Cell Stem Cell

How allelic variants in lineage-regulating transcription factors drive diverging human developmental outcomes remains poorly understood. This is partly due to the lack of human model systems. Here, we used vessel organoids from human induced pluripotent stem cells (hiPSCs) to resolve variant-specific functions of Forkhead Box F1 (FOXF1), a critical regulator of mesoderm and vascular development. Using three patient-derived hiPSC lines harboring unique FOXF1 variants, we show that heterozygous variants cause capillary maldevelopment of varying severity. Single-nucleus multiomic analysis revealed variant-specific mechanisms - a severe variant impairs differentiation of nascent mesoderm to lateral plate mesoderm and disrupts vascular progenitor specification, while moderate variants permit mesoderm differentiation but rewire vascular progenitor states and function. Restoration of wild-type FOXF1 via lipid nanoparticle-mediated mRNA delivery rescued capillary formation in a variant- and developmental-stage-dependent manner. Together, these findings demonstrate that different variants disrupt stage-specific FOXF1 functions in human mesoderm-to-vascular development, underscoring the importance of variant-specific therapeutic strategies. HIGHLIGHTS O_LIHuman vessel organoids reveal variant-specific roles of FOXF1 in mesoderm patterning and capillary development. C_LIO_LISevere FOXF1 variant c.253T>A (p.F85I) impairs nascent mesoderm-to-lateral plate mesoderm differentiation and disrupts vascular progenitor specification. C_LIO_LI Moderate FOXF1 variants differentially rewire endothelial and mural progenitor cell states and function. C_LIO_LILipid nanoparticle-mediated FOXF1 mRNA delivery rescues capillary formation in a variant- and developmental-stage-dependent manner. C_LI

Human cardiac injury responses are governed by dynamic interacting processes that are difficult to resolve. Unlike adults, fetal mammalian hearts regenerate through coordinated remodeling and proliferation supported by a pro-regenerative immune environment, extracellular matrix (ECM), and immature cardiomyocytes, including trabecular subtypes. Here, we establish a modular human cardioid injury platform to dissect these interactions. We show that anti-inflammatory macrophages selectively migrate to the injury, clear debris, and promote ECM remodeling, whereas inflammatory macrophages suppress cardiomyocyte proliferation. Synergistic FGF2-NRG1 signaling induces trabecular identity and morphology in a hyaluronan-dependent manner, conferring enhanced injury repair, characterized by cytoskeletal remodeling and cardiomyocyte proliferation mediated by YAP and WNT signaling. Exogenous YAP, but not WNT, is sufficient to promote repair in non-trabecular cardioids. These findings uncover coordinated immune-ECM-cardiomyocyte interactions governing human fetal regenerative competence and mechanistically resolve remodeling and proliferative components of cardiac repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/735236v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@7ff079org.highwire.dtl.DTLVardef@184d5bdorg.highwire.dtl.DTLVardef@1ec775borg.highwire.dtl.DTLVardef@190008e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Brown adipocytes (BAs) hold therapeutic promise for obesity and metabolic diseases. While interscapular BAs derive from Pax3+/Myf5+ dermomyotome, peri-aortic BAs are inferred from an unknown Pax3+/Myf5- somitic origin. Here, we identify human endotome as an MYF5-independent source of peri-aortic BAs. Through interrogating public mouse organogenesis and in-house human trunk embryoid single-cell data, we show that the early endotome cells are MYF5-independent and are primed by TGF-{beta}-induced epithelial-to-mesenchymal transition. Mechanistically, endotome-to-BA specification requires sequential BMP inhibition and Wnt activation. This roadmap results in UCP1-expressing and metabolically active BAs that transcriptionally resemble in vivo peri-aortic BAT. The multipotent endotome cells also give rise to vascular smooth muscle and endothelial cells, offering a self-sufficient source for BAT vasculature. Endotome-derived BAs show accelerated differentiation, reduced heterogeneity, and sustained Wnt activity. Thus, the endotome provides a versatile platform for generating BAs and supporting vasculature, with implications for cell-based therapy and tissue engineering in metabolic disease.

Microtubule-stabilizing agents consistently improve functional recovery after spinal cord injury (SCI), yet the structural mechanism underlying their shared therapeutic effects remains unclear. Here, we find that chemically distinct stabilizers converge on preservation of ciliary integrity within central canal-associated cells, including ependymal cells and cerebrospinal fluid-contacting neurons. Using complementary SCI models, including complete transection and crush injury, we observe that maintenance of ciliary architecture is associated with reduced glial scarring, improved tissue continuity, and enhanced locomotor recovery. Single-cell transcriptomic analysis further identifies these cell populations as prominent responders to microtubule stabilization, with ciliogenesis-related programs selectively preserved. Importantly, pharmacological disruption of cilia-associated signaling attenuates recovery, whereas promoting ciliogenesis partially recapitulates therapeutic effects, identifying ciliary integrity as a critical cilia-associated structural dependency that contributes to microtubule-stabilizer-mediated spinal cord repair. Together, these findings identify a cilia-dependent central canal regenerative niche as a candidate structural checkpoint linking microtubule stabilization to functional recovery after SCI and identify ciliogenesis as a therapeutically actionable target for SCI. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/733052v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@320578org.highwire.dtl.DTLVardef@5240dorg.highwire.dtl.DTLVardef@181f251org.highwire.dtl.DTLVardef@4bacd1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Systemic aging and metabolic overload remodel the vasculature; however, how endothelial cells integrate these stresses across organs remains unclear. Using multi-organ single-cell and spatial transcriptomics with functional validation, we mapped endothelial and hematopoietic responses in adipose tissue, skeletal muscle, liver, and heart. Organ-specific endothelial transcriptional features were relatively preserved, whereas chronic stress selectively reconfigured regulatory programs: aging induced a conserved Irf/Stat-centered endothelial program, while high-fat diet engaged organ-biased lipid and remodeling programs. Spatial analysis revealed perivascular niches centered on aging-associated interferon-stimulated endothelial activation, with neighboring immune and stromal cells expressing C3 and LRP1-associated signals. Rather than simply amplifying inflammation, these niches contained mechanisms that restrained IFN activation, as C3 depletion upregulated vascular IRF7 expression. In parallel, the IFN downstream effector BST2 promoted anti-inflammatory macrophage differentiation and suppressed atherosclerosis. These findings define vascular inflammaging as an organ-resolved niche process in which endothelial IFN activation is coupled to local inflammatory restraint. HighlightsO_LIAging induces a shared endothelial type I IFN program across organs. C_LIO_LIA high-fat diet triggers organ-biased endothelial remodeling programs. C_LIO_LIPerivascular interferon niches couple inflammation with local restraint. C_LIO_LIIFN-induced endothelial BST2 promotes CD200R-associated macrophage regulatory features. C_LI

Epithelial cells can encode prior damage into lasting epigenetic and functional states, enabling a primed response to future insults. In the pancreas, acute injury induces reversible acinar cell reprogramming toward a progenitor-like identity that persists beyond repair, supporting resilience to recurrent injury but creating a permissive state for malignant transformation. Given the central role of the tissue niche in stem cell regulation, we investigated microenvironmental adaptations that sustain this primed epithelial state. Using genetic mouse models and ex vivo organoid co-cultures, we identify a sex-specific sensory neural memory after pancreatitis that sustains long-term epithelial plasticity through a CGRP-dependent neuron-epithelial axis. We show that sex differences in acute inflammation drive neutrophil-dependent suppression of neural activation in females, decoupling neural memory from epithelial plasticity after repair. In males, neural memory promotes post-injury plasticity, revealing tissue memory as coordinated adaptation between epithelial progenitors and their niche.

Following injury, predisposition towards regenerative repair and away from degenerative remodeling is central to organismal health, yet the upstream determinants that instruct this fate choice remain poorly understood. Here, we identify mesothelial cell plasticity as a central determinant between regeneration and degeneration by comparing mouse models of pneumonectomy (PNX) versus chronic lung allograft dysfunction (CLAD). While mesothelial cells expand in both settings, following PNX, these cells undergo differentiation through multiple transitional states, culminating in an inflammatory population that orchestrates monocyte recruitment and subsequent tissue regeneration. In contrast, in CLAD, mesothelial cells enrich in an extracellular matrix-enriched state that lacks pro-regenerative signaling capacity. Using single cell epigenomic profiling and in vivo genetics, we define a mesothelium-specific TWIST1-CCL2 axis that governs mesothelium plasticity and signaling, monocyte recruitment and lung regrowth. Together, these findings demonstrate that context-dependent reprogramming of a pleural population, known to protect organs, can be leveraged to drive regeneration.

Human genetics implicates innate immunity as a key modifier of tau toxicity, yet human-specific neuroimmune mechanisms remain difficult to test in vivo. Here, we developed HuMiNAX, the first humanized iPSC-based neuroimmune xenograft model of tau-associated neurodegeneration, enabling human microglia to interact with human neurons and astrocytes in the adult mouse brain. In HuMiNAX, tau seeding induced aggregation only in mutation-carrying human neural grafts, causing neuron loss and inflammatory activation of human microglia. Progranulin-overexpressing human microglia dampened tau-associated inflammation, preserved neurons, and restored neuronal gene-expression and RNA-splicing programs, supporting microglial control of neuronal resilience. CRISPRi knockdown of the human-specific lncRNA HNRNPK-AS1 also protected neurons in HuMiNAX. These findings establish HuMiNAX as a human neuroimmune model of tauopathy and identify microglial and RNA-mediated strategies of neuronal resilience.

A central unresolved and highly contested question in cardiac biology is whether the adult mammalian heart, believed to have a very limited endogenous cardiomyocyte (CM) regenerative capacity, can be coaxed into an effective regenerative response after acute CM loss. Using TgMyh6MCM:R26stop-DTA mice, we show that selective diffuse ablation of [~]15% of left ventricular CMs causes acute heart failure but is followed by complete structural and functional recovery within 28 days. Recovery is accomplished by robust generation of new mononucleated CMs, replacing [~]1/10 of the left ventricular CM compartment. This CM regeneration is produced by the activation of resident cardiac stem cells (CSCs), which exit quiescence, proliferate, produce new CMs, and subsequently return to quiescence. Depletion of the putative CSCs blocks repair, whereas transplantation of either clonogenic or primary CSCs through the systemic circulation fully restores myocardial regeneration and function, establishing that the CSCs home, nest and differentiate in the damaged myocardium and, therefore, are the main effectors of regeneration in this setting. Mechanistically, we show that miR-221-dependent repression of p57 governs the transition from quiescence--to activation--to differentiation--to quiescence of the CSCs, defining a reversible regulatory program which, under the proper conditions, endows the adult myocardium with robust CM regenerative competence.

Wolman disease (WD), the severe infantile form of lysosomal acid lipase deficiency, is a rare metabolic disorder caused by inactivating mutations in the LIPA gene. Although WD is characterized by profound hepatic dysfunction, experimental human systems capable of modelling multicellular liver pathology and supporting therapeutic testing remain limited. Here, we generated an isogenic human model of WD by introducing LIPA loss-of-function mutations into induced pluripotent stem cells and differentiating them into multicellular human liver organoids (HLO). LIPA-deficient HLO preserved hepatic lineage specification while recapitulating key biochemical and cellular features of WD, including loss of LIPA activity, lysosomal expansion, lipid accumulation, and activation of inflammatory and fibrogenic programs. Single-cell RNA sequencing resolved cell-type-specific disease states across hepatocyte-, stromal-, and biliary-like populations, revealing the emergence of a reactive biliary program consistent with ductular reaction, a complex tissue response associated with chronic liver injury. Importantly, this reactive biliary phenotype was supported by targeted gene-expression analysis in WD liver organoids and independently validated in liver tissue from mouse models and WD patients. Isolated LIPA-deficient cholangiocyte organoids failed to reproduce the DR-associated program, indicating that this response depends on multicellular interactions within the hepatic microenvironment rather than on biliary cell-autonomous dysfunction alone. Consistently, hepatocyte-directed AAV-mediated restoration of LIPA expression attenuated metabolic stress, inflammatory and fibrogenic programs, and suppressed ductular reaction both in organoids and in vivo. Together, these findings establish multicellular human liver organoids as a physiologically relevant platform for modelling emergent tissue-level responses in WD and for evaluating therapeutic rescue strategies in a human context.

HighlightsCERAMIC enables continuous and high-capacity lineage tracing of liver tumor initiation Fatty liver-associated hepatocytes acquire regenerative and premalignant cell states before malignant transformation Lineage reconstruction identifies Hep_Bi-zonal cells as the cellular origin of liver tumor initiation Transcriptional and regulatory programs distinguish tumor-fated hepatocytes from failed-to-transform lineages Peroxisomal metabolism is required for progenitor-state formation and liver tumor initiation Spatial remodeling identifies a macrophage niche associated with tumor-fated hepatocytes Dual ontogenies and functional specialization of lipid-associated macrophages shape the tumor-fated hepatocyte niche Fatty liver disease predisposes to primary liver cancer, yet the lineage routes and niche mechanisms that select rare tumor-fated hepatocytes remain unclear. Here we developed CERAMIC, a high-capacity CRISPR-Cas9 lineage recorder that co-recovers editing scars and transcriptomes from single cells, and applied it to an AKT/NRAS-driven model of MASLD-associated liver tumor initiation. Longitudinal lineage, single-cell and spatial analyses revealed a hierarchical trajectory in which Hep_Bi-zonal cells, rather than Hep_CVlike cells, generated regenerative and neoplastic hepatocyte progenitor states that progressed toward both hepatocellular carcinoma and intrahepatic cholangiocarcinoma lineages. Tumor-fated cells preferentially expanded along a remodeled midlobular-periportal axis and depended on ACOX1-mediated peroxisomal beta-oxidation to withstand lipotoxic and oxidative stress. Spatial and lineage analyses further identified a sequential lipid-associated macrophage niche, in which monocyte-derived LAMs engaged tumor-fated hepatocytes through an LGALS9-P4HB axis, and P4HB inhibition suppressed tumor expansion. These findings define liver tumor initiation as a lineage-restricted process licensed by peroxisomal metabolic adaptation and macrophage-derived niche signals.

Hematopoietic stem cell (HSC) longevity critically depends on maintaining a deep dormant state, yet the molecular mechanisms that preserve this rare and functionally essential population remain poorly understood. Here, we identify the deubiquitinase USP7 as a key regulator of long-term HSC dormancy. Using a Usp7+/- mouse model, we uncover selective depletion of hematopoietic stem and progenitor cells (HSPCs), which is associated with impaired long-term repopulation capacity. Strikingly, H2B-GFP label-retention assays reveal a profound loss of dormant HSCs in Usp7+/- mice, demonstrating a failure to maintain the most quiescent stem cell fraction in vivo. Consistently, single-cell RNA sequencing shows erosion of the transcriptional dormancy program, linking USP7 activity to the preservation of stem cell identity at both functional and molecular levels. Mechanistically, ultra-low-input proteomic profiling and biochemical approaches identify HMGA2 as a novel USP7 substrate, suggesting that ubiquitin-dependent regulation of chromatin architecture contributes to the control of HSC dormancy. Together, our findings establish USP7 as a critical regulator of HSC dormancy, revealing a previously unrecognized post-translational mechanism controlling stem cell longevity, with implications for aging, regeneration, and hematopoietic disorders.

Inflammatory memory has emerged as a fundamental principle by which prior injury shapes future tissue responses, yet whether sensory neurons participate in long-term tissue memory remains unknown. Here, we show that vagal sensory neurons acquire a durable, experience-dependent state following gastric injury or Helicobacter pylori infection, leading to enhanced regeneration, metaplasia, and tumor progression upon re-injury. This neuronal program is stable, functionally transferable, and sufficient to drive epithelial responses in vivo. Mechanistically, injury-activated ILC2s establish sensory neuronal memory through IL-13-dependent epigenetic remodeling, inducing SMYD4-mediated H3K4 trimethylation and promoting CGRP-dependent activation of gastric epithelial cells. Together, our findings support a model in which tissue memory is not restricted to epithelial or immune compartments but emerges through coordinated long-term adaptations across multiple cellular systems. Within this framework, sensory neurons provide a persistent substrate for recall responses, linking prior inflammatory experience to sustained epithelial plasticity and cancer susceptibility. HIGHLIGHTSO_LISensory neurons function as a durable compartment of tissue memory. C_LIO_LICGRP-RAMP1 signaling couples neuronal memory to gastric stem cells. C_LIO_LIILC2-derived IL-13 establishes sensory neuronal memory programs. C_LIO_LISMYD4-mediated H3K4me3 stabilizes long-term neuronal memory. C_LIO_LINeuronal memory promotes gastric regeneration and tumor susceptibility. C_LI

Hematopoietic stem cells (HSCs) generated from induced pluripotent stem cells (iPSCs) offer a promising patient-specific alternative to allogeneic transplantation, yet current differentiation protocols fail to fully recapitulate in vivo HSC maturation. During mouse development, yolk sac (YS)-derived macrophages populate the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence, but the mechanisms by which they support ex vivo hematopoietic stem and progenitor cell (HSPC) generation remain poorly defined. Bulk RNA sequencing revealed that mature AGM CD206 macrophages upregulate pro-inflammatory cytokines and the adhesion molecule F4/80. Using F4/80 knockout embryos, we identify a previously unreported, niche-specific role for F4/80 in restraining the frequency and colony-forming activity of HSPC subsets in the AGM, while supporting endothelial cell maintenance; this effect was absent in the YS. Lineage-tracing with a Cdh5-CreERT2;Rosa26LSL-tdTomato pulse-chase system confirmed that both CD206 and CD206- AGM cells originate from early YS-derived endothelial precursors, with no evidence of local macrophage generation within the AGM. Functional co-culture assays further demonstrated that the ability of CD206 macrophages to enhance the progenitor potential of hemogenic endothelium is AGM-specific and not an intrinsic, ontogeny-determined property, as YS macrophages failed to confer the same benefit even when paired with AGM endothelial cells, and AGM macrophages were ineffective with YS endothelium. Differential expression and NicheNet ligand-receptor interaction analyses identified a small set of AGM-restricted macrophage genes - including Mmp2, Nrep, Ccl2, and Cxcl16 - which are predicted to interact with both endothelial and cluster cells during endothelial-to-hematopoietic transition. Together, these findings establish that AGM macrophages acquire niche-specific transcriptional and functional properties upon entry into the aortic microenvironment, independent of their YS origin, and identify candidate macrophage-derived factors and a novel regulatory role for F4/80 in shaping HSPC output. These insights may guide the refinement of iPSC-based HSC differentiation protocols through the targeted, temporally controlled addition of macrophage-associated signals.

Hematopoietic stem cells (HSCs) in the adult mouse can be prospectively isolated to near-purity through phenotypic markers, enabling detailed analysis of stem cell function. Homeobox B5 (Hoxb5) was previously identified as a definitive marker of long-term (LT) HSCs in adult bone marrow1. In contrast, fetal HSCs have not been purified to the same extent. Here, we show that Hoxb5 is expressed in fetal liver (FL) HSCs at embryonic day (E) 12.5-16.5 using a single-color tri-mCherry reporter driven by endogenous Hoxb5 regulation. Prospective purification by stringent multiparameter flow cytometry revealed Hoxb5 FL-HSCs to exhibit robust, multilineage reconstitution upon serial transplantation. Quantitative assays reveal that Hoxb5 enriches FL-HSCs to near-single-cell purity, analogous to its role in the adult bone marrow, underscoring its reliability in distinguishing LT-HSCs throughout hematopoietic ontogeny. Notably, Hoxb5 expression is not exclusive to FL-HSCs, as it is also detected across the fetal liver hematopoietic hierarchy and in fetal liver endothelial cells, suggesting developmental stage-specific regulation of its expression. In addition, single-cell RNA sequencing of FL-HSCs identified distinct transcriptional states defined by Hoxb5 expression. These findings establish Hoxb5 as a robust marker for enhancing the purification of fetal liver phenotypic HSCs (pHSC) and provide a framework for dissecting the molecular regulation of HSC ontogeny.

Mucosal cannabidiol formulations are known regulators of the glioblastoma microenvironment, yet the underlying origin point triggering this stroma-remodeling efficacy remains entirely unknown. Here, by mapping innate cell trafficking pathways, we define a novel baseline neuro-immune-microbiome axis in orthotopic glioblastoma, characterized by diverse microbial communities, likely seeded via blood-brain barrier disruption, paired with dense infiltration of host mast cells and mature, crystalloid-containing eosinophils. Localized intranasal administration of a synthetic cannabidiol formulation achieved striking therapeutic efficacy, driving dramatic tumor regression. Mechanistically, high-throughput 16S rRNA sequencing and quantitative flow cytometry revealed this progression was subverted by taming the tumor ecosystem; cannabidiol restricted chaotic microbial diversity, selectively filtering the landscape toward Delftia and depleting Archaea, while simultaneously suppressing hyper-inflammatory host mast cell and eosinophil populations. This study builds upon established innate trafficking frameworks to present the first therapeutically targetable stromal-microbial axis in neuro-oncology.

Upon treatment, cancer cells engage non-genetic adaptations, including tolerance and subsequent persistence, to survive therapy. Eliciting programmed cancer cell death in these persister cells (PCs) remains a primary goal in oncology. We found that ferroptosis is the programmed cell death mechanism most deregulated in persisters by some of the most widely used therapeutic regimens, including platinum-based therapies, which combined with ferroptosis inducers ablate persister colorectal cancer cells. Conversely, persisters emerging from topoisomerase inhibitor regimens withstand ferroptosis and ferroptotic inducers, increasing instead intracellular iron concentration. We found that topoisomerase inhibitors trigger the Xc- antiporter axis (via SLC7A11 and CD44) increasing both intracellular cystine, to activate GPX4, and extracellular glutamate. Glutamate then engages the NMDA receptors (NMDARs), which are essential in neurotransmission but recently reported to be deregulated also in cancer cells. In PCs, NMDARs stimulate intracellular Ca2+ uptake and trigger the AKT/NFE2L2 axis, thereby engaging a cytoprotective program to cope with oxidative stress. Furthermore, we found that NFE2L2 increases the distance between the endoplasmic reticulum and mitochondria while reducing mitochondrial ROS in PCs. The synergistic inhibition of both the standard (Xc- antiporter) and this novel NMDAR/NFE2L2 axis resensitizes PCs to ferroptosis. These data provide new opportunities to improve the efficacy of widely used therapeutic regimens. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/738168v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1f32868org.highwire.dtl.DTLVardef@e1c7eborg.highwire.dtl.DTLVardef@10c4d25org.highwire.dtl.DTLVardef@9ce6d6_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIPersister cells induced by FOLFOX are sensitive to ferroptosis while resistant upon FOLFIRI treatment C_LIO_LIIncreased extracellular glutamate activates a NMDAR/NFE2L2 axis C_LIO_LINFE2L2 copes with oxidative stress by inhibiting juxtaposition between ER and mitochondria C_LIO_LIInhibition of Xc- antiporter alongside NMDAR/NFE2L2 is required to trigger ferroptosis in FOLFIRI persister cells C_LI

Adult long-term hematopoietic stem cells (LT-HSCs) are classically defined by self-renewal, multilineage regenerative capacity, and relative quiescence, but how and when lifelong LT-HSCs are established during development remains unclear. Here, we demonstrate that Hoxb5 fetal liver HSCs exhibit bona fide LT-HSC activity, including long-term multilineage reconstitution and serial transplantation capacity, whereas Hoxb5- fetal liver HSCs display limited regenerative potential. Embryonic lineage tracing further demonstrates that E14.5 Hoxb5-expressing hematopoietic cells contribute broadly to adult hematopoiesis, including the adult HSC compartment, and give rise to functional adult LT-HSCs. Across developmental stages, single-cell transcriptional profiling revealed that fetal Hoxb5 HSCs remain highly proliferative while maintaining canonical LT-HSC transcriptional programs and superior repopulating activity relative to predominantly quiescent adult Hoxb5 HSCs. Fetal Hoxb5 HSCs also exhibited elevated ITGA4-mediated adhesion programs, and disruption of the ITGA4-VCAM1 axis impaired engraftment following transplantation. Together, these findings establish a developmental continuum linking fetal and adult LT-HSCs and identify enhanced ITGA4-mediated adhesion as a defining feature of fetal LT-HSCs.

Macrophages are innate immune cells of embryonic or adult origin with tissue specific roles in homeostasis, disease surveillance, and wound repair that can be co-opted to promote tumor growth and spread1-11. An understanding of the specific roles of macrophage subsets in lung tumor initiation and progression could promote new therapeutic approaches for this deadly disease. Here, we show that KRASG12D mutations in lung epithelium drive proliferation of resident, embryonically-derived alveolar macrophages, which then promote tumor cell proliferation and protection from ferroptosis, leading to tumor progression. Using genetically engineered mouse models of mutant KRASG12D non-small cell lung cancer12,13, we found that alveolar macrophages accumulate by proliferation in response to tumor cell-secreted IL-34, recapitulating events observed in late embryonic lung development. Tumor alveolar macrophages in turn drive IGF-1-dependent tumor cell proliferation. Neutralization or deletion of IL-34 suppresses IGF-1 expression, reduces macrophage and tumor cell proliferation and inhibits tumor progression. High IL34 and IGF1 correlate with poor survival in KRASG12D/V lung adenocarcinomas and in other solid tumors, indicating that bi-directional proliferative signaling between resident macrophages and tumor cells can drive human lung tumor progression. These studies identify resident macrophage-tumor cell interactions as key interception points for lung cancer therapy.

Rectal cancers are often treated with neoadjuvant chemoradiotherapy (CRT), yet 85% of patients do not achieve a pathological complete response. To identify the molecular determinants of CRT response, we profiled the single-cell signalling, DNA-damage, cell-cycle, apoptotic, and cell-fate responses of 2,769 patient-derived organoid cultures treated with CRT, cancer-associated fibroblasts (CAFs), and signal-rewiring agents. We find that CRT response is determined by stem cell-fate. CRT triggers comparable DNA-damage in isogenic proliferative (proCSC) and revival (revCSC) colonic stem cells, but proCSC retain damage and die whereas revCSC resolve damage and persist. Both CRT and CAFs drive proCSC to a common treatment-resistant revCSC fate and high revCSC predicts worse survival in patients. Pharmacologically constraining stem-cell plasticity increases CRT sensitivity, and Spatial Perturbation of ARrayed Tumour Assembloids (SPARTA) confirms YAP/TEAD inhibition improves chemotherapy responses in human stromal-tumour models. These results suggest that cancer cell-fate, not genotoxic damage itself, ultimately governs response to standard-of-care chemoradiotherapy. HIGHLIGHTSO_LIRectal cancer stem cell-fate determines chemoradiotherapy-induced apoptosis C_LIO_LIproCSCs retain DNA-damage and die, whereas revCSCs repair damage and persist C_LIO_LICAFs and chemoradiotherapy converge on a common chemo-radioresistant revCSC state C_LIO_LISPARTA reveals TEAD inhibition blocks DNA-repair persisters in stromal assembloids C_LI