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The implications of the early phases of human telencephalic development, involving neural stem cells (NSCs), in the etiology of cortical disorders remain elusive. Here, we explore the expression dynamics of cortical and neuropsychiatric disorder-associated genes in datasets generated from human NSCs across telencephalic fate transitions in vitro and in vivo. We identify risk genes expressed in brain organizers and sequential gene regulatory networks throughout corticogenesis, revealing disease-specific critical phases when NSCs may be more vulnerable to gene dysfunction and converging signaling across multiple diseases. Further, we simulate the impact of risk transcription factor (TF) depletions on neural cell trajectories traversing human corticogenesis and observe a spatiotemporal-dependent effect for each perturbation. Finally, single-cell transcriptomics of autism-affected patient-derived NSCs in vitro reveals recurrent expression alteration of TFs orchestrating brain patterning and NSC lineage commitment. This work opens perspectives to explore human brain dysfunction at early phases of development. The implications of early telencephalic development in cortical disorders remain elusive. Here, the authors define risk gene dynamics and perturbation effects in neural stem cells, revealing vulnerability phases during early human corticogenesis.

CD84, a member of the signaling lymphocyte activation molecule immunoglobulin superfamily, has been identified as playing a significant role in regulating various immune cell activities. However, its intrinsic role in cancer cells remains largely unknown. We aim to explore the direct role of CD84 in acute myeloid leukemia (AML) progression and to clarify the underlying molecular mechanisms involved in nonhomologous end-joining (NHEJ) repair. Herein, we found that CD84 is frequently upregulated in various types of AML and leukemia initiation cells (LICs)-enriched cells. Knockdown or blocking of CD84 significantly inhibits the growth and induces the apoptosis of AML cells. Moreover, knockdown of CD84 significantly delays AML progression and prolongs the survival of the xenografted mice in vivo. Mechanistically, CD84 promotes the expression of NHEJ core factors by recruiting SAP and activating the AKT signaling pathway. Knockdown of CD84 inhibits NHEJ repair in AML cells via regulating the expression of NHEJ core factors, including PRKDC, LIG4, XRCC5, and DCLRE1C. Subsequently, this leads to double-strand breaks accumulation and cell apoptosis. Importantly, CD84 is required for the proliferation and self-renewal of human LICs. In conclusion, CD84 plays important roles in AML growth and progression through promoting NHEJ repair. Targeting CD84 may be a potential approach for inhibiting AML development and eliminating LICs.

AbstractIncreased soluble FMS-like tyrosine kinase 1 (sFLT1) levels have been associated with preeclampsia, chronic kidney diseases, and kidney transplant rejection. However, lower levels of sFLT1 exhibit beneficial properties in various processes, such as the organization of the actin cytoskeleton in podocytes and immune regulation in healthy pregnancy. Therefore, understanding the transcriptional regulation of sFLT-1 and preserving appropriate expression levels are critical for effective treatment of preeclampsia and other diseases. Cytotrophoblasts (CTBs) were isolated from three first-trimester placentas and differentiated into extravillous trophoblasts (EVTs) for 6 days. RNA was extracted at different time points and used for RNA sequencing. Differentially expressed genes (DEGs) and transcription factors (DETFs) were analyzed. Transcription factor (TF) enrichment analysis and pathway analysis were performed on DEGs screened from EVTs and CTBs. TF inhibitors were added to primary CTBs directly or during CTB to EVT differentiation to confirm the regulatory effect of TFs on sFLT1 expression. In total, 197 TFs were differentially expressed between CTBs and EVTs, among which 15 DETFs (EPAS1, ETS1, TBX3, CEBPB, FLI1, TEAD4, GATA4, TBX2, LMX1B, ARNT, FOXM1, ERF, PRDM1, TFAP2A, and NR2F2) that potentially regulate sFLT1 expression were predicted by ChEA3 and KnockTF software. The mRNA levels of 15 DETFs were validated upon CTBs differentiation into both EVTs and syncytiotrophoblasts. The regulatory effects of FOXM1 and CEBPB were confirmed in vitro experiments, and their expression patterns were validated during CTBs differentiation into EVTs and in first-trimester placentas. Pathway analysis showed that FLT1 was involved in P13K-Akt, Rap1, MAPK, Ras, and HIF-1 signaling pathways, focal adhesion, and cytokine–cytokine receptor interaction. Protein–protein interaction analysis showed that FLT4, PDGFB, TGFB1, IL6R, TNFRSF1B, CSF1R, and TGFB2 interact with FLT1. The identified TFs can serve as therapeutic targets in preeclampsia to keep the sFLT1 levels within appropriate limits.

BackgroundThe spread of SARS-CoV-2 Omicron variant and its subvariants, highly transmissible but responsible of milder disease, has increased unreported infection cases. Identifying unaware infected individuals is crucial for estimating the true prevalence of infection and evaluating the breadth of hybrid immunity. In this study, this challenge was addressed by applying several Machine Learning approaches.MethodsA group of 116 participants, vaccinated against SARS-CoV-2, was enrolled in the IMMUNO_COV study at Siena University Hospital, Italy. Blood samples were collected before and six months after third vaccine dose. Machine Learning analysis, involving dimensionality reduction techniques, unsupervised clustering methods and classification models, were applied to serological data including antibody responses specific for wild type SARS-CoV-2 strain as well as Delta, Omicron BA.1 and Omicron BA.2 variants. Spike- and nucleocapsid-specific B cells were also assessed in each participant.ResultsUsing dimensionality reduction and unsupervised clustering, participants are grouped into high- and low-responders, with infected participants mainly distributed within the high-responders. Implementation of a consensus-based approach, including k-NN, RF, and SVM models, identifies 14 participants unaware of previous infection. Their immunological profile, characterized by strong spike- and nucleocapsid-specific humoral and B cell responses, significantly differs from that of non-infected participants.ConclusionsMachine Learning approaches are applied to identify participants unaware of prior infection and to dissect their hybrid immunity profiles. Based on serological data, this cost-effective method can be a valuable tool for estimating the true prevalence of infection, improving comprehension of immune responses elicited by vaccination alone or combined with infection, and tailoring public health interventions. Plain Language SummaryWith the spread of the Omicron variants, SARS-CoV-2 infections have become more contagious but less symptomatic, resulting in many individuals becoming infected without knowing it. Using advanced computer-based techniques, applied on easily accessible blood test results, such as antibodies and immune cells levels, we developed a model capable of identifying individuals who had been previously infected but were unaware of their infection. These individuals exhibited immune responses more closely resembling those of infected individuals than non-infected ones. The application of this model not only enables the profiling of immune responses induced by mRNA vaccination alone or in combination with infection, but also provides a more accurate picture of virus spread and supports targeted prioritization of vaccine allocation. Montesi, Costagli et al. use Machine Learning algorithms to develop a consensus based approach to dissect hybrid and vaccine-induced immunity following a third dose of mRNA SARS-CoV-2 vaccine. This method can be a valuable tool for estimating the true prevalence of infection, improving comprehension of immune responses elicited by vaccination alone or combined with infection, and tailoring public health interventions.

SummaryPrimary human cells cultured in long-term expandable 3D organoid format have great promise as potential regenerative cellular therapies, but their immunogenicity has not yet been fully characterized. In this study, we use in vitro co-cultures and in vivo humanized mouse experimental models to examine autologous and allogeneic immune response to human primary cholangiocyte organoids (PCOs) as treatment for bile duct disorders. Our data demonstrate that PCOs upregulate the expression of human leukocyte antigen (HLA)-I and HLA-II in inflammatory conditions. The allogeneic immune response to PCOs is driven by both HLA-I and HLA-II and is substantially ameliorated by donor-recipient HLA matching. While allogeneic cells display evolving stages of immune rejection in vivo, autologous PCOs induce a low-level immune infiltration into the graft site possibly influenced by acquired mutations in culture, cell viability, and culture matrix. Our findings have important implications for the design and clinical translation of autologous and allogeneic organoid cellular therapies. Graphical abstract Highlights•Human primary cholangiocyte organoids (PCOs) upregulate HLA-I/II in inflammatory conditions•Allogeneic immune response is substantially ameliorated by donor-recipient HLA matching•Autologous PCOs induce a low-level immune infiltration into the graft site•Humanized mice distinguish early/vigorous and late/resolved stages of allogeneic response Petrus-Reurer et al. use in vitro co-cultures and in vivo humanized mice to examine autologous and allogeneic immune response to human primary cholangiocyte organoids (PCOs). The authors show that autologous cells induce a low-level immune infiltration into the graft and allogeneic cells display HLA-driven evolving stages of immune rejection.

BackgroundKidney transplant (KT) candidates with very high calculated panel reactive alloantibody (cPRA >95%) have limited chances to receive an HLA-matched transplant unless they undergo pretransplant desensitization.ObjectiveTo assess the efficacy of immunoadsorption (IA) in desensitizing pretransplant KT candidates with high cPRA and positive crossmatch.Materials and methodsThis was a single-center retrospective cohort study involving highly HLA-sensitized patients (cPRA >85%). Forty-nine patients underwent HLA-incompatible (HLAi) KT, of whom 25 (51%) received kidneys from deceased donors. Of these 49 patients, 23 had either a positive complement-dependent cytotoxic cross-match (CDC) and/or a positive flow cytometry cross-match (FCM). The remaining 26 patients had donor-specific anti-HLA (DSAs) detectable only by Luminex (CDC and FCM cross-matches were negative). Only CDC-positive and FCM-positive patients were desensitized. These 49 patients were compared with 160 patients who had cPRA >85% but underwent HLA-compatible (HLAc) KT, i.e., without pretransplant DSAs.Pretransplant desensitization included IA sessions, rituximab, tacrolimus, steroids, and mycophenolate mofetil. Induction therapy consisted of antithymocyte globulins.ResultsThe mean follow-up duration was 7.4 ± 4 years. At 1-year and at last follow-up, 43 patient and death-censored graft survival rates were similar between HLAc and HLAi patients. However, HLAi patients experienced significantly more biopsy-proven rejections compared to HLAc patients. These rejections were predominantly antibody-mediated. Finally, the rate of infectious complications was similar between HLAc and HLAi patients.ConclusionIA in addition to immunosuppression is an effective option for desensitizing HLAi patients, yielding favorable long-term outcomes.

Single-cell profiling protein-chromatin interactions is often constrained by complex workflows, high cost, or dependence on specialized equipment. We present indexed tagmentation-based single-cell CUT&Tag-sequencing (IT-scC&T-seq), a modular, plate-based strategy using three-round combinatorial barcoding. IT-scC&T-seq robustly profiles histone modifications and transcription factors with high specificity and throughput, supporting simultaneous analysis of multiple samples and epitopes. Notably, it enables sensitive single-cell mapping of lamina-associated domains, low-abundance chromatin features previously difficult to resolve. Applied to adult mouse mammary gland, the method reveals cell-type-specific chromatin landscapes and lineage-regulatory dynamics. Together, IT-scC&T-seq provides a scalable, cost-effective, and broadly accessible approach for high-resolution chromatin profiling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03661-z.

Glucose transporter 4 (GLUT4) expression on white adipocytes is critical for facilitating cellular uptake of blood glucose, failure of which promotes hyperglycemia. Matrix metalloproteinases (MMPs) play a crucial role in remodeling the white adipose tissue (WAT) during obesity. MMPs have multiple protein substrates, and surprisingly, it is unknown if they can directly target GLUT4 on the adipocyte surface and impair glucose uptake. We identified MMP2 as the highly active gelatinase, a class of MMP, in the gonadal WAT of high-fat diet-induced obese mice. In vitro, metabolic studies in 3T3-L1 adipocytes revealed MMP2 attenuated glucose uptake and glycolysis, which were recovered by an MMP2 inhibitor. In silico structural Analysis using AlphaFold identified a putative MMP2 cleavage site on the extracellular domain of GLUT4. Further, in a substrate competition assay, a peptide mimicking the MMP2 cleavage site on GLUT4 attenuated the cleavage of an MMP substrate by MMP2. Altogether, our results suggest a novel mechanism of impaired glucose utilization by adipocytes, which may contribute to hyperglycemia during obesity.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06252-x.