracrine mechanisms, and immunomodulation. We evaluate current therapeutic strategies, including local transplantation, endogenous recruitment, cell-free therapies, and molecular modulation. We also discuss key knowledge gaps and technical challenges hindering clinical translation, such as MSC heterogeneity and optimal delivery parameters. We propose that emerging tools like single-cell sequencing and artificial intelligence will be crucial for developing personalized, targeted interventions to accelerate bone regeneration. Ultimately, a deeper understanding of MSC mechanobiology is essential to translate their therapeutic potential into improved clinical outcomes in distraction osteogenesis.
Skeletal muscle regeneration is predominantly driven by satellite cells, yet the contributions of other resident progenitor populations remain unclear. Using Mcam-CreER; R26-tdTomato mice, we combined lineage tracing with RNA sequencing and cell transplantation assays to evaluate Mcam+ mural cell fates and myogenic potential. Mcam⁺ mural cells exhibit distinct fates across postnatal development and injury contexts. During the postnatal period, these cells function as myogenic progenitors and contribute to dystrophin⁺ myofibers. In adulthood, they maintain a stable vascular identity under homeostasis. However, adult Mcam⁺ mural cells transdifferentiate into PDGFRα⁺ fibroblasts and incorporate into the fibrotic stroma following cardiotoxin-induced skeletal muscle injury. Notably, RNA sequencing demonstrates that neonatal Mcam⁺ mural cells exhibit a transcriptional program enriched for myogenic and mesenchymal signatures. When transplanted into injured muscle, these mural cells engrafted and differentiated into myofibers. Collectively, our results identify Mcam⁺ mural cells as a bipotent progenitor population exhibiting distinct fates during muscle growth and repair, underscoring their therapeutic potential in muscle regeneration.
Mesenchymal stem cell (MSC)-based therapy has shown promising cardioprotective effects in myocardial infarction (MI), yet the therapeutic utility of aged MSCs (AMSCs) is substantially compromised by replicative senescence and functional decline during in vitro expansion. Apelin is implicated in cardiovascular homeostasis and stem cell maintenance. However, whether sustained Apelin signaling can preserve the proliferative capacity and paracrine functionality of AMSCs during extended culture remains incompletely understood. Mouse AMSCs were genetically modified to overexpress Apelin (AMSCsApelin) or an empty control vector (AMSCsNC). Cellular proliferation, senescence, and multipotent differentiation were assessed across passages. Single-cell RNA sequencing (scRNA-seq) was performed to characterize Apelin-associated changes in cellular composition. Exosomes derived from these cells (Exo-AMSCsApelin, Exo-AMSCsNC, and Exo-AMSCs) were isolated and characterized, and their uptake and cytoprotective effects were evaluated in H9c2 cells and HUVECs under hypoxia/serum deprivation in vitro. Therapeutic efficacy was further assessed in a mouse MI model. Exosomal miRNA sequencing and functional assays were used to identify and validate key miRNAs mediating the therapeutic effects. Apelin overexpression preserved the proliferative capacity and differentiation potential of AMSCs while attenuating replicative senescence during extended culture. scRNA-seq analysis revealed that Apelin selectively expanded subpopulations enriched for proliferative features and exosome biogenesis–associated gene programs in AMSCsApelin. Correspondingly, exosomes derived from AMSCsApelin displayed increased secretion yields, enhanced uptake efficiency and superior cytoprotective effects in vitro. In vivo, administration of Exo-AMSCsApelin significantly improved cardiac function, reduced fibrotic remodeling, and enhanced neovascularization following MI. miRNA profiling identified miR-486a-5p as the most enriched miRNA in Exo-AMSCsApelin, and functional assays demonstrated that miR-486a-5p modulated cardiomyocyte survival and endothelial angiogenic responses, at least in part through suppressing Pten. Apelin reprograms the cellular composition of aged MSCs and enhances the quantitative and qualitative properties of their secreted exosomes. This coordinated regulation promotes the generation of miR-486a-5p–enriched exosomes with augmented cardioprotective capacity. These findings identify Apelin as a key regulator of the cellular–paracrine axis in aged MSCs and provide a rationale for optimizing MSC-derived exosome–based strategies for cardiac repair.
