Cell Stem Cell

Genetically engineered human induced pluripotent stem cells (hiPSCs) represent a promising platform for regenerative medicine and next-generation immunotherapies. While recent advances enable stroma-free differentiation of hiPSCs into mature CD3TCR{beta} cytotoxic T lymphocytes (CTLs), overall efficiency remains limited. Here, we identify small-molecule modulators that enhance T cell output, particularly at the ProT cell stage. Targeted and stage-specific inhibition of AHR, DOT1L, or GSK3 drives robust maturation from ProT to CD4 immature single-positive (ISP) cells, markedly increasing CD4CD8 populations and augmenting CTL production of up to 2000 fold. hiPSC-derived T (iT) cells matured under these conditions display superior activity in cytotoxicity assays using AMG-701 (BCMAxCD3) or Blinatumomab (CD19xCD3). These effects were reproducible across independent hiPSC lines, diverse hematopoietic progenitor generation methods, and multiple stroma-free differentiation platforms, and were further validated in cord blood CD34 cells. Notably, AHR inhibition enhanced T cell development and promoted B lymphopoiesis, revealing shared regulatory pathways in lymphoid lineage specification. We also demonstrate that the Oct4-activating compound OAC1 functions as a weak AHR inhibitor, partially recapitulating the effects of canonical AHR blockers in both cellular and zebrafish AHR reporter systems. Collectively, our findings define key molecular circuits governing human lymphoid differentiation and establish practical strategies to optimize the yield and function of hiPSC-derived cytotoxic T cells. This work advances the development of both universal and autologous hiPSC-derived T cell therapies, offering a path forward even for patient-specific hiPSC lines with suboptimal T cell differentiation potential.

Following spinal cord injury, endogenous neural stem cells (NSCs) derived from ependymal cells become activated but fail to functionally regenerate the tissue, largely because the injury microenvironment constrains their differentiation toward glial fates. Dissecting how specific niche components drive these outcomes has remained challenging in vivo, and current neural organoid models predominantly recapitulate embryonic neurodevelopment rather than the adult injury context. Here we describe neuroids - a modular organoid system built from injury-activated adult spinal cord ependymal NSCs that spontaneously differentiate into neurons, astrocytes, and to some degree oligodendrocytes within a self-organised 3D structure. Using a bottom-up approach, we reconstruct the injury niche by incorporating meningeal fibroblasts and primary adult microglia, individually and in combination. Fibroblasts accumulate in the organoid core, deposit extracellular matrix (ECM), and trigger reactive astrocyte responses mirroring in vivo scar organisation, while microglia integrate throughout, adopt heterogeneous activation states, and remain functionally active. Their combined incorporation further enhances ECM deposition and promotes oligodendrocyte lineage commitment, suggesting cooperative niche interactions. Single-nucleus multiome profiling and trajectory inference show that these injury-like conditions shift NSC differentiation away from neuronal programs toward proliferative and astroglial states, recapitulating NSC behaviour after injury in vivo. Ligand-receptor analysis implicates microglia-derived TGF{beta}, WNT, and ECM-associated signals as candidate drivers of this gliogenic bias. Together, neuroids provide a tractable platform to study how the adult injury niche regulates endogenous NSC fate, and to identify strategies that simultaneously redirect these cells toward regeneration while targeting the fibrotic scar - two barriers that together prevent functional recovery after spinal cord injury.

The skin epidermis is maintained by spatially organized stem cell populations with distinct cellular dynamics; however, how inflammation affects this heterogeneity remains largely unknown. Here, we demonstrate that acute skin inflammation alters epidermal stem cell compartments through IL-1-mediated suppression of canonical Wnt signaling. Lineage tracing in inflamed mouse skin revealed that slow-cycling Dlx1+ epidermal stem cell clones persist, whereas fast-cycling Slc1a3+ clones decline through enhanced differentiation and lineage conversion, driving the reorganization of epidermal stem cell compartments. IL-1 signaling is both necessary and sufficient for this change: administration of IL-1/{beta} recapitulates these effects, while transgenic induction of the IL-1 decoy receptor preserves the balance of stem cell populations. IL-1 suppresses canonical Wnt activity in both the mouse epidermis and human keratinocytes, and Wnt ligand administration restores the fast-cycling compartment in vivo. Together, these results identify a reversible IL-1-Wnt axis that governs inflammation-induced stem cell plasticity and spatial tissue remodeling. HighlightO_LIInflammation induces reversible remodeling of epidermal stem cell compartments C_LIO_LIDistinct epidermal stem cell populations exhibit differential responses to inflammation C_LIO_LIIL-1 suppresses canonical Wnt signaling, thereby biasing fast-cycling stem cell behavior C_LIO_LIReactivation of Wnt signaling restores stem cell population balance under inflammatory conditions C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/704488v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@e430e5org.highwire.dtl.DTLVardef@1464550org.highwire.dtl.DTLVardef@70ba1borg.highwire.dtl.DTLVardef@ca502c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Donor organ shortage remains the major barrier to transplantation resulting in deaths on the waiting list. For lungs, aspiration-related injury is a common cause of donor organ discard and increases the risk of primary graft dysfunction. Currently, no effective therapies exist to repair damaged donor lungs prior to transplantation. Here, we investigated whether mesenchymal stromal cells (MSCs) from bone marrow or full-term amniotic fluid could restore severely injured donor lungs in a porcine model integrating ex vivo lung perfusion, transplantation and post-transplant follow-up (n=48; 24 donors, 24 recipients). MSCs were administered either once during ex vivo lung perfusion or repeatedly across lung perfusion and the early post-transplant period and compared with placebo treated controls. A single dose conferred only partial benefit, whereas repeated dosing restored graft function, normalized gas exchange and haemodynamics, and prevented graft dysfunction. MSCs from both sources were similarly effective in repeated regimens. These findings identify dosing schedule, rather than cell source, as key determinant of durable organ rescue and support perfusion-guided cell therapy as potentially generalizable regenerative strategy across solid-organ transplantation.

The lungs are highly susceptible to chronic disease in advanced age, likely due to the uniquely compromised repair function of alveolar type II (AT2) cells, facultative progenitor cells that maintain the gas exchange surface. Using aging mouse models, single-cell sequencing, and ex vivo organoid assays, we found that homeostatic aged AT2 cells exhibited an Interferon {gamma} (IFN{gamma}) response associated with IFN{gamma}+ CD8+ T cells in tertiary lymphoid structures (TLS). Aged AT2 cells exhibit impaired regeneration in organoid assays and lost markers of an IFN{gamma} response outside the lung microenvironment, demonstrating that elevated local IFN{gamma} influences the state of AT2 cells. Neutralization of IFN{gamma} signaling and immunoproteasome knockout mice with attenuated IFN{gamma} levels partially rescued aged AT2 cell regeneration. Our findings demonstrate that local IFN{gamma} signaling in aging lungs actively represses alveolar regeneration, establishing chronic inflammatory signaling as a cause of age-related decline in the lung. Halting chronic inflammatory processes restored alveolar regeneration and may provide a means to improve lung health in old age.

Mammalian body plan formation arises from the self-organization of pluripotent cells through conserved morphogenetic processes that are difficult to study in vivo. Stem cell-based embryo models (SEMs) offer accessible three-dimensional systems to investigate these events but are currently limited to mouse and human cells and largely recapitulate posterior embryonic structures. In addition, no in-vitro models exist for post-gastrulation development in ungulate species, whose early development differs from that of rodents and primates. Here, we establish SEMs for two common ungulates, sheep and pig, using pluripotent stem cell-derived aggregates. We generate ovine and porcine gastruloids that recapitulate key features of gastrulation, including germ layer specification, symmetry breaking, and axial elongation. We further develop ovine trunk-like structures (oTLSs) that robustly model post-gastrulation trunk development, exhibiting sustained elongation, neuromesodermal progenitor maintenance, segmented somite formation, and a central neural tube-like axis. Time-resolved single-cell RNA sequencing combined with immunostaining reveals coordinated emergence of neural, mesodermal, and intermediate mesodermal lineages arranged along an anteroposterior axis. Notably, oTLSs generate dorsal neural derivatives, anterior neuronal populations, and renal primordia, representing an expansion in the lineage repertoire reported for existing trunk models. Together, this work extends SEMs to livestock species and establishes a platform for comparative mammalian developmental studies, with potential applications in fundamental research, veterinary toxicology, and agricultural biotechnology.

Chronic lung diseases such as pulmonary fibrosis are characterized by the irreversible loss of alveolar type 1 (AT1) cells, yet the mechanisms governing human alveolar stem cell self-renewal and differentiation remain poorly defined. Here, we identify a lung endothelial niche that sustains the self-renewal of human alveolar type 2 (AT2) stem cells through MAPK signaling, enabling robust long-term expansion while preserving stem cell fate. Although YAP activation initiates AT1 transcriptional programs, it is insufficient to complete lineage maturation. We show that MAPK inhibition together with LATS inhibition promotes nuclear translocation of YAP, enhancing AT1 differentiation. Expanded human AT2 stem cells engraft in fibrotic lungs and contribute to alveolar regeneration while undergoing directed differentiation within diseased human lung tissue. Together, our findings define a niche-controlled signaling mechanism governing human alveolar stem cell fate and advance our understanding of alveolar regeneration.

Trophoblast stem cells (TSCs) provide a tractable system for interrogating the signaling pathways that govern extraembryonic lineage commitment. Although trophoblast specification has been extensively characterized in humans and rodents, comparable tools and molecular frameworks remain poorly defined in pigs. Here, we identify defined biochemical conditions that enable the conversion of porcine extended pluripotent stem cells (pEPSCs) into TSCs. Pharmacological inhibition experiments demonstrate that coordinated repression of TGF-{beta}/Activin and MEK/ERK signaling is sufficient to induce and maintain a stable trophoblast transcriptional program. Under these conditions, cells robustly upregulate core trophoblast regulators, including CDX2, GATA3, KRT7/18, HAND1, and ELF5, while concomitantly suppressing pluripotency- and hypoblast-associated gene networks. Bulk transcriptomic profiling reveals extensive lineage reprogramming, with enrichment of pathways related to cell adhesion, extracellular matrix organization, and placental development. Functional in vivo assays further show that induced trophoblast-like cells form small, non-teratomatous lesions that express extraembryonic markers, whereas parental pEPSCs generate teratomas that contain derivatives of all three germ layers. Together, these findings establish that combined inhibition of TGF-{beta}/Activin and MEK/ERK signaling is sufficient to specify porcine trophoblast identity from pluripotent stem cells and provide a biochemical framework for dissecting conserved and species-specific mechanisms underlying trophoblast specification and placental development. HighlightsO_LIDefined signaling conditions enabling stable conversion of porcine extended pluripotent stem cells (EPSCs) into trophoblast-like stem cells (TSC). C_LIO_LIDual inhibition of TGF-{beta}/Activin and ERK pathways drives robust trophoblast commitment. C_LIO_LITranscriptional reprogramming reveals conserved trophectoderm regulatory networks distinct from pluripotency and hypoblast states. C_LIO_LIInduced porcine TSCs display restricted in vivo potential, consistent with trophoblast identity. C_LI

Clonal hematopoiesis (CH) is an age-related phenomenon driven by the expansion of mutant hematopoietic stem cell (HSC) clones, most commonly harboring mutations in DNMT3A. While inflammation is known to promote CH, the upstream triggers of this inflammatory state remain unclear. We show that aging selectively upregulates retrotransposons in the non-hematopoietic cell compartment of the murine bone marrow, particularly in mesenchymal stromal cells. Using in vivo competitive transplant models, we demonstrate that retrotransposon-induced inflammation cell-extrinsically promotes Dnmt3a-mutant HSC expansion. This competitive advantage arises from mutant HSC resistance to inflammation-driven cell cycle perturbations. Mechanistically, we show that retrotransposon activation induces type I interferon signaling via viral mimicry, such that stromal knockdown of either Irf3 or Sting abrogates the competitive advantage of Dnmt3a-mutant HSCs. Our findings establish stromal-selective retrotransposon reactivation as a previously unrecognized, non-cell-autonomous source of inflammation that contributes to age-associated CH.

Chronic kidney disease (CKD) is a major global health challenge affecting >840 million people, with rising prevalence driven by common conditions including diabetes, hypertension, obesity, and drug toxicity. The proximal tubule (PT) is central to kidney function yet is highly injury-prone, particularly within the S3 segment which plays key roles in drug clearance and CKD pathology. Despite decades of in vitro PT model development, achieving accurate functional and structural segmentation of the PT with distinct PT sub-cell types has remained challenging. Here, we report an enrichment of S3-like cells within PT-enhanced kidney organoids (PT-EKO) that confers segment-specific functionality and injury susceptibility. This advanced platform facilitated physiologically relevant modelling of hyperglycaemia-induced damage, including rapid detection of injury biomarker Kidney Injury Molecule-1 (KIM-1). Integrating with both static and organ-on-chip culture systems, the translational potential of S3-enriched PT-EKO was underscored by its amenability to scale-up via cryopreservation of day 13 progenitors with retained differentiation capacity. PT-EKO applications were further broadened as an expandable high-yield source of isolatable PT cells, retaining PT characteristics across multiple passages and cryopreservation. Together, these findings present a high-fidelity platform for modelling tubular injury and advancing translational applications including CKD drug development, cell-specific nephrotoxicity testing and cellular therapies. SIGNIFICANCE STATEMENTAdvancing stem cell-based proximal nephron models, here we identify proximal tubule-enhanced kidney organoids (PT-EKO) as an enriched source of the nephrons most injury-prone cell type; S3 proximal tubule cells. This advanced platform provides physiologically-relevant modelling of tubular injury and a scalable source of high-quality PT cells, paving the way for more accurate modelling, screening, and cellular therapy applications.

Leukemia stem cells (LSCs) contain the highest capacity for leukemia-reinitiation and therapy-resistance across all leukemic cells, but our understanding of their molecular and cellular properties remains limited due to their relative rarity and ineffective cell culture systems to maintain their purity at scale. Here, we develop Polymer-based Leukemic STem-cell Cultures (PLSTCs) and demonstrate their capacity to derive and propagate large numbers of Npm1cA/Flt3ITD acute myeloid leukemia (AML) stem cells at high purities. Compared to traditional cultures, PLSTCs show more than 1000-fold enrichment in functional LSCs based on single-cell gene expression signatures and leukemia-initiating assays. Tracing LSC clones with genomic LARRY barcodes during ex vivo expansion, we reveal that PLSTCs can sustain a diversity of self-renewing LSC states with stable, heritable transcriptional programs. Using dynamic state-fate analysis, we characterize clonal programs that are linked with enhanced ex vivo self-renewal, in vivo leukemia initiation, and therapeutic response to induction chemotherapy. LSC clones primed to resist treatment were enriched for a rare cell state that underwent a fate-switch and produced megakaryocytic-erythroid-like leukemic cells that expanded in the spleen. Targeting LSC programs through pooled CRISPR and single-cell sequencing (CROPseq) in PLSTCs, we reveal that chondroitin-sulfate synthesis is required to maintain a primitive LSC state and leukemic recovery from chemotherapy. In sum, our studies showcase the powerful application of scalable leukemic stem-cell expansion cultures and dynamic state-fate analysis of AML LSCs. We anticipate these systems will accelerate our understanding and interception of stem cell plasticity in cancer.

Extracellular vesicles (EVs) are increasingly recognized for their roles in orchestrating embryonic development. Emerging preclinical evidence further suggests that EVs from young organisms possess innate regenerative potential for adult or injured tissues. Here we show that small extracellular vesicles (sEVs) isolated from the mouse embryonic cortex exert neuroprotective effects in vitro and in vivo. Proteomic profiling revealed that embryonic sEVs are enriched with effectors of receptor tyrosine kinase activation, anti-inflammatory responses, and protein synthesis. Notably, we identified BDNF as a surface-bound cargo on embryonic sEVs, displaying superior stability and receptor activation kinetics than its non-vesicular form. Phospho-proteomic analysis further revealed that sEVmediated neuroprotection is driven primarily by the CaMKII signaling axis, which targets downstream effectors of microtubule stability, synaptic plasticity, and membrane-cytoskeleton interactions. Critically, embryonic sEVs, but not those from aged mice, restored microtubule stability and mitochondrial respiration in aged neurons in vitro. Our findings identify embryonic cortical sEVs as significant regulators of neuronal resilience and provide a molecular blueprint for EV-based strategies in neurodegeneration and aging research.

Parvalbumin-expressing cortical interneurons play a critical role in maintaining the balance between excitatory and inhibitory signalling and are essential for cognition, with dysfunction implicated in numerous brain disorders. Although human pluripotent stem cells have enabled the generation of diverse human neuronal types in vitro, including cortical interneurons, parvalbumin-expressing interneurons - unlike somatostatin-expressing interneurons - remain difficult to generate reliably and consistently. Here, we demonstrate the efficient and reproducible generation of parvalbumin-expressing cortical interneurons in vitro within 50 days of differentiation. Parvalbumin mRNA and protein were detected without forced gene expression, cell sorting, rodent co-culture or intracerebral transplantation, approaches commonly required by previous protocols. Single-cell transcriptomic analyses validated neuronal identity and authenticity, revealing enrichment for gene expression signatures of parvalbumin-expressing cortical interneurons in vivo. Together, these findings establish a robust method that facilitates interneuron research by enabling the reliable generation of authentic human parvalbumin-expressing cortical interneurons within a short time frame. eTOC blurbAzzouni et al. present a rapid and reproducible protocol for generating authentic human parvalbumin-expressing cortical interneurons from pluripotent stem cells in just 50 days, without forced gene expression or co-culture. Single-cell transcriptomics confirm robust acquisition of in vivo-like PVALB interneuron identity, enabling new opportunities for human interneuron research. HighlightO_LIOptimising SHH and WNT modulators enables consistent PVALB interneuron generation. C_LIO_LI10% of cells express PVALB mRNA within 50 days of 2D differentiation from hPSCs. C_LIO_LIPVALB expression occurs without gene forcing, sorting, co-culture or grafting. C_LIO_LIComparison of gene expression to in vivo interneurons confirms PVALB authenticity. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/710579v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1a53d13org.highwire.dtl.DTLVardef@14cd131org.highwire.dtl.DTLVardef@3a0f9corg.highwire.dtl.DTLVardef@1d7034d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Solid-organ transplantation in aging recipients represents a unique opportunity to study how age-related immunity in the context of non-specific immunosuppression strategies balances infection, malignancy, and rejection. Heart transplantation is an exemplar platform, as routine endomyocardial biopsy for rejection surveillance is the clinical "gold standard" regardless of clinical status. Here, we undertook the largest granular study to date to characterize the association between increasing recipient age at heart transplantation with acute allograft rejection and age-related cell-specific transcriptomic changes in circulating immune cells. This single-center retrospective cohort study evaluated individuals undergoing heart transplantation between July 2013 and December 2023 at Vanderbilt University Medical Center. Eligible participants were aged [≥]18 years. A subset of individuals underwent single-cell RNA-sequencing of circulating immune cells. Among 799 adults, each one standard deviation increase in recipient age was associated with a [~]17% lower odds of allograft rejection (adjusted OR 0.83, 95% CI 0.71-0.98). In 40 individuals who underwent single-cell RNA-sequencing of circulating immune cells, increasing recipient age was associated with increases in CD4+ and CD8+ memory T cell subsets, monocytes, and NK cells. Furthermore, genes upregulated with increasing recipient age were associated with enrichment for pathways involved in immunosenescence and chronic low-grade inflammation while downregulated genes suggested decreased protein synthesis. These findings have clinical implications for an aging transplant population and support a more personalized approach to immunosuppression.

Colonic stem cells reside in a microenvironment enriched in epidermal growth factor, which is essential for their survival and can activate both PI3K-AKT and MAPK-ERK pathways. This predicts co-activation of both pathways within the growth factor-high stem cell compartment at the base of crypts. However, in patient-derived human colonic organoids and normal human tissue, stem cells maintain robust AKT activity while suppressing ERK signaling despite active EGFR engagement. As stem cells differentiate, they activate pulsatile Erk signaling, which is essential for migration, survival, and maintenance of barrier function. We show that AKT-dependent phosphorylation of Raf-1 at serine 259 establishes a post-receptor checkpoint that maintains ERK temporal dynamics in stem cells. Acute activation of ERK in stem cells triggers rapid global differentiation. Disruption of the ERK checkpoint via mutation of serine 259 leads to sustained AKT and ERK co-activation in stem cells. Unlike ERK/AKT coactivation driven by apoptosis, co-activation in the stem cell compartment results in the emergence of a neoplastic, architecturally disorganized cell population dominating the cell fate profile. Incredibly, introducing brief ERK pulses through Akt inhibition or ERK activation triggers re-differentiation of neoplastic cells. Consistent with duration-dependent MAPK encoding principles, these data demonstrate that regardless of baseline signaling amplitude, ERK signaling dynamics are epistatic to total kinase signaling load in human colonic stem cells.

During fetal brain development, temporally defined alternative splicing (AS) programs control human neural stem cell (hNSC) self-renewal and differentiation, thereby regulating neurogenesis and gliogenesis. Polypyrimidine tract-binding protein 1 (PTBP1) is a master regulator of AS during neurogenesis; however, its functional role and dynamics in hNSCs remain largely unexplored. Here, we investigate the cellular and molecular functions, nucleocytoplasmic distribution, and intercellular trafficking of PTBP1 in primary hNSCs. We found that PTBP1 knockdown (KD) alters self-renewal capacity, mitochondrial dynamics and membrane potential, lipid droplet abundance, and PTBP2 expression. RNA sequencing analysis revealed that PTBP1 depletion affects the expression profiling of hundreds of coding and non-coding genes, collectively orchestrating a neuronal differentiation program. Super-resolution {tau}-STED microscopy and live-cell imaging demonstrated that PTBP1 localizes not only to the nucleus but also to the cytoplasm, tunneling nanotubes (TNTs), migrasomes, and extracellular vesicles (EVs). Co-culture experiments and EV uptake assays showed that cytosolic PTBP1 can be transferred between hNSCs and delivered to the nuclei of recipient cells via TNTs and EVs. Moreover, EVs were found to contain specific and previously uncharacterized PTBP1 isoforms and were efficiently transferred to PTBP1-KD cells, rescuing their proliferative capacity. Analysis of the mouse brain reveals the presence of PTBP1 in the V-SVZ and within TNT-like structures connecting NSCs, suggesting a role for TNT-mediated PTBP1 trafficking in vivo. Together, these findings uncover previously unrecognized roles for PTBP1 in hNSCs and provide the first evidence that PTBP1 can be transferred between hNSCs via TNTs and EVs, revealing a novel mechanism by which hNSCs may regulate fetal neurogenesis. Graphical AbstractA: PTBP1 regulates hNSC fate by controlling cell proliferation, lipid droplet dynamics, mitochondrial function, and post-transcriptional programs involved in neuronal commitment. B: Cytosolic PTBP1 is transferred between hNSCs via tunneling nanotubes (TNTs) and extracellular vesicles (EVs). Abbreviations: LV, lateral ventricle; aRG, apical radial glial cells; bRG, basal radial glial cells; V-SVZ, ventricular-subventricular zone; EVs, extracellular vesicles; TNTs, tunneling nanotubes.

Aging-related blood disorders are linked to defects in the regenerative and multilineage differentiation ability of hematopoietic stem and progenitor cells (HSPCs). While remodeling of the bone marrow (BM) microenvironment where HSPCs reside is known to contribute to these age-associated defects, the underlying factors and mechanisms remain poorly defined. Here, we discovered that the age-related decline of the neurotransmitter neuropeptide Y (NPY) in the BM is a critical driver of HSPC dysfunction. Using mouse models, we demonstrated that NPY levels decrease in the BM with age, and that genetic NPY overexpression or exogenous NPY administration in old mice substantially reverses aging-associated phenotypic and functional defects in HSPCs. Transcriptome analysis revealed that NPY supplementation in old mice restores aging-disrupted molecular pathways in their HSCs, including oxidative stress responses, myeloid differentiation, stemness, mitochondrial activity, and RhoA signaling. However, NPY genetic loss in young mice led to a decline in HSCs regenerative capacity and increased oxidative stress. Importantly, NPY levels also decline in elderly humans, and ex vivo treatment of human BM-derived HSPCs with NPY enhances their in vivo repopulating capacity. These results suggest that NPY supplementation or preservation of NPY-producing nerve fibers could be a therapeutic strategy to rejuvenate aged HSC function.

Stem-cell-based in vitro models offer promising potential to elucidate human brain cell functions and interactions under physiological and pathological conditions. However, harnessing this potential is impaired by low reproducibility, maturity, or cell-type diversity of existing models. Especially, prolonged incorporation of mature microglia and studies of neuroinflammation have proven challenging. Here, we developed a 3D cortical brain tissue model (3BTM) containing neurons, astrocytes, and microglia with high reproducibility, maturity, and viability. 3BTMs show morphological, functional, and proteomic maturation of all cell types, leading to high similarity to their in vivo counterparts. Incorporated microglia survive for over 6 months and display mature morphology, functions, and gene expression. Importantly, when engineered to model Alzheimers disease pathology, 3BTMs recapitulate key disease hallmarks including amyloid deposition, increased phospho-Tau levels, and neuroinflammation, with microglia shifting their transcriptional landscape to disease-relevant signatures. Together, our model offers unprecedented possibilities for studying physiological and pathological states of human brain tissue and translational applications.

Aging and genetic risk shape the molecular programs that confer cellular vulnerability in Alzheimers disease (AD), but whether these programs differ between clinical symptoms and neuropathological burden remains unclear. Using single-nucleus RNA sequencing (snRNA-seq) from the dorsolateral prefrontal cortex of 48 donors either with AD or controls ([~]70,000 nuclei), we integrated transcriptomic, genetic, and demographic data to identify shared and cell-type-specific molecular predictors of AD. Across six major brain cell types and 41 fine-grained subclusters, we uncovered robust pan-cell-type predictors--including RASGEF1B, LINGO1, and ARL17B--and distinct cell-specific programs such as CRYAB in oligodendrocytes and IFI44L in microglia. Many of these genes have regulatory links to genome-wide significant AD loci and brain eQTLs, linking genetic susceptibility to transcriptional state. Pseudotime analyses revealed progressive activation of amyloid and neuroinflammatory pathways along disease trajectories, while comparative modeling of clinical versus neuropathological outcomes highlighted divergent molecular programs between symptom manifestation and amyloid plaque burden. Validation in an independent cohort (21 donors, [~]172,000 nuclei) confirmed the reproducibility of predictive features across cell types. By jointly modeling genetic, demographic, and transcriptomic axes, our study nominates high-confidence, genetically anchored molecular drivers of AD and prioritizes them for mechanistic investigation and therapeutic targeting in age-related neurodegenerative disease.

Processes that direct initial colonization and maturation of the bone marrow remain elusive, despite their importance to lifelong hematopoiesis. Bone marrow mesenchymal and stromal cell (BMSC) maturation must establish supportive hematopoietic niches prior to the recruitment and colonization of hematopoietic stem cells (HSCs). Here, we define the identity of murine BMSC progenitors and temporal nature of marrow colonization through a single cell atlas spanning late gestation through 18 months of age. We define progenitor cells and developmental trajectories for Cxcl12-abundant reticular (CAR) cells and osteoblasts within the marrow, including the emergence of direct and secreted signaling modalities that impact HSC quiescence and regenerative capacity. We further identify temporal changes in Early B Cell Factor (Ebf) 1-3 transcription factor expression and activity that correlate with niche establishment. These transcriptional activities relate to change to changes in systemic physiology, including metabolic, inflammatory, and hypoxic signaling, that direct CAR cell emergence after birth in mice. These findings provide unprecedented resolution to the colonization and maturation of the murine bone marrow environment.