Cell Stem Cell

Airway epithelial homeostasis relies on multiple specialized cell types, with club cells playing central roles in maintaining epithelial integrity and regulating inflammation. Environmental insults such as allergens, viral infections, or pollutants preferentially damage club cells, impairing epithelial repair and contributing to pulmonary diseases. However, the functional properties of club cells remain incompletely defined, and tractable human models are lacking. Herein, we establish a robust platform to differentiate human pluripotent stem cells (hPSCs) into club cells exhibiting their hallmark secretory features, appropriate epithelial organization, and functional properties. Single-cell transcriptomic analyses and lineage trajectory inference revealed unexpected epithelial plasticity: hPSC-derived club cells give rise to multiciliated epithelial cells through a deuterosomal intermediate--a previously uncharacterized trajectory. Additionally, a distinct club cell subset exhibited transcriptional features indicative of neuroendocrine and goblet cell differentiation potential. This study uncovers club cell plasticity and establishes a hPSC-based platform for studying airway development, regeneration and disease modeling.

Modeling human epithelia in vitro remains challenging because current systems do not fully preserve the combination of architecture, heterogeneity, clonal composition, and long-term dynamics shown in vivo. While 3D approaches such as organoids and organotypic cultures capture important aspects of lineage differentiation and niche signaling, they often lose stable organization over time, limiting studies of long-lasting processes such as clonal evolution and cell competition. Here, we present human epithelioids as continuous long-term, 3D epithelial cultures efficiently derived from eight adult human epithelia, including trachea, skin, buccal mucosa, esophagus, blader, urethra, submandibular gland and endometrium. Using immunostaining, electron microscopy, single-cell RNA sequencing, functional assays and somatic mutation analyses, we deeply characterized human epithelioids and confirmed that they recapitulate native architecture and cell diversity, sustain regenerative capacity, and preserve donor-specific mutational landscapes, establishing a robust and versatile platform for longitudinal interrogation of clonal evolution, tissue dynamics and responses to clinically relevant perturbations, including radiotherapy and chemotherapy, over extended timescales.

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.

Despite advances in single-cell profiling of the human placenta, the genetic programs governing its physiological remodeling throughout gestation remain incompletely understood; this limits the interpretation of trophoblast organoid models. Here, we reconstruct the human placenta in its uterine environment across gestation by integrating public single-cell data into a unified developmental framework developed through a specialized computational strategy. We resolve 100 cell subtypes, expanding the known cellular repertoire and uncovering extensive gestational dynamics. In the placental mesenchymal core, we define a stromal-vascular niche comprising previously unresolved fibroblast heterogeneity and vascular hierarchies (capillary, arterial, and venous). This niche undergoes reprogramming from early angiogenesis to vascular maturation at term and engages signaling programs that support villous homeostasis. Within the trophoblast lineage, we uncover differential progenitor dynamics: bipotent cytotrophoblast (CTB) progenitors persist throughout gestation, whereas extravillous trophoblast (EVT)-biased progenitors are almost absent at term, coinciding with differentiation into specialized states. Benchmarking trophoblast organoids against this reference shows distinct regional identities and developmental biases. Tissue-derived models recapitulate villous CTB whilst trophoblast stem cell-derived organoids resemble smooth chorion CTB; all models capture early gestational syncytiotrophoblast and progressive EVT differentiation. Together, this work provides a resource for understanding placental remodeling across gestation and guiding the use of in vitro models.

At birth, the intestine must rapidly adapt to enable nutritional function and immune microbial tolerance. Here, integrating single-cell multi-omics and spatial transcriptomics we define the circuits underpinning this process. We identify asynchronous developmental trajectories with postnatal epithelial reprogramming characterised by coordinated changes in metabolism, junctional structure and innate defence. At birth epithelial stem cells demonstrate dynamic enhancer remodelling, with accessibility often preceding transcription. Fetal stemness elements remain accessible despite reduced transcription across epithelial lineages, retaining plasticity potential. Post-natal epithelia experience sequential homing of myeloid cells followed by innate T cells with peri-epithelial B cells localising later in infancy. Using developmentally staged organoids, we show that epithelial responses to inflammatory stimuli are age-dependent and constrained in early life. We identify BHLHE40 as an early-life regulator that attenuates the impact of interferon- and NF-{kappa}B-driven signalling. Altogether we define the events driving epithelial licensing and barrier adaptation at birth and through infancy.

While it was once thought that neurogenesis is complete by birth, it is now apparent that the human brain continues to generate new neurons postnatally, at least into childhood. While much attention has been focused on postnatally-born neurons, their presumed progenitor - the postnatal neural stem cell (NSC) - remains poorly characterized. Using index sorting, we identify and prospectively isolate two subsets of NSCs from the postnatal human brain, and describe their differentiation dynamics using clonal barcoding and in vivo xenotransplantation. We demonstrate an A2B5+EGFR+ population biased towards interneuron and oligodendrocyte fates (NINO), and an A2B5-EGFRhi population biased towards an astrocyte fate (NAC). Profiling of human brains across lifespan shows that the frequency of NSCs declined exponentially across the first two decades of life, but stabilized thereafter, still present in the brains of donors as old as 90 years. Our study provides a framework for the functional study of postnatal human NSCs and their potential roles in development, aging, and disease.

Direct lineage reprogramming holds therapeutic promise but often depends on transcription factor overexpression, resulting in unstable phenotypes. Here, we describe a novel strategy to convert fibroblasts into endothelial-like cells by activating lysosomal activity. Constitutively active MEK2 induces an endothelial gene program via sustained MAPK/ERK signaling, leading to enhanced vacuolar ATPase (V-ATPase) activity, lysosomal acidification, extracellular matrix degradation, and angiogenic behavior. V-ATPase inhibition impairs these effects, whereas pharmacologic activation with EN6 recapitulates key features of reprogramming and promotes nuclear translocation of TFEB, a master lysosomal regulator. Consistently, TFEB overexpression-particularly a phospho-deficient mutant-boosts lysosomal function and endothelial gene expression. These findings define a MAPK-V-ATPase-TFEB axis that drives endothelial reprogramming and highlight the lysosome as a central hub for cell fate transitions, offering an organelle-centric framework for regenerative medicine. HighlightsO_LISustained MEK2 activation reprograms fibroblasts into endothelial-like cells C_LIO_LIMEK2 enhances lysosomal acidification by upregulating V-ATPase subunits C_LIO_LIV-ATPase drives acidification and ECM remodeling for endothelial reprogramming C_LIO_LIV-ATPase activation promotes TFEB nuclear entry and endothelial gene expression C_LI

The regulation of human hematopoietic stem cell (HSC) function through its native environment is virtually unknown. Cross-species chimeras, particularly humanized mice, are essential tools for investigating human HSC function in vivo. However, conventional models often require toxic conditioning that impairs niche and donor cell function, or simplify niche complexity. We utilize NSGW41 mice, which harbor a KIT receptor mutation, to achieve robust human leukocyte engraftment without prior treatment. We demonstrate that KIT-proficient human HSCs possess a clear advantage, effectively outcompeting endogenous murine stem cells and progenitors to establish stable, multilineage human hematopoiesis. Crucially, the murine niche undergoes significant plastic adaptation in response to humanization. We identify that mesenchymal stromal cells (MSCs) expand and undergo a transcriptional shift, transitioning from a mixed adipo- or osteo-primed state toward predominantly Lepr+ adipo-primed HSC-supporting cells. This adaptation is vital; the depletion of Lepr+ MSCs or the targeted deletion of Stem Cell Factor (SCF) from these cells leads to the mobilization or loss of human HSC engraftment, respectively. These findings provide compelling evidence for functional cross-species niche-HSC communication, identifying Lepr+MSCs as primary regulators of human HSC maintenance in xenotransplantation models. By mapping this molecular dialogue, our work establishes a physiological in vivo platform to study human HSC biology and evaluate niche-targeted therapeutic interventions to improve transplantation outcomes.

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

Astrocytes play essential roles in neuronal development, function, and disease, yet existing methods to derive astrocytes from human pluripotent stem cells (hPSCs) are complex and can involve months of in vitro maturation. We developed a genomic safe-harbor knock-in system for inducible expression of the astrogenic transcription factors NFIA, NFIB, and SOX9, enabling rapid and robust generation of functional induced astrocytes (iAstrocytes). Across five hPSC lines, NFIB-SOX9 and NFIA-NFIB-SOX9 combinations efficiently generated highly pure populations expressing astrocyte-specific and synaptogenic genes. iAstrocytes displayed cytokine-induced expression of complement factors C3 and C4 and were amenable to CRISPR interference (CRISPRi) gene expression knockdown. Optimization of culture conditions enabled survival of NFIB-SOX9 iAstrocytes in co-culture with human induced neurons (iNeurons). Through pharmacological and genetic perturbations, we uncovered a previously undescribed phenomenon in which co-culture with iAstrocytes promoted the development of synchronized iNeuron network calcium activity mediated by specific gap junction proteins. This rapid and genetically tractable iAstrocyte platform provides a robust model to dissect human genetic and environmental effects on astrocyte-neuron interactions.

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.

Aging is often associated with progressive tissue degeneration and chronic inflammation, yet the role of immune cells in mediating structural and functional decline in organs remains poorly defined. Here, we investigated immune-tissue interactions in the aged lung and identified emphysematous remodeling characterized by alveolar loss. Notably, aged lungs exhibited a marked expansion of tissue-resident lymphocytes (TRLs) with senescent features, accompanied by a significant reduction in alveolar stem/progenitor cell (AT2) abundance. In vivo adoptive T cell transfer and 3D immune-stem cell organoid assays revealed that these expanded TRLs suppressed AT2 growth via secretion of oncostatin M and interferon gamma. In vivo blockade of IL-7 receptor (IL-7R) reduced TRL accumulation in the lungs and ameliorated age-related emphysematous changes, including restoration of alveolar density. Our findings identify TRLs as key drivers of alveolar degeneration in aging and propose IL-7R inhibition as a therapeutic strategy to mitigate pulmonary decline. TeaserBlocking IL-7R clears harmful lymphocytes and helps rebuild the damaged air sacs of the aging lung.

Pluripotent stem cells (PSCs) have remarkable capacity for unlimited self-renewal and differentiation into all somatic lineages. Although translational regulation has been implicated in the maintenance of PSC identity, the specific mechanisms involved remain poorly understood. Here, we identified EIF3H, a conserved subunit of the eIF3 translation initiation complex, as an essential regulator of human primed PSC proliferation and differentiation. CRISPR interference-mediated knockdown of EIF3H markedly reduced colony size, impaired proliferation, and diminished differentiation potential in all three germ layers. Integrated transcriptomic and translatomic profiling revealed that EIF3H loss decreased the translation of metal ion-related genes. Notably, the targeted suppression of metallothionein genes encoding metal-binding proteins recapitulated the proliferative defects observed in EIF3H-deficient PSCs, demonstrating a functional requirement for EIF3H-mediated translation of this gene family. Taken together, these findings establish EIF3H as a critical translational regulator that sustains PSC self-renewal and differentiation by maintaining the expression of key metabolic and stress-response genes, providing new insights into the molecular basis of pluripotency.

Prenatal inflammation has been associated with an increased likelihood of the child developing neurodevelopmental conditions, such as autism spectrum disorder (ASD). Several pro-inflammatory cytokines are significantly upregulated and play critical roles during immune activation, with interleukin-6 (IL-6) being particularly prominent. However, the specific impact of elevated IL-6 levels on human neural development remains to be elucidated. To address this, we established a human pluripotent stem cell-based forebrain organoid model enriched for multiple interneuron lineages, validated through immunohistochemistry and transcriptomic alignment with human fetal reference datasets. We showed IL-6 responsiveness via the activation of the JAK/STAT pathway and characterized downstream effects using bulk and single-nucleus RNA sequencing (RNA-seq). Bulk RNA-seq at the end of IL-6 exposure uncovered activation of inflammatory pathways, upregulation of MHC-I machinery, and early disruption of GABAergic signaling programs. Notably, single-nucleus RNA-sequencing performed one month after IL-6 withdrawal revealed a persistent inflammatory transcriptional signature across interneuron development, accompanied by accelerated progression through maturation stages and altered interneuron subtype output. Together, these findings demonstrate that transient prenatal IL-6 exposure is sufficient to reshape human interneuron fate specification and maturation trajectories, providing mechanistic insights into neuroimmune contributions to ASD.

Human pluripotent stem cell (hPSC)-derived therapies are advancing rapidly toward clinical application, yet heterogeneity of transplanted cell populations remains a major barrier to safety, predictability and scalability. Existing strategies to mitigate this risk either incompletely eliminate proliferative cells or ablate the entire graft, thereby compromising therapeutic benefit. Here we present NeuroGuard, a lineage-selective suicide gene platform that decouples safety from efficacy by preserving functional neurons while enabling inducible elimination of all other cell types after transplantation. NeuroGuard integrates an inducible caspase-9 system with NEUROD1-driven Cre recombination, protecting post-mitotic neurons from apoptosis while rendering non-neuronal and proliferative populations susceptible to ablation. In vitro, activation of the system enriched neuronal content to >90% and increased dopaminergic neuron proportion >3-fold. Following transplantation of ventral midbrain progenitors, timed activation eliminated proliferative and glial populations, resulting in compact, neuron-enriched grafts without loss of dopaminergic neuron number, target innervation or behavioural recovery in Parkinsonian rodents. Single-cell transcriptomics confirmed selective removal of non-neuronal lineages while preserving neuronal identity and maturation programs. This work establishes a generalizable framework for post-engraftment editing of cell therapy composition, providing a versatile strategy to enhance the safety and functional predictability of regenerative therapies.

The incidence of emotional disorders in patients with idiopathic pulmonary fibrosis (IPF) is substantially higher than that in the general population, severely compromising their quality of life. However, the underlying mechanisms remain poorly understood. In this study with multi-omics, we demonstrated that sphingosine-1-phosphate (S1P) derived from IPF lungs drive anxiety and depressive-like behaviors. Mechanistically, circulating S1P in the blood bound to hippocampal S1PR1 to regulate the PI3K/PKA/CREB signaling pathway, leading to synapse damage, the activation of microglia and astrocytes, neuroinflammation and ferroptosis in the hippocampus. Pharmacological inhibition of Sphk1, a key enzyme in S1P synthesis, reduced serum S1P levels and alleviated IPF-induced anxiety and depressive-like behaviors. Similarly, selective inhibition of hippocampal S1P receptor signaling using Fingolimod also attenuated neuroinflammation and ferroptosis and ameliorated mood disorders in IPF models. Collectively, these findings demonstrate that metabolite S1P from fibrotic lungs serves as a mediator of lung-to-brain functional influence, providing new insights into the IPF comorbid mood disorders and potential therapeutic targets.

Brain organoids are three-dimensional cultures derived from human pluripotent or embryonic stem cells that recapitulate key genetic, biochemical, and molecular features of the human brain. They provide a powerful platform for studying human brain development and modeling genetic neurological disorders. However, their application to age-dependent neurodegenerative diseases remains limited, largely due to the absence of standardized methods for incorporating functional microglia, critical regulators of neuroinflammation and disease progression. Here, we describe a strategy for generating neuroimmune assembloids, brain organoids containing functional glial cells capable of mounting inflammatory responses. By introducing hematopoietic progenitor cells into developing brain organoids, we enable their in situ maturation into microglia-like cells that persist in culture for up to one month. These cells exhibit hallmark microglial behaviors, including morphological remodeling, migration, phagocytosis and transcriptional changes in response to inflammatory stimuli. Together, these immunocompetent-like brain organoids provide a promising and versatile platform for investigating neuroimmune interactions and neuroinflammatory mechanisms underlying age-related neurodegenerative diseases.