Cell Stem Cell

Transplantation is limited by how long organs survive outside the body. Existing preservation methods slow metabolism or sustain physiological support, but neither eliminates ischemia-reperfusion injury (IRI), which shortens storage and drives organ discard. Embryonic diapause and hibernation show that mammalian cells retain a latent capacity for reversible metabolic arrest. Screening the PI3K-mTOR axis, we identified a dual inhibitor that pauses embryonic stem cells reversibly at ambient oxygen and holds intact mouse embryos in arrest for 11 days. Combined with a mitochondria-targeted hydrogen sulfide donor, this yields TorPause: a two-component flush pairing metabolic pausing with mitochondrial protection, compatible with existing preservation protocols. In a rat liver transplantation model, TorPause reduced structural injury and improved recipient survival after severe warm ischemic storage, potentially expanding the usable donor pool.

Neuronal loss resulting from brain injury and neurodegenerative diseases currently lacks effective regenerative therapies. While neurogenic compounds and cell transplantation hold promise, clinical translation is hindered by poor graft integration and limited cell survival. Here, we developed a novel organoid-to-organoid transplantation model using human pluripotent stem cell-derived cortical organoids (hCOs) to evaluate a dual cell transplantation and pro-neurogenic strategy for brain repair. We demonstrate that pro-neurogenic treatment with ER272 stimulates early-stage hCO growth and enhances neurogenesis through a PKC- and Notch-dependent pathway. Mechanistically, ER272 upregulates genes associated with extracellular matrix, cell adhesion and growth factor signaling. Notably, combining early-stage hCO transplantation with ER272 treatment significantly enhanced graft integration and improved neuronal repair in an ischemic-like model of late-stage host hCOs. This novel human organoid-to-organoid platform offers a robust translational tool to test regenerative therapies and highlight the potential of combining cell transplantation with pro-neurogenic stimulation for the treatment of neurological conditions associated with neuronal loss.

Stem cell-derived islets (SC-islets) are a promising source for replacing the loss of insulin-secreting {beta} cells in insulin-dependent diabetes patients, but compared to their primary human counter parts, SC-islets are considered functionally immature and do not immediately regulate blood glucose after transplantation. To better understand the molecular underpinnings of {beta} cell maturation, we genetically engineered human pluripotent stem cells (hPSCs) to control the induction of the {beta} cell-maturing transcription factor MAFA or a MAFA S64F mutant variant associated with premature {beta} cell aging and senescence in SC-islets. We employ a combination of in vitro assays, multi-omics analyses, and murine in vivo transplantation studies to demonstrate that MAFA remodeling of the SC-islet transcriptional landscape promotes SC-islet biogenesis and metabolic maturation giving rise to greater SC-islet resilience and insulin secretory capacity. Parallel MAFA S64F studies highlight the importance for compensatory mechanisms for sustaining accelerated oxidative metabolism and enhanced insulin secretion without inducing intracellular stress responses and islet dysfunction. Overall, these results reveal MAFA-governed transcriptional changes that drive SC-islet functional maturation, which provides the foundation for maturing SC-islets in vitro, preserving {beta} cell integrity, and improving SC-islet cell replacement therapies.

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.

Understanding neuro-glial roles in homeostasis and disease requires scalable central nervous system (CNS) models, particularly as macroglia like astrocytes and Muller glia remain challenging to study in slow-developing human systems. Here, we generate and map the cellular diversity of mouse neural organoids spanning multiple brain regions and the retina. Benchmarking transcriptomic maturation against in vivo references captures postnatal neuro-macroglial states within a month. We show that macroglia are highly responsive to inflammatory cues and adopt specific transcriptomic states also found in vivo. Through a single-cell perturbation screen targeting 13 Muller-glia-specific pathways, we map how certain inflammatory cytokines drive degeneration-like stress programs in photoreceptors and distinct reactive states in Muller glia, including an Oncostatin M-driven chaperone-associated state. Mechanistically, we identify gp130-STAT3 signaling as a targetable driver of macroglial reactivity and photoreceptor distress. Together, these single-cell atlases provide a foundation for dissecting cellular crosstalk and neuro-glial pathology in the mature CNS.

Vascular senescence is a central driver of whole-body ageing, yet rescue strategies remain limited. Here we show that hydrogen sulfide (H2S) bioavailability declines in ageing and atherosclerosis and is associated with endothelial senescence. Developing a novel remote-control, endothelial-specific, mitochondria-targeted chemogenetic system, we spatiotemporally controlled H2S generation and thereby mitigated endothelial senescence-driven programs and reversed vascular remodeling in aged mice.

A central unresolved problem in acute kidney injury (AKI) is how a transient epithelial insult is converted into persistent fibrotic repair that drives progression toward chronic kidney disease. Although tubular, immune and stromal responses are individually well recognized, the mechanistically continuous relay linking these three compartments has remained unresolved. Here we define a directional information-conversion pathway spanning injured tubular epithelial cells, macrophages and fibroblasts. Time-resolved single-cell profiling identified a TREM2/SPP1 macrophage state that emerged as post-ischemic repair became fibrotic. Tubular injury remodeled phosphatidylserine (PS) species, increased oxidized PS and PS externalization, and generated a membrane-lipid context for TREM2 sensing, supported by PS masking, defined liposomes and orthogonal binding assays. Structure-function analysis separated R47-dependent phospholipid recognition from K183-dependent DAP12 coupling, linking membrane damage to SYK activation and NF-kB/AP-1-driven Spp1 transcription. Macrophage-derived SPP1 then activated ITGAV/ITGB1-associated FAK-SMAD2/3 signaling, fibroblast contraction and matrix production. Crucially, repair-phase SPP1 neutralization and post-injury Cx3cr1-lineage Trem2 deletion attenuated fibrosis, with delayed Trem2 deletion protective in both ischemic and obstructive injury. Human single-cell, spatial, urinary and tissue analyses showed cross-species and cross-scale convergence. These findings resolve how transient tubular membrane damage is successively transformed across three cellular compartments into persistent, yet interruptible, fibrotic repair.

How adult stem cell populations coordinate tissue regeneration across physiological and pathological states remains unclear. Hair follicles contain epithelial and mesenchymal stem cells that codetermine hair regeneration. Here, we integrate a multimodal spatiotemporal atlas with functional validation to define the mesenchymal-epithelial stem cell dynamics governing human hair follicle homeostasis, aging, and androgenetic alopecia. Aging is characterized by passive depletion of hDSC-derived regenerative cues, whereas AGA involves a shift of hDSC toward an inhibitory signaling hub that promotes HFSC dysfunction. We further pinpoint hDSC-secreted PTN as a key regenerative factor that promotes hair growth and counteracts androgen-induced growth suppression. This study provides a holistic framework of adult stem cell crosstalk across physiological and pathological states, offering a mechanistic roadmap to reinstate tissue regeneration.

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.

TNF is the primary age-related cytokine responsible for hyperreactive platelet formation. In mouse models, increased TNF induced abnormal megakaryocyte development and platelet activity. Here, we extend these findings to demonstrate that TNF drives this thrombotic phenotype through expansion of the hematopoietic stem and progenitor cell (HSPCs) compartment. Using HSPC-specific TNF receptor labeling and single cell RNA sequencing, we found that TNF receptors are absent from megakaryocytes and their progenitors (MkPs) indicating that TNF does not directly act on these cells. Chronic TNF exposure expanded HSPCs in the bone marrow and extramedullary tissues - and these expanded HSPCs retained functional repopulation capacity. Using species-specificity TNF receptor activation, we further demonstrate that TNFR1 signaling is sufficient to induce platelet hyperreactivity independent of HSPC expansion. Unlike emergency hematopoiesis, chronic TNF promoted megakaryopoiesis through the canonical hematopoietic hierarchy as demonstrated by lineage-tracing studies. Mechanistically, chronic TNF induced a distinct transcriptional program in single cell RNA sequencing of HSPCs and megakaryocytes. Together, these findings establish that chronic TNF promotes hyperreactive platelet formation not through direct effects on megakaryocytes or MkPs but by expanding and transcriptionally reprogramming HSPCs, thereby imprinting a TNF dependent program that persists through megakaryopoiesis and ultimately produces hyperreactive platelets.

Brown adipose tissue (BAT) increases energy expenditure but is scarce in adults, yet most strategies focus on activating existing thermogenic adipocytes rather than recruiting new ones. We integrate human single-nucleus transcriptomics with protein-interaction and knowledge-graph analyses to identify stage-specific, druggable programmes governing brown adipogenesis. This pipeline resolved four ordered transcriptional states and three transitions, revealing an amyloid-{beta}/APP regulatory layer and nominating APP as a central signalling hub. Most prioritised compounds altered differentiation or metabolic phenotype, several substantially. Phenserine (APP-lowering) and sacubitril (neprilysin inhibitor) promoted differentiation through distinct mechanisms and stages and could be combined sequentially for benefit. Phenserine increased differentiation across three human models and, in mice, expanded BAT mass and increased thermogenic capacity. Together, these findings reveal targetable control points in human brown adipocyte recruitment, nominate repurposable drugs for BAT expansion, and establish a generalisable framework coupling single-cell transcriptomics with network-based drug repurposing across staged biological systems.

Intervertebral disc (IVD) degeneration, a major cause of low back pain, is driven by the disruption of nucleus pulposus (NP) tissue homeostasis. The NP arises from embryonic notochord cells that differentiate and adapt to the hypoxic, matrix-rich environment of the developing disc. Recapitulating the developmental trajectory from notochordal progenitors to NP-like cells in vitro remains challenging, as most protocols bypass early specification steps and fail to capture this understudied lineage. Here, using a three-dimensional (3D) system, we reconstruct this developmental path from human pluripotent stem cells into mesendoderm progenitor cells (MEPCs), notochord-like cells (NLCs), and NP-like cells, guided by single-cell transcriptomics and spatial profiling. We identify MEPCs as a distinct progenitor population with a unique gene signature and conserved regulatory networks (GSC, KLF8, LHX1, MIXL1) shared with early organizer and mesendodermal cells. Self-organization in MEPCs-derived aggregates induced spontaneous expression of NOTO, a key transcription factor orchestrating notochordal identity, and enhanced commitment to notochordal fate. Long-term culture in a medium containing TGF-{beta}1 and GDF-5 promoted maturation into SOX9+, KRT8+, CD109+ NP-like cells exhibiting robust matrix deposition and prominent LAMP1-positive intracytoplasmic vacuoles. This scalable, physiologically relevant model enables fundamental studies of human disc biology and early degenerative processes, while advancing regenerative strategies.

Glioblastoma is a highly aggressive brain tumour driven by therapy-resistant glioblastoma stem cells (GSCs) that switch between dormant and proliferative states through poorly defined programs. In this study, we identify SAT1, encoding the rate-limiting polyamine-catabolic enzyme SSAT1, as enriched in quiescent GSCs, linking metabolic rewiring to dormancy. Pharmacologic induction of SSAT1 with the polyamine analogue N1,N11-Diethylnorspermine (DENSpm) triggers a chemoprotective, reversible growth arrest in patient-derived GSCs, independent of stemness, differentiation or senescence. SAT1-induced dormancy coincides with reduced eIF5A hypusination and global protein translation, and a marked rise in double-stranded RNA that activates type I interferon signalling. Knocking down the mitochondrial antiviral-signalling protein, essential for antiviral innate immunity, partially reverses this growth arrest, establishing viral mimicry as functionally required for dormancy. Single-cell and cohort analyses across GSCs and neural stem cells demonstrate that susceptibility to metabolically induced quiescence is set by an intrinsic inflammatory tone and lysosomal dependency, defining a context-dependent metabolic-immune axis which can be targeted to prevent GBM progression.

Aging is the strongest risk factor for Alzheimer disease (AD), yet existing human neuronal models either erase age-associated features during reprogramming or fail to recapitulate three-dimensional organization of human neuronal tissue. Here, we establish directly induced neuronal spheroids (iSpheroids), a scalable three-dimensional platform generated by direct conversion of primary fibroblasts without pluripotency. iSpheroids generate long-lived, electrophysiologically active neurons from young, unimpaired aged, and sporadic AD donors while preserving donor-specific epigenetic age. Single-nucleus RNA sequencing revealed approximately 96% of cells acquired a neuronal identity and were distributed across multiple neuronal states, including a diversity of neurons. The platform supports incorporation of astrocytes and microglia and generation from non-human primate fibroblasts. AD iSpheroids spontaneously recapitulated neuronal degeneration, amyloid-beta; accumulation, progressive Tau pathology, and metabolic dysfunction. Integrated proteomic, metabolomic, and lipidomic profiling distinguished physiological aging from AD and revealed disease-associated metabolic remodeling. Oxaliplatin shifted AD iSpheroids toward oxidative metabolism, whereas a multi-compound screen identified trametinib as a modulator of inflammatory and age-associated programs.

Definitive hematopoietic stem cells (HSCs) emerge predominantly along the ventral surface of the dorsal aorta in the embryonic aorta-gonad-mesonephros (AGM) region, yet the spatial organization of the niche underlying this developmental asymmetry remains poorly understood. Defining the native niche of pre-HSCs has also been hindered by their rarity, transient nature and lack of a single definitive marker for their identification in situ. Here, we integrated single-cell and spatial transcriptomics to construct a spatiotemporal atlas of the niche in the AGM. Neural cells and chondrogenic mesenchyme predominated dorsally, whereas N-cadherin mesenchymal stromal cells (N-cad+ MSCs) formed a ventral layer between the aortic endothelium and mesonephric cells. BMP and SHH signaling showed overall dorsal enrichment, whereas WNT, NOTCH and KIT signaling were enriched ventrally, with these cellular and signaling patterns dynamically remodeled across HSC ontogeny. Imaging-based spatial transcriptomics validated the findings and further enabled the identification of individual pre-HSCs in situ and revealed a multicellular niche comprising endothelial cells, N-cad+ MSCs, intra-aortic hematopoietic cluster cells and macrophages. TGF{beta} and NOTCH signals were primarily supplied locally, whereas major sources of BMP and WNT were more distant. Functional perturbation identified niche-derived CD200 as an immune-regulatory signal promoting HSC development, validating a spatially predicted niche interaction. Together, our findings link tissue-scale spatial organization with the local multicellular niche supporting the preferential development of HSCs along the ventral aorta.

Pathological cardiac fibrosis and hypertrophy, key contributors to adverse remodeling that drives heart failure (HF), are not adequately targeted by current therapies. We identified tRNA-Asp-GTC-3'tDR, an ischemia-induced tRNA-derived small RNA (tDR), as an endogenous regulator of both remodeling processes across cardiac fibroblasts and cardiomyocytes. In murine models of myocardial ischemia- and pressure overload-induced HF, cardiac delivery of tRNA-Asp-GTC-3'tDR reduced fibrosis and hypertrophy, preserved cardiac function, and improved survival, with limited three doses conferring durable protection after ischemia, whereas silencing it worsened both. Strikingly, tRNA-Asp-GTC-3'tDR acts through distinct mechanisms in different cell types: in fibroblasts, it assembles stress granules that sequester ribosome-loaded profibrotic transcripts, whereas in cardiomyocytes it engages pseudouridine synthase PUS7 to activate RNA autophagy. Its regulation and functions were confirmed in human cardiac cells and tissues. These findings demonstrate that a single tDR coordinates cell-type-specific protective programs in the heart, suggesting a new therapeutic axis to mitigate HF progression.

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.

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.

Tissue homeostasis requires mechanisms that preserve quiescence while allowing rapid activation after injury, but how tissues actively restrain injury responses during homeostasis remains poorly understood. Here we identify alveolar type 1 (AT1) cells as organizers of the distal lung niche through tonic expression of PDGFA, which maintains alveolar fibroblast identity and restrains myofibroblast activation. Cell type-specific ablation revealed that loss of AT1s, but not other major alveolar cell populations, was sufficient to evoke spontaneous fibroblast activation and tissue-wide responses that recapitulate injury. In silico ligand-receptor analysis, ex vivo screening, and conditional genetic models identified AT1-derived PDGFA as a key homeostatic signal. Loss of AT1-derived PDGFA caused spontaneous myofibroblast induction and tissue remodeling, whereas increasing PDGFA promoted fibroblast proliferation while suppressing myofibroblast differentiation. Integrated phosphoproteomic, transcriptomic, and chromatin accessibility analyses showed that PDGFA engages SHP2-ERK signaling, suppresses MRTFA-associated SRF programs, and promotes E2F-associated cell-cycle programs, thereby opposing the pro-fibrotic effects of TGF{beta}-driven fibroblast activation. Restoring PDGFA signaling abrogates myofibroblast features from human idiopathic pulmonary fibrotic lungs and attenuated fibrotic remodeling in vivo. These findings define a tonic AT1-derived PDGFA signal that maintains alveolar homeostasis and offers a therapeutic target to prevent or revert lung fibrosis.

Neuroinflammation drives neurodegeneration and cognitive decline in aging, but the signals that spread this inflammation across the CNS remain unclear. Here, we show in mice that advanced age and experimental autoimmune encephalomyelitis (EAE) both result in elevated cytosolic mtDNA in microglia, which stimulates the production and export of cGAMP from these cells. Meanwhile, ENPP1, the dominant cGAMP hydrolase in the brain and spinal cord, is downregulated in both EAE mice and MS patients, releasing the brake on extracellular cGAMP accumulation. In Enpp1H362A mice, which selectively lack cGAMP hydrolysis activity, unrestrained extracellular cGAMP worsens EAE-associated paralysis and drives premature aging phenotypes including impaired motor coordination (rotarod, pole test) and reduced exploration (open field test) in a manner dependent on STING. Single-cell RNA sequencing pinpoints microglia, astrocytes, and inhibitory neurons as the principal cGAMP responders that activate STING-dependent interferon-stimulated gene induction in response to elevated extracellular cGAMP. Mechanistically, microglia import cGAMP through the LRRC8A:C channel, while astrocytes and neurons import cGAMP via the glutamate antiporter system xc-, reinforced by SLC1A2-mediated glutamate uptake in astrocytes and SLC38A1/2-fueled glutamine-to-glutamate synthesis in neurons. In isolated primary astrocytes and neurons, high intracellular glutamate and system xc--driven redox demands favor cGAMP uptake, raising the possibility that neurotransmitter cycling and redox burden shape cellular vulnerability to cGAMP immunotransmission in vivo. Together, these findings position ENPP1 as a protective brake on neuroinflammation in aging and MS, nominating cGAS inhibition and CNS ENPP1 enhancement as therapeutic strategies.