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Neural crest stem cells (NCSCs) compose a highly migratory, multipotent, stem cell population arising from the neural plate border of the embryonic ectoderm. Investigating the development of NCSCs is critical in understanding both embryonic development and abnormal events that underlie neurocristopathies. Suggested seeding densities in in vitro human induced pluripotent stem cells (hiPSCs) differentiation protocols, varying between 10,000 cells/cm 2 and 200,000 cells/cm 2 , demonstrate a lack of consensus on the optimal conditions to obtain NCSCs. Aiming to maximize the differentiation efficiency of hiPSCs towards the NCSCs lineage, we investigated the effect of the initial seeding density on NCSCs lineage commitment, both in fibroblast- and human peripheral blood mononuclear cell (PBMC)-derived hiPSCs. Cultures were characterized with gene and protein expression analysis assessing stemness ( OCT3/4 and NANOG ), neural crest identity ( SNAI2 and SOX10 ) and neuroectoderm identity ( PAX6 and SOX1 ). We demonstrate that reaching a confluent monolayer of cells by the end of the differentiating protocol is crucial to obtaining NCSCs from hiPSCs. To achieve this, our results indicated 17,000 cells/cm 2 is the optimal initial seeding density. Under this protocol, a confluent monolayer was reached after 8 days of differentiation and an average of 89% SOX10 positive cells were obtained. The fold change of SNAI2 and SOX10 expression was 11-fold and 17-fold higher, respectively, in cultures seeded with 17,000 cells/cm 2 , compared to the highest tested density of 200,000 cells/cm 2 . In contrast, seeding 200,000 cells/cm 2 induced neuroectoderm-like cells, confirmed by an average of 45% of cells marking positive for PAX6. With this work, we demonstrate the importance of achieving cellular confluency during NCSCs differentiation.

Sac1 is a conserved phosphoinositide phosphatase, whose loss-of-function compromises cell and organism viability. Here, we employ acute auxin-inducible Sac1 degradation to identify its immediate downstream effectors in human cells. Most of Sac1 is degraded in ~1 h, paralleled by increased PI(4)P and decreased cholesterol in the trans-Golgi network (TGN) during the following hour, and superseded by Golgi fragmentation, impaired glycosylation, and selective degradation of TGN proteins by ~4 h. The TGN disintegration results from its acute deacidification caused by disassembly of the Golgi V-ATPase. Mechanistically, Sac1 mediated TGN membrane composition maintains an assembly-promoting conformation of the V0a2 subunit. Key phenotypes of acute Sac1 degradation are recapitulated in human differentiated trophoblasts, causing processing defects of chorionic gonadotropin, in line with loss-of-function intolerance of the human SACM1L gene. Collectively, our findings reveal that the assembly of the Golgi V-ATPase is controlled by the TGN membrane via Sac1 fuelled lipid exchange. This study employs auxin-inducible degradation of Sac1. The authors reveal that acute Sac1 depletion changes the Golgi membrane lipid composition, causing disassembly of the Golgi V-ATPase and eventually resulting in cargo processing defects.

BackgroundA growing body of evidence from primate embryos as well as in vitro systems supports the notion that amnion and primordial germ cell (PGC) lineage progressing cells share a common precursor.ResultsTo gain comprehensive transcriptomic insights into this critical but poorly understood precursor and its progeny, we examine the evolving transcriptome of a developing human pluripotent stem cell-derived model of amnion and PGC formation at the single cell level. This analysis reveals several continuous amniotic fate progressing states with state-specific markers. Additionally, a progenitor-like cell, that displays bi-potential characteristics for amnion and PGC-like cell lineages and is marked by CLDN10, is identified. Strikingly, we find that expression of CLDN10 is restricted to the amnion-epiblast boundary region in our human post-implantation amniotic sac model as well as in peri-gastrula cynomolgus macaque embryos; moreover, this boundary region presents amnion and PGC progenitor-like transcriptional characteristics. Furthermore, our loss of function analysis shows that CLDN10 promotes amniotic but suppresses PGC-like fate.ConclusionsOverall, based on the single cell transcriptomic resource in this study, we identify a CLDN10+ amnion and PGC progenitor-like population at the amnion-epiblast boundary of the primate peri-gastrula, and present additional molecular clues as to how amnion and PGC may be formed at the amnion-epiblast boundary in human peri-gastrula. Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03751-y.

The aim of this study is to establish an in vitro co-culture system to model allograft rejection using kidney organoids system derived from human induced pluripotent stem cells (hiPSCs). We co-cultured kidney organoids derived from wild-type hiPSCs with HLA-mismatched peripheral blood mononuclear cells (PBMCs) from healthy controls (HC) for 24 h. To assess allogeneic rejection modeling, we measured the expression of HLA molecules, (HLA-ABC and HLA-DR), and evaluated cellular damage in the kidney organoids. Additionally, we analyzed the distribution of T cells and their subsets within the co-cultured PBMCs. The immunosuppressive effect of tacrolimus was also evaluated in this co-culture system. Transcriptomic analysis, conducted using RNA sequencing, identified molecules associated with allogeneic rejection. When kidney organoids were co-cultured with alloreactive PBMCs for 24 h, HLA-ABC and HLA-DR expression significantly increased in kidney organoid cells. Additionally, kidney organoids showed reduced cell viability and increased apoptosis compared to syngeneic controls, as assessed by flow cytometry and Annexin V/PI staining. However, treatment with tacrolimus reduced HLA expression in a dose-dependent manner, highlighting the diminished alloimmune responses. Further analysis of PBMC subsets revealed shifts in T helper (TH) and cytotoxic T cell (TC) populations under allogeneic conditions, including increased effector TH and TC cells. Transcriptomic analysis through RNA sequencing identified 256 differentially expressed genes (DEGs), with notable immune-related pathways such as NF-kappa B and TNF signaling involved in allograft rejection. These results provide evidence that a co-culture system with allogeneic kidney organoids and PBMCs can potentially model transplant rejection in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05867-7.

Immune rejection is one of the most serious challenges in allogeneic transplantation, including allogeneic induced pluripotent stem cell (allo-iPSC)-derived cell therapy. Beta-2-Microglobulin gene-knockout, human leukocyte antigen (HLA) class I-deficient iPSCs can evade immune rejection by host T cells, which occurs due to HLA mismatches. However, natural killer (NK) cells recognize HLA class Ⅰ-deficient cells and reject them, which is known as the missing-self response. Introducing chimeric HLA-E protein to HLA class Ⅰ-deficient iPSCs suppresses the missing-self response of NK cells expressing the inhibitory receptor NKG2A; however, technology to suppress NKG2A-negative NK cells is still required. Here, we developed novel agonists for the other inhibitory receptor, killer immunoglobulin receptor (KIR), on NK cells. We found that antibodies that bind to activating KIR enhance NK cell activation and developed selective agonists for inhibitory KIRs (KIR2DL1, KIR2DL2/3, and KIR3DL1). Introducing these selective inhibitory KIR agonists on T cells and HLA class Ⅰ-deficient iPSCs allowed them to evade immune rejection by NK cells. Additionally, we identified an NKG2A-selective agonist as an alternative to chimeric HLA-E, which stimulates the activating receptor NKG2C. This technology enhances immune tolerance in allo-iPSCs and facilitates the development of various iPSC-derived regenerative medicines. The online version contains supplementary material available at 10.1038/s41598-025-18394-z. Subject terms: Allotransplantation, NK cells

Small non‐coding microRNAs (miRNAs) play essential roles in Alzheimer's disease (AD) pathogenesis. Repressor element 1‐silencing transcription factor (REST) is involved in AD, though its regulation remains unclear. We performed real‐time quantitative polymerase chain reaction (qPCR) in autopsied brain tissues to determine miR‐153‐3p and AD associations. A reporter‐based assay measured the activity of REST mRNA 3′‐untranslated region (3′‐UTR). Induced pluripotent stem cells (iPSC)‐derived neurons and human cell lines were applied to determine miR‐153‐3p regulation of endogenous proteins. Elevation of miR‐153‐3p is associated with a reduced probability of AD, while elevated REST is associated with a greater probability of AD. The 3′‐UTR functional assay pinpointed the miR‐153‐3p binding sites. miR‐153‐3p treatment reduced REST, amyloid precursor protein (APP), and α‐synuclein (SNCA) 3′‐UTR activities and protein levels. miR‐153‐3p treatment altered REST and neuronal differentiation in iPSC‐derived neuronal stem cells. RNA‐sequencing and proteomics revealed miR‐153‐3p‐associated networks. miR‐153‐3p reduces REST, APP, and SNCA expression, pointing toward its therapeutic and biomarker potential in neurodegenerative diseases. With the increased emphasis on comorbidities of Alzheimer's disease (AD) and other neurodegenerative diseases, we identified that miR‐153‐3p, as a master regulator, reduced a group of neurodegeneration related proteins: REST, amyloid precursor protein (APP) and α‐synuclein (SNCA) levels. The elevation of miR‐153‐3p levels is associated with reduced probability of AD in posterior cingulate cortex (PCC), while REST, by contrast, is associated with a greater probability of AD. miR‐153‐3p treatment alters REST protein levels and neuronal differentiation in induced pluripotent stem cells (iPSC) derived neuronal cells. RNA sequencing proteomics and interactome analysis revealed the role of miR‐153‐3p in axonal guidance.

Alzheimer's disease (AD) is characterized by tau pathology, leading to neurodegeneration. Astrocytes regulate central nervous system homeostasis and influence AD progression. The APOE3‐Christchurch (APOE3‐Ch) variant is linked to AD resilience, but its protective mechanisms remain unclear. Human induced pluripotent stem cell–derived astrocytes (APOE3‐Ch and wild type) were used to assess tau uptake, clearance, lipid metabolism, and transcriptomic adaptations. Fluorescently labeled 2N4R‐P301L tau oligomers were tracked, and pathway‐specific inhibitors dissected tau clearance mechanisms. Lipidomic and transcriptomic analyses were performed to identify genotype‐specific adaptations. APOE3‐Ch astrocytes exhibited enhanced tau uptake via heparan sulfate proteoglycan‐ and lipoprotein receptor‐related protein 1‐mediated pathways and superior clearance through lysosomal and proteasomal degradation. They exported less tau, limiting propagation. Transcriptomic analyses revealed upregulation of genes involved in cell projection assembly and endocytosis. Lipidomic profiling showed reduced ceramides and gamma‐linolenic acid, linked to decreased neuroinflammation and ferroptosis. APOE3‐Ch astrocytes promote tau clearance and metabolic adaptations, providing insights into genetic resilience in AD and potential therapeutic targets. APOE3‐Christchurch (APOE3‐Ch) astrocytes exhibit significantly increased tau internalization compared to wild‐type astrocytes, facilitated by upregulated heparan sulfate proteoglycan and low‐density lipoprotein receptor‐related protein 1 pathways. APOE3‐Ch astrocytes demonstrate more efficient tau degradation via both lysosomal and proteasomal pathways, while exporting significantly less tau, potentially reducing tau propagation in the central nervous system. APOE3‐Ch astrocytes show upregulation of genes involved in cell projection assembly and endocytosis, suggesting structural and functional modifications that enhance tau processing. Lipidomic profiling reveals reduced ceramide levels and gamma‐linolenic acid downregulation in APOE3‐Ch astrocytes, alterations linked to reduced neuroinflammatory and ferroptotic activity, contributing to the protective phenotype.

Type 1 interferons (IFN-Is) exhibit significant antiviral, antitumor, and immunoregulatory properties, demonstrating substantial therapeutic potential. However, IFN-Is are pleiotropic cytokines, and the available data on their effect under specific pathological conditions are inconclusive. Furthermore, the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types, including human immune cells. In this study, we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4, SOX2, TRA 1-60, and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline, all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line, 126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types, as well as for generating different types of cells with controllable IFN-β expression.

Seven female individuals with multiple congenital anomalies, developmental delay and/or intellectual disability have been found to have a genetic variant of uncertain significance in the mediator complex subunit 12 gene ( MED12 c.3412C>T, p.Arg1138Trp). The functional consequence of this genetic variant in disease is undetermined, and insight into disease mechanism is required. We identified a de novo MED12 p.Arg1138Trp variant in a female patient and compared disease phenotypes with six female individuals identified in the literature. To investigate affected biological pathways, we derived two induced pluripotent stem cell (iPSC) lines from the patient: one expressing wildtype MED12 and the other expressing the MED12 p.Arg1138Trp variant. We performed neural disease modelling, transcriptomics and protein analysis, comparing healthy and variant cells. When comparing the two cell lines, we identified altered gene expression in neural cells expressing the variant, including genes regulating RNA polymerase II activity, transcription, pre-mRNA processing, and neural development. We also noted a decrease in MED12L expression. Pathway analysis indicated temporal delays in axon development, forebrain differentiation, and neural cell specification with significant upregulation of pre-ribosome complex gene pathways. In a human neural model, expression of MED12 p.Arg1138Trp altered neural cell development and dysregulated the pre-ribosome complex providing functional evidence of disease aetiology and mechanism in MED12-related disorders. The online version contains supplementary material available at 10.1186/s10020-025-01365-5.

IntroductionExposure to teratogenic compounds during pregnancy can lead to significant birth defects. Given the considerable variation in drug responses across species, along with the financial and ethical challenges associated with animal testing, the development of advanced human-based in vitro assays is imperative for effectively identifying potential human teratogens. Previously, we developed a human induced pluripotent stem cells (hiPSCs)-based biomarker assay, ReproTracker, that follows the differentiation of hiPSCs into hepatocytes and cardiomyocytes. The assay combines morphological profiling with the assessment of time-dependent expression patterns of cell-specific biomarkers to detect developmental toxicity responses.MethodsTo further increase the predictability of the assay in identifying potential teratogens, we added differentiation of hiPSCs towards neural rosette-like cells. We evaluated the performance of the extended assay with a set of 51 well-known in vivo teratogens and non-teratogens, including the compounds listed in the ICH S5 reference list.ResultsThe optimized assay correctly identified (neuro)developmental toxicants that were not detected in the hepatocyte and cardiomyocyte differentiation assays. These compounds selectively downregulated gene and protein expression of the neuroectodermal marker PAX6 and/or neural rosette marker NESTIN in a concentration-dependent manner and disrupted the differentiation of hiPSCs towards neural rosette-like cells. Overall, based on the current dataset, the addition of neural commitment improved the assay accuracy (from 72.55% to 86.27%) and sensitivity (from 67.50% to 87.50%), when compared to the previously described assay.DiscussionIn summary, trilineage differentiation expanded the spectrum of teratogenic agents detectable by ReproTracker, making the assay an invaluable tool for early in vitro teratogenicity screening.

Prion diseases, such as sporadic Creutzfeldt-Jakob Disease (sCJD), are neurodegenerative disorders caused by misfolding of the prion protein (PrP). The D178N mutation in the PrP gene causes Fatal Familial Insomnia (FFI). Here we show that both sCJD and FFI prions can infect human cerebral organoids with or without the D178N mutation, and that the resulting infection is dictated by the inoculating prion and not the host organoid genotype.

Biomolecular condensates are found throughout a diversity of eukaryotic cell types and cellular compartments, playing roles in various cellular functions. A given protein generally forms functionally and compositionally heterogeneous condensates, but the underlying regulatory mechanisms are unknown. Here, we found that different RNA motifs modulate the formation of heterogeneous mRNA-protein condensates via riboregulation. Fragile X-related 1 (FXR1), an RNA-binding protein interacting with nuclear pores, assembles distinct localized subcellular mRNP condensates linked to cytosolic accumulation of G-quadruplex-containing pluripotent mRNAs and the localized translation of nucleoporin mRNAs at nuclear pores. The diverse locations of FXR1 condensates depend on the unique RNA-protein interaction modules of its two RNA binding domains, and the opposing effects of different RNA motifs on the affinity of FXR1 for nuclear pores. Notably, reduced FXR1 levels and impaired nuclear pore function lead to the nuclear accumulation of transcribed RNAs, facilitating fate transition in human embryonic stem cells. Preventing this decline would result in impaired hESC differentiation. Subject terms: RNA metabolism, Embryonic stem cells, RNA, RNA transport