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.

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.

Post-traumatic osteoarthritis (PTOA) is a common long-term consequence of joint injury and a major cause of chronic pain and disability, yet no disease-modifying therapies are currently available. A central barrier to effective intervention is the persistence of maladaptive synovial inflammation, driven in part by macrophage-mediated signaling that sustains tissue degeneration and pain. Here, we developed a scalable, chemically defined platform to generate human induced pluripotent stem cell (iPSC)-derived anti-inflammatory macrophages (iMac-M2) as an off-the-shelf cell therapy designed to restore joint immune homeostasis after injury. These cells maintained a stable anti-inflammatory phenotype and function under osteoarthritis-relevant inflammatory conditions and suppressed inflammatory and catabolic responses in human joint cell co-culture systems. In a preclinical model of PTOA, intra-articular delivery of iMac-M2 after injury improved functional and structural outcomes while modulating synovial inflammatory and pain-associated transcriptional programs. Treatment was well tolerated, with no evidence of systemic immune activation or ectopic tissue formation. Together, these findings support iPSC-derived macrophage therapy as a clinically translatable immunomodulatory strategy to interrupt early inflammatory drivers of PTOA and preserve joint health following injury. One Sentence SummaryAn iPSC-derived macrophage therapy restores joint balance, protects cartilage, and relieves pain after traumatic joint injury.

Mesenchymal stem cell (MSC) heterogeneity and conventional phenotypic criteria limitations represent major bottlenecks in therapeutic manufacturing. Here, we present a framework to prospectively identify naturally superior MSCs by shifting from superficial markers to the digital quantification of fundamental epigenetic flaws in inferior clones. We show that intrinsic MSC functional decline is driven by targeted hypermethylation of poised enhancers, causing paradoxical derepression of developmental genes. We term this process poised enhancer decommissioning (PEnD). By isolating this universal decay axis from donor-specific immunological variability, we translate this complex epigenetic state into a streamlined transcriptomic signature: the Poised Enhancer-related Gene Expression (PErGE) score. Overcoming the limitations of standard in vitro differentiation assays, our approach enables accurate, donor-independent prediction of long-term proliferative potential. Together, our findings establish a mechanism-based biomarker of cellular aging, and provide a readily applicable tool to improve the quality control of next-generation MSC-based therapies.

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.

Oligodendrocytes play essential roles in central nervous system development and homeostasis, and their dysfunction is a hallmark of numerous neurological disorders. However, human in vitro systems that support oligodendrocyte lineage progression while enabling the study of disease-relevant states remain limited. Here, we establish human spinal cord organoids (hSpO) and cortico-motor assembloids as platforms to model oligodendrocyte development, neuron-glia interactions, and cytokine-induced dysfunction. We show that hSpO generate oligodendrocyte lineage populations that transcriptionally resemble those found in the developing human spinal cord, and oligodendrocyte progenitor cells that exhibit physiologically-relevant functional properties, including migration and monosynaptic input from neurons. Exposure of assembloids to pro-inflammatory cytokines induces transcriptional changes across the oligodendrocyte lineage, characterized by altered lineage progression and acquisition of disease-associated gene expression programs that mirror signatures observed in multiple sclerosis patient tissue. Together, this work establishes hSpO and assembloids as in vitro systems for studying oligodendrocyte lineage development and disease-associated states in a human multi-cellular context.

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.

Rejuvenating aging human skin is a major therapeutic goal, but objective, quantitative measures of intrinsic aging are limited. We performed a cross-sectional histological study of UV-protected buttock and abdominal skin in adults spanning multiple decades of life to identify features that reliably track age. Epidermal thickness measured between rete ridges was unchanged, but rete ridge size declined linearly with age: ridges became shorter and thinner in both sites, though rete ridge number decreased only in the abdomen. Consistent with these structural changes, proliferative cells (Ki67+) per ridge and expression of integrin {beta}4 (ITGB4), a putative stem-cell marker, were reduced in aged skin. We combined these biomarkers into a predictive model that estimated skin age more accurately than any single marker. To test whether the model detects longitudinal change, we analyzed aged abdominal skin before and after xenografting onto young or aged mice, a procedure previously reported to rejuvenate human skin in young but not aged recipient mice. Both individual biomarkers and the imaging model indicated rejuvenation regardless of host age; however, notably, engraftment efficiency was lower in aged hosts, with surviving grafts showing younger histological phenotypes. These results provide quantitative criteria for assessing intrinsic skin aging and suggest that the process of engraftment itself is sufficient to induce rejuvenation-like changes.

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.

The hindlimbs and genital tubercle arise from a bipotent progenitor population within the posterior lateral plate mesoderm (pLPM) and share common genetic programmes, reflecting their deep evolutionary homology. Perturbations of this shared developmental programme underlie several congenital conditions. Yet most insights into the divergence of pLPM fates come from traditional model organisms, emphasising the need for a human-based system. Here, we report the WNT-FGF-BMP-dependent differentiation of human pluripotent stem cells (hPSCs) into pLPM-derived hindlimb/genital tubercle mesenchymal progenitors (HGTps). We show that BMP signalling plays a pivotal role in driving transcriptomic changes reminiscent of the trunk-to-tail transition--a major reorganisation of the embryonic body plan that initiates pLPM/HGTp specification. We further show that retinoic acid signalling exerts a biphasic effect on LPM specification: early exposure blocks trunk-to-tail transition-like transcriptome changes, whereas late exposure enhances genital tubercle mesenchymal fate by suppressing alternative pLPM derivatives. Strikingly, differentiating genital tubercle mesenchyme self-organises with an overlying epithelium resembling in vivo counterparts. Through xenografting approaches, we show that human HGTp-derived spheroids contribute to the genital tubercle region of the chick embryo revealing their developmental potency. Collectively, our work establishes a platform for the reverse engineering and disease modelling of human HGTps.

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.

Muscle-derived stem/progenitor cells (MDSPCs) are an adult stem cell population with demonstrated regenerative and rejuvenative potential distinct from other muscle progenitor cells. However, their molecular identity and developmental status remain poorly defined. Using single-cell transcriptomics and proteomics, we comprehensively profiled murine MDSPCs across age groups. We show that MDSPCs exist along a transcriptional continuum of maturation--ranging from metabolically active, proliferative early-stage cells to late-stage, lineage-committed myogenic populations. While lacking canonical pluripotency markers, early-stage MDSPCs express gene programs associated with embryonic progenitor identity, suggesting a non-canonical, multipotent-like state. These features distinguish them from both satellite cells and committed myoblasts. Aging reshapes this continuum by reducing stemness-associated signatures while enhancing differentiation programs and oxidative stress. Our identification of distinct MDSPC states provide critical insights into mechanisms that underly tissue regeneration and aging. These findings offer a blueprint for development of future regenerative therapies to combat age-related functional decline.

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

The developmental program governing meibomian gland (MG) morphogenesis and proliferation remains poorly understood, largely due to the lack of physiologically relevant model systems. Here, we established a novel high-fidelity, three-dimensional organoids model derived from mouse meibomian gland (mMGO) epithelium. Transcriptomic and phenotypic analyses demonstrated that mMGOs faithfully recapitulate postnatal gland development in vivo, including dynamic transcription program, branching morphogenesis, lineage differentiation, and functional lipid accumulation. Leveraging this model, we identified the Hippo-YAP pathway as a pivotal regulator of MG epithelial proliferation and homeostasis for the first time. YAP inhibition severely impaired organoids growth, while pharmacological inhibition of Hippo pathway with XMU-MP-1 enhanced proliferation and progenitor clonogenicity. Crucially, in inflammation-induced atrophic organoids, XMU-MP-1 treatment rescued YAP nuclear localization and stimulated regrowth and functional restoration. Our study provided new mechanistic insights and a robust organoids platform for MG development research, and nominated targeted Hippo pathway inhibition as a promising strategy to reverse glandular atrophy in meibomian gland dysfunction.

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.

Tumor-associated macrophages (TAM) exert essential functions during the immune response to cancer. However, investigations of TAM within a native human tumor microenvironment (TME) have been impeded by a lack of appropriate model systems. Here, patient-derived organoids (PDO) from air-liquid interface (ALI)-grown tumor fragments, containing a human TME that encompassed stroma and immune subsets, robustly preserved TAM that were maintained by endogenous CSF-1 and appropriately responded to polarization signals. Antibody blockade of the CD47 regulatory checkpoint in organoids stimulated phagocytosis and remodeled TAM cytokine secretion profiles that were confirmed in anti-CD47 phase I trial patients. Amongst PDO histologies screened, anti-CD47 tumor killing was notable in clear cell renal cell carcinoma (ccRCC) which was associated with increased TAM infiltration. PDO contained diverse previously described TAM subsets; however, anti-CD47 reprogrammed organoid TAM toward an immunosuppressive SPP1+ phenotype, highlighting a negative feedback mechanism. Our findings uncover a resistance circuit engaged by macrophage checkpoint blockade and position ALI PDO as a robust translational platform for dissecting human macrophage biology and informing precision immunotherapy.

Emerging evidence indicates that a subset of cancer cells enriched for stemness-related gene signatures possess distinct immunomodulatory capacities, enabling these tumor-initiating stem cells (tSCs) to more effectively evade or resist anti-tumor immunity. Despite these advances, the tSC-specific molecular circuits orchestrating their specialized immune privilege program are not well defined. Here, in squamous cell carcinomas of the skin and oral cavity, we comprehensively delineate the unique immune-evasive properties of tSCs and dissect the transcriptional regulation shaping their immunomodulatory programs. By integrating transcriptome profiling, chromatin landscape mapping, genetic perturbation, and single-cell RNA sequencing, we found that the tSC-specific immune program is broadly governed by SOX2, a stemness-associated transcription factor. We demonstrate that SOX2 enables tSCs to sustain immature tumor-associated neutrophils (TANs) and subsequently trigger these myeloid cells to foster the development of tumor-associated macrophages (TAMs). This SOX2-directed tSC-TAN-TAM axis establishes a localized immunosuppressive niche for protecting tSC. SIGNIFICANCEHere, we uncover SOX2 as a master regulator that orchestrates conserved immune modulatory circuits in tSCs to sustain pro-tumor myeloid cell states. These findings place tSCs at the apex of immune landscape remodeling, asserting a central role of stemness-associated program in organizing the immunosuppressive tumor microenvironment.