Stem Cell Reports

BackgroundGains of chromosome 20q11.21 are among the most common culture-acquired abnormalities in human pluripotent stem cells (hPSC), conferring a well-defined survival advantage while altering differentiation capacity. However, it remains unclear whether this advantage persists during differentiation, how the aneuploidy alters ectodermal and retinal pigment epithelium (RPE) lineage specification, and which genes within the minimal amplicon drive these effects. MethodsWe used three isogenic human embryonic stem cell line pairs (wild-type and 20q11.21 gain) and assessed their behaviour in two neuroectoderm differentiation systems: directed neuroectoderm induction (dual SMAD inhibition) and long-term spontaneous RPE differentiation. Competitive dynamics were measured in mixed cultures, and lineage outcomes were analysed using immunostaining, gene expression profiling and single-cell RNA sequencing. To identify driver genes, we generated BCL2L1 and ID1 overexpression lines and tested their effects under both directed and spontaneous differentiation conditions. ResultsAcross all lines and conditions, 20q cells expanded from a minor fraction to dominate mixed cultures, indicating that their competitive advantage persists beyond the undifferentiated state. Despite this dominance, pure 20q cells failed to specify to neuroectoderm or RPE. Single-cell transcriptomics revealed consistent diversion toward non-neural ectodermal and extraembryonic fates. Mechanistically, overexpression of BCL2L1 and ID1 alone or in combination impaired neuroectoderm specification, while synergistic effect of both genes promoted non-neural ectodermal outcomes under directed differentiation conditions. In spontaneous differentiation, both genes could disrupt differentiation. ConclusionsThe 20q11.21 gain couples a persistent survival advantage with a disruption of neural and RPE lineage competence, redirecting cells toward alternative ectodermal and extraembryonic fates. These effects arise from the combined action of two dosage-sensitive genes BCL2L1 and ID1 within the amplicon, illustrating how regional gene dosage can reshape developmental signalling responses in hPSC.

Development of motoneurons from stem cells is characterized by a change from glycolytic to oxidative metabolism. Since this transition remains poorly understood, we examined it at five distinct differentiation stages from hiPSC to motoneuron. While a direct comparison of hiPSCs and mature motoneurons confirmed the expected glycolytic-to-oxidative shift, the intermediate stages showed that the conversion was not monotonic. After an initial drop of glycolysis at the hiPSC-to-neuroepithelial transition, late neuroepithelial cells showed intermittent peaks of the glycolytic marker lactate dehydrogenase A and the metabolic regulator TIGAR. Furthermore, the lactate-produced-to-glucose-consumed ratio remained elevated. A fully oxidative phenotype was only assumed upon progress from neural progenitors to motoneurons, portrayed by a definitive drop of the lactate-produced-to-glucose-consumed ratio, an increase of mitochondrial membrane charging, and shifts from lactate dehydrogenase A to B, from pyruvate dehydrogenase to anaplerotic pyruvate carboxylase, and from Mitofusin 1 to 2. Together, our data show that metabolic maturation in human motoneurons does not occur as a simple switch. Instead, it unfolds through distinct stages in a directional yet nonlinear manner.

Despite lacking a robust interferon response, pluripotent stem cells remain highly resistant to viral infection, in part through the constitutive expression of immune genes traditionally classified as interferon-stimulated genes. While interferon signaling has been shown to be incompatible with the maintenance of pluripotency, the molecular mechanisms underlying this relationship remain poorly understood. Here, we investigate the transcriptional response of human embryonic stem cells (hESCs) to infection with a potent activator of the interferon response, an influenza A virus mutant lacking the viral NS1 protein. Single-cell RNA sequencing revealed that while most hESCs remain unresponsive to infection, a distinct subpopulation expresses type I and III interferons. Notably, only interferon-expressing cells mounted a robust antiviral response, characterized by strong induction of interferon-stimulated genes. In contrast to the bulk hESC population, interferon responding cells exhibited reduced expression of core pluripotency factors as well as negative regulators of interferon signaling, such as SOCS1 and SPRY4. Depletion of SOCS1 enabled hESCs to respond robustly to interferon stimulation, showing that this negative regulator is a key suppressor of interferon signaling in pluripotent stem cells. We further show that SOCS1 and additional negative regulators of IFN signaling are intrinsically expressed in hESCs and are transcriptionally controlled by pluripotency factors, such as NANOG, SOX2 and OCT4. Together, our findings support a model in which pluripotency factors regulate intrinsic immune gene expression, including negative regulators of interferon signaling, thereby suppressing canonical interferon signaling to preserve pluripotency while maintaining antiviral resistance. IMPORTANCEBy combining single-cell transcriptomics with functional studies, we demonstrate that the pluripotency transcriptional program active in pluripotent stem cells coordinately regulates pluripotency factors, antiviral genes, and negative regulators of interferon signaling. This integrated control enables pluripotent stem cells to achieve effective protection against viral infection while preserving their differentiation potential, providing new insights into how innate immunity is selectively constrained in pluripotent stem cells. These findings have important implications for stem cell-based therapies, where transient modulation of antiviral responses without disrupting pluripotency could improve therapeutic efficacy. More broadly, this work advances our understanding of interferon regulation that could inform the development of antiviral strategies that enhance protective immune responses while limiting harmful or unwanted inflammatory signaling.

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

The establishment of genetic circuits in pluripotent stem cells (PSCs) allows to model and manipulate developmental events. However, prototyping complex circuitry remains challenging, due to limitations in screening circuit components and transgene silencing. Here, we introduce KELPE: PSCs with two silencing-resistant insulated genomic landing pads targeted to genomic safe harbour sites. KELPE cells enable the stable integration of multiple transgenes into the same genomic region, facilitating fair comparisons of genetic circuit components. We demonstrate this by fine-tuning "synthetic neighbour-labelling" technologies. We first generate optimised PUFFFIN PSCs, which report on cell-cell interactions by fluorescently labelling wild-type neighbours. We then generate new synNotch "receiver" PSCs, which can trigger expression of any transgene following interaction with a synthetic ligand presented by "sender" cells of interest. We describe an optimised circuit syntax that abolishes ligand-independent transgene induction in receiver PSCs, and showcase this by synthetically programming cell death in receiver cells engineered to express a toxin following interaction with sender cells. In summary, we describe a new cell line that facilitates silencing-resistant transgene expression and prototyping of synthetic biology tools in a developmentally-relevant model.

Prolonged cytopenias are a frequent complication of chimeric antigen receptor (CAR) T-cell therapies and are associated with increased infection risk and non-relapse mortality. Although inflammatory cytokines released during CAR-T cell activation have been implicated in immune effector cell-associated hematotoxicity (ICAHT), their direct effects on human hematopoietic stem and progenitor cells' (HSPCs) function remains incompletely understood. Here, we established a reductionist model of CAR-T-associated hematotoxicity using conditioned media (CM) derived from activated CD19 CAR-T cells. Sustained exposure of human HSPCs to CAR-T-derived inflammatory secretome impaired HSPC expansion and reduced long-term repopulating capacity in xenotransplantation assays. In contrast, short-term exposure did not abrogate HSPC function, indicating that brief inflammatory signals can initiate durable reprogramming events, with functional consequences emerging during subsequent proliferative expansion. Mechanistically, CAR-T CM induced IFN gamma- (IFNg) and TNF alpha- (TNFa) responsive transcriptional programs in HSPCs and promoted inflammatory myeloid skewing without evidence of apoptosis-dependent stem cell loss. Combined inhibition of IFNg and TNFa restored HSPC expansion, normalized lineage output, reversed inflammatory transcriptional signatures, and rescued in vivo repopulating capacity without impairing CAR-T cytotoxic activity. These findings demonstrate that CAR-T-derived inflammatory signaling can directly impair human HSC function and identify dual IFNg/TNFa blockade as a potential strategy to mitigate CAR-T-associated hematotoxicity while preserving antitumor efficacy.

Microglia are the resident immune cells of the central nervous system and play key roles in the healthy brain during development and adulthood, as well as during neurodegenerative diseases - including Parkinsons disease (PD). Yet the role of microglia in PD pathogenesis has not been fully elucidated. Limitations of 2D cell culture and animal models in simulating human microglia in the brain parenchyma have contributed to this knowledge gap. Human midbrain organoids (hMOs) provide a promising model that can recapitulate elements of PD pathology but lack microglial cells. Here we adapt protocols for the differentiation of hMOs and human iPSC-derived microglia (iMG) to generate iMG-hMO assembloids. Within assembloids, integrated iMG (intMG) express canonical microglia markers and induce the release of cytokines and chemokines. Transcriptomic profiling by single cell RNA sequencing reveals that intMG adopt a more mature and inflammation-responsive state compared to 2D iMG. The integration of microglia results in increased signaling through inflammatory and trophic pathways that drive altered transcriptional signatures of dopaminergic neurons and astrocytes within assembloids. Overall, iMG-hMO assembloids have the potential to more faithfully model the role of microglia and neuroinflammation in PD pathogenesis.

AimsThe activation of inflammatory cells, particularly macrophages, plays a pivotal role in the pathogenesis of cardiac remodelling and heart failure. Emerging evidence indicates that extracellular traps released from inflammatory immune cells contribute to the progression of various pathologies. However, the clinical relevance and mechanistic role of macrophage extracellular traps (METs) in heart failure remain to be elucidated. Methods and ResultsEndomyocardial biopsy specimens from 69 patients with heart failure were analysed by fluorescent immunostaining to identify and quantify METs. The numbers of METs per myocardial tissue area in patients with heart failure showed a negative correlation with left ventricular (LV) ejection fraction and a positive correlation with LV end-diastolic diameter. Patients with higher MET counts had significantly lower event-free survival from the composite cardiac events. In a murine model of pressure overload by transverse aortic constriction (TAC), METs were most abundantly observed at 3 days post-TAC and remained detectable throughout the 4-week observation period. In vitro, time-dependent MET formation was induced by an intrinsic trigger of mitochondrial DNA in bone marrow-derived macrophages from wild-type (WT) mice, but not in peptidyl arginine deiminase 4 (PAD4)-deficient macrophages, indicating that PAD4 activity is indispensable for MET formation. The recipient mice transplanted with bone marrow cells from PAD4 knockout mice showed more preserved cardiac function, reduced myocardial fibrosis, and improved survival in response to TAC, compared to those transplanted with WT mice. Ex vivo analyses demonstrated that conditioned medium containing METs from WT macrophages induced fibroblast-to-myofibroblast transition via Toll-like receptor 4 signalling. ConclusionsPAD4-dependent MET formation from bone marrow-derived macrophages represents a novel driver of cardiac remodelling. Targeting MET formation may offer a potential therapeutic strategy for heart failure. Translational PerspectiveMacrophage extracellular traps (METs) are abundant in myocardial tissue from patients with heart failure with reduced ejection fraction and are associated with adverse left ventricular remodelling and worse clinical outcomes. These findings support myocardial MET burden as a potential tissue biomarker to improve risk stratification in heart failure patients. In mice, pressure overload induces MET formation, and hematopoietic PAD4 deficiency suppresses myocardial METs, attenuates fibrosis, preserves cardiac function, and improves survival. Mechanistically, mitochondrial DNA-enriched cardiomyocyte-derived exophers trigger PAD4-dependent METs, which activate cardiac fibroblasts through TLR4 signalling. Suppressing METs represents a potential therapeutic strategy to attenuate the progression of heart failure. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/711858v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1c6a637org.highwire.dtl.DTLVardef@caa356org.highwire.dtl.DTLVardef@1a994bforg.highwire.dtl.DTLVardef@6493bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDespite effective antiretroviral therapy, HIV-1 remains a global health challenge. Most people living with HIV (PWH) are diagnosed in the chronic stage and around half globally are diagnosed late. Within the group with a chronic diagnosis, insight into the effect of time of therapy initiation on reservoir dynamics and immune reconstitution from diagnosis onwards is limited. MethodsIn this prospective cohort study of PWH diagnosed during the chronic stage (from Fiebig VI) were stratified into late (<350 CD4+ T cells/mm3 or an AIDS-defining illness) or non-late diagnosis groups. We analyzed the viral reservoir by IPDA, SQuHIVLa and FISH-Flow, and the immune compartment with the AIM assay and a 45-color spectral flow cytometry panel in the first year after ART initiation. FindingsAlthough proviral DNA decreased in the first year of ART, the inducible reservoir remained stable. PWH with a late diagnosis had a significantly higher inducible reservoir and lower CD4+ T-cell counts than the non-late HIV diagnosis group. A year after ART initiation, the group with a late diagnosis showed a higher abundance of exhausted CD8+ T cells, higher expression of activation/exhaustion markers, and a lower naive CD4+ T-cell abundance than the non-late diagnosis group. Moreover, activation and exhaustion marker expression on T cells correlated significantly with CD4+ T cell count pre-ART. InterpretationOur results show that late diagnosis is associated with a persistently higher inducible viral reservoir and impaired immune recovery. These findings underline the importance of early diagnosis and treatment, and rationalize the use of late diagnosis as a covariate in future studies. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPrior research has demonstrated that initiation of antiretroviral therapy (ART) during the acute stage of HIV-1 infection limits reservoir seeding, reduces immune activation and preserves immune function. Therefore, studies have focused on reservoir and immune dynamics in this acutely diagnosed group. However, only 8,4% of diagnoses in Europe are made at an acute stage. Despite the fact that the far majority of diagnoses are made at chronic infection, heterogeneity within this group has been overlooked. In clinical settings, late HIV-1 diagnosis, defined as CD4+ T cell count <350 or AIDS-defining illness, has been shown to lead to more opportunistic infections, slower time to viral suppression and increased mortality when compared to a non-late (but still chronic) diagnosis. Yet, the reservoir and immune dynamics in these groups remains poorly characterized. Added value of this studyIn this study, PBMCs from a prospective cohort of people with a chronic HIV diagnosis in the first year after ART initiation were analyzed using integrated approaches - reservoir quantification and in depth immunophenotyping to simultaneously characterize reservoir dynamics and the immune compartment. We show a significant decrease in the intact viral reservoir for all participants within the first year of ART, whereas the inducible reservoir remained stable. A comparison was performed between the group with late and non-late (but still chronic) diagnosis, shedding light on the heterogeneity within the chronic diagnosis group. The late diagnosis group had a significantly higher inducible reservoir and immune exhaustion, both in marker expression as well as exhausted T cell abundance. All but four of these activation and exhaustion markers differentially expressed between the two groups correlate with CD4+ T cell count pre-ART, highlighting the large heterogeneity in this group. Implications of all the available evidenceTogether, these findings provide insight into the reservoir and immune dynamics within the first year after ART initiation. The data can especially inform both reservoir-targeting strategies that intervene at or shortly after ART initiation, as well as strategies that aim to harness the immune system. Moreover, these results reinforce the importance of early diagnosis, even after the acute stage. The differences in the reservoir and immune compartments between the late and non-late diagnosis groups underscore the need for the use of late diagnosis or time to diagnosis as a covariate in future cure studies.

Cellular therapies, toxicity screening, and regenerative medicine depend on selecting mammalian cell types with optimal lifespan, persistence post-transplant, immunogenicity, and chemical resilience. This review synthesizes data from over 50 immune, parenchymal, stem, and emerging engineered cell populations--including gamma-delta T cells, iNKT cells, CAR-macrophages, and hypoimmune iPSC derivatives--drawing from in vivo lifespan studies (including 1{blacksquare}C birth-dating and deuterium labeling), engraftment dynamics, immune rejection risk, and stress sensitivity profiles. We introduce a Programmability & Persistence Score (PPS; 0-20) that integrates these features into a unified metric, complemented by Pareto frontier analysis to visualize multi-objective trade-offs. High-PPS cell types (e.g., HLA-matched HSCs, hypoimmune iPSCs, chondrocytes) are suited for long-term regenerative applications, while low-PPS sentinels (e.g., neutrophils, enterocytes) serve acute assays. We discuss mathematical extensions including multi-criteria decision analysis, fuzzy membership functions, and Bayesian frameworks that address limitations of linear additive scoring. Together, these integrated profiles support cell selection for gene editing, organ-on-chip systems, in vivo cell programming, and immunotherapy, bridging cell biology with translational engineering.

Primordial germ cells (PGCs) are the population of cells that, in the human embryo, specify day 12 post-fertilization, and form the precursor cells for the future egg or sperm cells. Although in vitro differentiation of PGCs from human stem cells has been achieved, these primordial germ cell-like cells (hPGCLCs) fail to further mature. The reason for this is unclear. Previous studies in mice revealed that several specific metabolic changes occur during the maturation of these cells, which are essential for their developmental progress. However, very little is known about the metabolic profile of human primordial germ cells. In the severe scarcity of human PGCs, hPGCLCs serve as a research model to study PGC formation. To investigate this, we differentiated hPGCLCs using induced-pluripotent stem cells and performed a mass spectrometry analysis to establish their metabolome and proteome. These cells revealed distinct metabolic profile, with changes particularly at the proteome level. This included a shift between canonical and non-canonical citric acid cycle in hPGCLC, downregulation of late-stage glycolysis and reduction of nucleotide de novo synthesis. By providing an integrative map of these metabolic networks, we aim to provide insight on the influence of metabolism on human PGC development that could help improve methods for in vitro differentiation and maturation hPGCLCs.

Hirschsprung disease (HSCR) is a deadly congenital disorder where the enteric nervous system (ENS) is absent from the distal bowel. Current surgical treatment is generally life-saving but is often accompanied by long-term bowel problems and comorbidities. As alternative, we are developing a regenerative therapy based on rectal administration of Glial cell-derived neurotrophic factor (GDNF). We previously showed that administering GDNF enemas to HSCR mice soon after birth is sufficient to permanently induce a new ENS from tissue-resident neural progenitors. Here, we elucidate the underlying mechanism using single-cell transcriptomics, signal transduction inhibitors and genetic cell lineage tracing tools. We found that the neurogenic effect of GDNF is mediated by NCAM1 (Neural cell adhesion molecule 1), rather than by its canonical signaling receptor RET (Rearranged during transfection). We also unveiled the existence of multiple neuronal differentiation pathways that involve a larger than expected repertoire of tissue-resident neural progenitors, including a surprising one not derived from the usual neural crest. These data support feasibility of GDNF-based therapy in most human patients, even those bearing a RET variant. This work also has far-reaching implications for the choice of ENS progenitor source to use when developing cell transplantation-based therapeutic approaches.

The distinct features of neonatal megakaryocytes, high proliferation and inefficient platelet production, have clinical repercussions. A diminished capacity for stress thrombopoiesis, the response to acute drops in platelet counts, contributes to the high prevalence of thrombocytopenia in premature infants and to impaired platelet recovery after umbilical cord blood stem cell transplantation. High proliferation also promotes leukemogenesis in babies with Down Syndrome (DS). The transcriptional coactivator Mkl1/MrtfA participates in programming the ontogenic shift from fetal/neonatal to adult-type megakaryopoiesis; in this activity it is opposed by the DS-associated kinase Dyrk1a. In a screen for downstream ontogenic effectors in human progenitors, we identified the kinesin Kifc3 as a factor selectively decreased in adult megakaryocytes and whose knockdown in neonatal megakaryocytes induced adult-type morphogenesis with augmented platelet release. Kifc3 acts as a minus-end directed motor for centrosomal delivery of various cargos. Centrosomal release of Cep192 has recently been found induce cellular process extensions through actin remodeling, reminiscent of megakaryocyte platelet release. In our studies, Cep192 showed striking upregulation and dispersion in adult vs neonatal megakaryocytes, and Kifc3 knockdown recapitulated this effect in neonatal megakaryocytes. A role for Cep192 in promoting megakaryocyte morphogenesis, distinct from its role in centrosome biogenesis, was demonstrated in vitro and in vivo. In silico screening for Kifc3 inhibitors identified a small molecule that affected neonatal megakaryocytes similarly to Kifc3 knockdown, indicating feasibility for therapeutic argeting of the Kifc3-Cep192 pathway in clinical conditions associated with fetal-type megakaryopoiesis. Key PointsO_LIThe motor protein Kifc3 dictates megakaryocyte ontogeny in association with its control of the centrosomal actin-remodeling factor Cep192. C_LIO_LIKnockdown or small molecule targeting of Kifc3 enhances neonatal megakaryocyte morphogenesis and thrombopoiesis. C_LI

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.

Epidermal Growth factor (EGF) signaling is associated with (oncogenic) proliferation. Conversely, EGF-family ligands are able to trigger a differentiation program in cultured cells, an effect attributed to ligand affinity and EGFR phosphorylation. How EGF/EGFR driven proliferation-differentiation dynamics underlie tissue self-renewal has not been addressed. We show that culturing mouse small intestinal organoids (mSIOs) without EGF enhanced EGFR expression and base phosphorylation while maintaining a balanced development of proliferative crypts and differentiated villi. Addition of EGF or EREG triggers receptor endocytosis, reducing cell-surface and expression levels. While EGF promoted crypt proliferation, EREG promoted both proliferation and villus differentiation compared to untreated controls. Removal or re-introduction of EGF or EREG proved sufficient to induce development comparable to constant presence of ligands over 96h. Sub-saturating concentrations of EGF led to increased villus differentiation, resembling EREG treatments, suggesting that control over EGFR endocytic cycle ultimately regulates the balance of proliferation and differentiation in mSIOs SummaryExpression and signaling competency at the plasma membrane of EGFR drives crypt proliferation vs villus differentiation by medium ligand-composition, aiding mouse intestinal organoids self-renewal and regeneration.

Early human development involves dynamic transitions in cell identity, including transient transcriptional modulation and stable lineage commitment. Distinguishing these types of gene expression changes is challenging and can be further exacerbated by genetic and experimental heterogeneity in the context of human pluripotent stem cell (hPSC) research. To address this challenge and help uncover transcriptional changes indicative of true developmental state, we establish a curated, cross-platform marker framework for robust identification of pluripotency and early germ-layer identity. Starting from an unbiased RNA-seq discovery set, we systematically validate candidate markers across qPCR, bulk and single-cell RNA sequencing, and quantitative proteomics platforms, yielding a refined panel of 67 markers (20 for the undifferentiated state, 17 for endoderm, 15 for ectoderm, and 15 for mesoderm). We show that this framework reliably identifies early developmental states across heterogeneous datasets, generalizes to in vivo human embryo cell types, and preserves lineage identity despite substantial transcriptional variability. Furthermore, we demonstrate concordant protein-level expression for a subset of markers, supported by deep proteomic profiling of the reference line KOLF2.1J. To enable broad application, we introduce DeepDiff, a web-based resource integrating the validated markers, allowing automated fate classification in a user-friendly interface. Together, this work provides a standardized framework for defining early human developmental identity and disentangling lineage commitment from context-dependent modulation.

Generating genetically identical mammalian oocytes is challenging due to stochastic meiotic recombination. Here, we established parthenogenetic double-haploid embryonic stem cells (PG-DhESCs) possessing complete homozygosity. By employing blastocyst complementation in Prdm14-deficient embryos, we generated chimeric females that produced oocytes derived exclusively from these donor cells. Fertilization of these oocytes yielded viable, fertile, maternally semi-cloned (MSC) mice of both sexes. Although DNA methylation was largely restored during gametogenesis, subtle epigenetic defects correlated with increased body weight in MSC offspring. This study establishes a robust platform combining PG-DhESCs with blastocyst complementation to generate isogenic mammalian oocytes, overcoming traditional limitations in mammalian cloning.

BackgroundCompetitive transplantation is essential for defining intrinsic repopulating capacity of murine hematopoietic stem and progenitor cells (HSPCs), yet comparable assays for human cells have been limited by the lack of a robust in vivo platform. MethodsHere, we describe a novel competitive transplantation method in humanized NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice that enables simultaneous engraftment and longitudinal tracking of distinct human grafts within a shared microenvironment. ResultsUsing human leukocyte antigen-mismatched donor CD34+ cells, this method facilitates standard flow cytometry panels to track multiple donor cell chimerism, lineage output, and HSPC composition. The experimental framework may be adapted to different mouse models, conditioning strategies, donor sources, and treatments. ConclusionsOverall, this humanized competitive repopulation assay fills a critical translational gap and offers a flexible foundation for advancing mechanistic discovery in human hematopoietic biology and improving clinical strategies for stem cell transplantation.

Naked mole-rats (NMRs, Heterocephalus glaber) display unusual longevity and resistance to age-related decline, and accumulating evidence suggests that their autophagy-lysosome pathway (ALP) is regulated differently from that of conventional mammalian models. However, most studies in NMR cells have relied on static biochemical or ultrastructural readouts, leaving the dynamic organisation of autophagy in living cells poorly defined. Here, we establish a stable tandem fluorescent autophagy reporter in NMR skin fibroblasts using an mCherry-EGFP-LC3NMR construct to enable live-cell, single-cell resolution analysis of ALP dynamics. Under basal conditions, NMR skin fibroblasts exhibit a greater abundance of LC3-positive structures than HeLa cells, together with a mixed population of autophagosomes and autolysosomes, indicating a distinct steady-state organisation of the ALP. Chloroquine (CQ)-induced lysosomal stress caused the expected accumulation of LC3-positive structures but also triggered the formation of large cytoplasmic vacuoles in NMR skin fibroblasts. Importantly, this vacuolation was not associated with acute cytotoxicity and progressively resolved following CQ removal, accompanied by reorganisation of LC3-positive compartments and recovery of lysosomal acidity. Electron microscopy showed that CQ-induced vacuoles are membrane-bound, containing internal material and co-existing with multiple ALP-related vesicular compartments. Primary NMR skin fibroblasts display a similar vacuolation phenotype, indicating that this response is not an artefact of immortalisation or reporter expression. Together, these findings establish a live-cell platform for analysing autophagy in NMR cells and identify a distinctive, reversible vacuolation response to lysosomal stress, consistent with dynamic remodelling of the lysosomal system within NMR skin fibroblasts.

Delivery of cells or vectors in advanced therapies is probably the major challenge for genetic disorders that affect a large part of the body such as Duchenne Muscular Dystrophy (DMD). Here, we describe a novel approach for systemic cell delivery based upon an implantable bio-scaffold composed of aligned polycaprolactone nanofibers coated with laminin, able to support adhesion and extensive proliferation of mesoderm cells both in vitro and when implanted subcutaneously in a DMD mouse model. The scaffold is rapidly vascularised leading to cell entering the circulation and colonising multiple distal organs, including distant skeletal muscles and heart. Cells survive in colonized muscles and differentiate into muscle fibres that produce well detectable levels of dystrophin and -sarcoglycan. These results are game changing for cell therapy, as they allow colonization of life essential but "difficult to reach" muscles such as diaphragm and heart while avoiding invasive catheterization. Once optimised, this approach will rapidly enter clinical experimentation for DMD, other muscular dystrophies, and possibly other genetic disorders of the mesoderm. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/715524v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@11dfd34org.highwire.dtl.DTLVardef@1da6599org.highwire.dtl.DTLVardef@14427f0org.highwire.dtl.DTLVardef@19a242a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Study design and therapeutic outcome. Muscle biopsies were obtained from Duchenne muscular dystrophy (DMD) patients to isolate human DMD mesangioblasts (DMD-hMabs). Cells were genetically corrected using a lentivirus carrying a snRNA able to induce exon skipping (U7snRNA), generating U7-hMabs (1). U7-hMabs were seeded onto laminin-coated polycaprolactone (Lam-PCL) nanofiber scaffolds and implanted into the back muscle of DMD-NSG mice. This platform enabled systemic distribution of hMabs cells through circulation, resulting in engraftment across multiple muscle groups, including tibialis anterior, triceps, diaphragm and heart. C_FIG