Studying epithelial dysfunction in respiratory diseases requires physiologically relevant human models that faithfully recapitulate airway structure and function. Primary human nasal epithelial cells (hNECs) represent an accessible and clinically validated system, widely used for mutation-specific drug theratyping. However, the broader potential of hNECs as an integrated experimental platform remains underexplored. Here, we establish a customized hNEC-based platform spanning ex vivo, in vitro, and stem cell-based applications, with direct relevance to precision medicine. Using freshly fixed nasal brushings, we developed a quantitative single-cell metric to assess Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) localization at the ionocyte plasma membrane, identifying a candidate ex vivo biomarker sensitive to disease status and pharmacological modulation in cystic fibrosis (CF). In parallel, transcriptomic profiling demonstrated that air-liquid interface-differentiated hNECs closely recapitulate the molecular features of native nasal epithelium. Functionally, differentiated hNECs reproduced key properties of airway surface liquid (ASL) physiology and its dysregulation under inflammatory and CF conditions. Finally, we show that basal hNECs provide a robust and accessible source for induced pluripotent stem cell (iPSC) generation. hNEC-derived iPSCs exhibited canonical pluripotency features, differentiated efficiently into germ layer derivatives and lung progenitors, and displayed transcriptomic profiles indistinguishable from iPSCs generated from other somatic sources, supporting their suitability for patient-specific disease modeling. Collectively, these findings position hNECs as an integrated, patient-specific platform linking ex vivo biomarker discovery, in vitro functional assays, and stem cell–based modeling, thereby expanding their utility for precision medicine.

Induced glutamatergic neurons (iGluNeurons) generated by Neurogenin-2 (NGN2) overexpression in human pluripotent stem cells are a powerful model for studying human neuronal maturation and function; however, NGN2-based protocols still lack standardized culture conditions that critically affect neuronal development and function. Three key factors have been identified by previous literature, namely the composition of extracellular matrix coating, the initial plating density, and the choice of culture medium, but the differential effects of their combination have not been thoroughly analyzed. Here, we investigated the combinatorial effects of these three variables, testing eight distinct culture conditions resulting from the combinations of two coatings (poly-L-ornithine and polyethyleneimine), two media (BrainPhys and Neurobasal), and two cell densities (4800 and 1200 cells/mm²). We assessed electrophysiological properties at the single-cell and network levels, characterized morphofunctional and proteomic features across multiple developmental stages. Electrophysiological data indicate that medium composition and plating density, rather than substrate coating, determine neuronal maturation dynamics, with BrainPhys and high density promoting rapid but transient maturation while Neurobasal and low density supporting gradual and sustained network development. Morphofunctional analyzes of synapses and the axon initial segment, together with neuronal maturation markers, support an early BrainPhys-driven acceleration of development that is later exceeded by Neurobasal. To enable accurate proteome profiling of the iGluNeuron system-comprising human neurons and rat astrocytes-we developed a robust taxonomic filtering algorithm that selectively identifies human-specific proteins. This approach confirmed the presence of a conserved core of NGN2-driven differentiation pathways across all settings, in addition to condition-specific signatures. Finally, in the optimal conditions identified through our experimental analyzes, robust spontaneous and evoked synaptic activity was observed. These results provide a framework for optimizing iGluNeuron cultures, balancing rapid maturation and long-term functional stability, and establishing a benchmark for human neuronal models in disease research and drug screening.

IntroductionPACC1 (also named TMEM206) encodes a proton-activated chloride channel implicated in acid-induced cell death, but its tissue distribution, cellular expression, and isoform-specific roles are incompletely understood.MethodsWe mapped PACC1 expression and splicing across normal human tissues, with emphasis on the central nervous system, using RNAscope in situ hybridization, quantitative cell-type-specific co-detection, RT-PCR, and isoform-specific in situ probes. Functional properties of PACC1 splice variants were assessed by reconstituting each isoform in PACC1-deficient cells.ResultsPACC1 was broadly expressed across tissues, with especially high and uniform expression in the brain. Quantitative analyses revealed PACC1 localization in both neurons and astrocytes, with higher abundance in astrocytes. Two major splice variants, PACC1-V1 and PACC1-V2, were investigated, distinguished by exon 2 inclusion and exhibiting distinct tissue and developmental expression patterns. Functional assays indicated isoform-specific differences: PACC1-V2 predominantly localized to endosomes and prevented endosomal hyperacidification, whereas PACC1-V1 accumulated at the plasma membrane and enhanced acid-induced cell death.ConclusionAlternative splicing governs PACC1 channel trafficking and function. Isoform-specific behavior suggests distinct roles for PACC1 variants in cell development and responses to acid stress, particularly within the nervous system.

Zinc finger E-box-binding homeobox 2 (ZEB2) is a key transcription factor involved in multiple aspects of nervous system development, including neuronal specification, migration, and differentiation. Loss-of-function variants in ZEB2 cause Mowat-Wilson syndrome (MWS), a severe neurodevelopmental disorder characterized by intellectual disability, epilepsy, and brain structural abnormalities. In this study, we generated and characterized induced pluripotent stem cell (iPSC) lines from MWS patients carrying pathogenic ZEB2 variants. Patient-derived iPSCs retained full pluripotency and were capable of differentiating into all three germ layers, including neural lineages. Upon directed differentiation into neural progenitor cells (NPCs) and early neurons, we identified distinctive transcriptional alterations affecting neuroepithelial-to-radial glia transition and lineage specification. RNA-seq analysis revealed dysregulation of genes involved in cytoskeletal remodeling, extracellular matrix organization, and cell motility. Functional holographic live imaging confirmed a significant increase in motility behavior in MWS NPCs and early neurons, suggesting that altered cell dynamics may underlie aberrant neural circuit formation. Despite these changes, early neuronal markers such as MAP2 were expressed at comparable levels in MWS and control cells. Together, these findings uncover novel cellular and molecular phenotypes associated with ZEB2 deficiency and provide insight into how disrupted progenitor behavior and transcriptional mis-regulation may contribute to the neurodevelopmental features of MWS.

CAPRIN1 gene encodes a RNA-binding protein, abundant in the brain where it plays a crucial role, regulating the transport and translation of mRNAs of synaptic proteins.CAPRIN1 haploinsufficiency causes a neurodevelopmental disorder characterized by language impairment/speech delay, intellectual disability, attention deficit, hyperactivity disorder, and autism spectrum disorder. To understand the pathogenesis of this disorder and in view of future treatment, we generated human induced pluripotent stem cells (iPSCs) from a patient carrying the c.1744C>T CAPRIN1 variant. The line show marker expression for the pluripotency and the capacity to differentiate into the three germ layers.

Progress of human brain in vitro models stands as a keystone in neurological and psychiatric research, addressing the limitations posed by species-specific differences in animal models. The generation of human neurons from induced pluripotent stem cells (iPSCs) using transcription factor reprogramming protocols has been shown to reduce heterogeneity and improve consistency across different stem cell lines. Despite notable advancements, the current protocols still exhibit several shortcomings. This study focuses on standardizing and optimizing the procedure for iPSC-derived glutamatergic neurons generation through the inducible overexpression of Neurogenin-2. Noteworthy refinements include stringent scrutiny of genomic rearrangements post-fibroblast reprogramming, selection of a homogeneously integrated NGN2-cassettes population, and the incorporation of an intermediate step during neuronal differentiation to store neuronal progenitors. The neural culture showed a high degree of neuronal maturation and consistency, as shown by single-cell and network electrophysiological recordings. These advancements aim to provide more reliable tools for disease modelling and drug screening in neurological disorders.

BACKGROUND:Distal arthrogryposis with impaired proprioception and touch (DAIPT) is a rare autosomal recessive neurological disease characterized by progressive alteration of mechanosensation. DAIPT is caused by loss of function variants in the PIEZO2 gene that encodes an ionic channel involved in mechanotransduction signaling. Our study started from the case of an 11-year-old boy with skeletal and neuromuscular features suggestive of DAIPT. METHODS:Exome sequencing was performed on the trio. The identified variants in PIEZO2 were validated by Sanger sequencing. Functional assays of the variants were performed by minigene assay in HEK-293 cells and on patient-derived cells using NMD inhibitors. RESULTS:Trio exome sequencing revealed the presence of two novel variants in the PIEZO2 gene: a nonsense variant (c.1924G>T; p.Glu642*) and an intronic variant of uncertain significance (c.2170-15A>G). Functional analysis demonstrated that the intronic variant disrupts splicing, leading to premature stop codon formation and possible mRNA targeting to nonsense-mediated mRNA decay (NMD). Molecular study in patient-derived fibroblasts with specific NMD inhibitors shows that transcripts derived from both alleles are degraded by NMD, thus confirming the effect of the nonsense variant and enabling reclassification of the VUS. CONCLUSION:We present the phenotypic and genetic description of a patient with features suggestive of DAIPT carrying novel biallelic variants in PIEZO2, one of which could be reclassified as pathogenic after functional assays. This study also provides a detailed review of all the published patients with DAIPT and expands the phenotypic and genetic understanding of DAIPT, aiding in diagnosis, genetic counseling, and clinical management.

ZEB2 is a protein-coding gene belonging to a very restricted family of transcription factors. ZEB2 acts mainly as a transcription repressor, is expressed in various tissues and its role is fundamental for the correct development of the nervous system. The best-known clinical picture associated with ZEB2 mutations is Mowat-Wilson syndrome, caused mostly by haploinsufficiency and characterized by possible multi-organ malformations, dysmorphic features, intellectual disability, and epilepsy. In this study we report the generation of IGGi004-A and IGGi005-A, iPSC clones from two patients carrying different heterozygous mutations in ZEB2, which can be used for disease modelling, pathophysiological studies and therapeutics testing.

Sotos syndrome (SoS) is a neurodevelopmental disorder that results from NSD1 mutations that cause haploinsufficiency of NSD1. Here, we generated an induced pluripotent stem cell (iPSC) line from fibroblasts of a SoS patient carrying the pathogenic variant (c.1633delA). The cell line shows typical iPSC morphology, high expression of pluripotent markers, normal karyotype, and it differentiates into three germ layers in vitro. This line is a valuable resource for studying pathological pathways involved in SoS.

The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement.

WDR44 prevents ciliogenesis initiation by regulating RAB11-dependent vesicle trafficking. Here, we describe male patients with missense and nonsense variants within the WD40 repeats (WDR) of WDR44, an X-linked gene product, who display ciliopathy-related developmental phenotypes that we can model in zebrafish. The patient phenotypic spectrum includes developmental delay/intellectual disability, hypotonia, distinct craniofacial features and variable presence of brain, renal, cardiac and musculoskeletal abnormalities. We demonstrate that WDR44 variants associated with more severe disease impair ciliogenesis initiation and ciliary signaling. Because WDR44 negatively regulates ciliogenesis, it was surprising that pathogenic missense variants showed reduced abundance, which we link to misfolding of WDR autonomous repeats and degradation by the proteasome. We discover that disease severity correlates with increased RAB11 binding, which we propose drives ciliogenesis initiation dysregulation. Finally, we discover interdomain interactions between the WDR and NH 2 -terminal region that contains the RAB11 binding domain (RBD) and show patient variants disrupt this association. This study provides new insights into WDR44 WDR structure and characterizes a new syndrome that could result from impaired ciliogenesis.

The human PLAA gene encodes Phospholipase-A2-Activating-Protein (PLAA) involved in trafficking of membrane proteins. Through its PUL domain (PLAP, Ufd3p, and Lub1p), PLAA interacts with p97/VCP modulating synaptic vesicles recycling. Although few families carrying biallelic PLAA variants were reported with progressive neurodegeneration, consequences of monoallelic PLAA variants have not been elucidated. Using exome or genome sequencing we identified PLAA de-novo missense variants, affecting conserved residues within the PUL domain, in children affected with neurodevelopmental disorders (NDDs), including psychomotor regression, intellectual disability (ID) and autism spectrum disorders (ASDs). Computational and in-vitro studies of the identified variants revealed abnormal chain arrangements at C-terminal and reduced PLAA-p97/VCP interaction, respectively. These findings expand both allelic and phenotypic heterogeneity associated to PLAA-related neurological disorders, highlighting perturbed vesicle recycling as a potential disease mechanism in NDDs due to genetic defects of PLAA.

Immune dysregulation in Inborn Errors of Immunity (IEI) shows a broad phenotype, including autoimmune disorders, benign lymphoproliferation, and malignancies, driven by an increasing number of implicated genes. Recent findings suggest that childhood cancer survivors (CCSs) may exhibit immunological abnormalities potentially linked to an underlying IEI, along with a well-known increased risk of subsequent malignancies due to prior cancer treatments. We describe a patient with two composite heterozygous pathogenic variants in the interleukin-2-inducible T-cell kinase (ITK) gene and a history of multiple tumors, including recurrent Epstein-Barr virus (EBV)-related nodular sclerosis and Hodgkin's lymphoma (NSHL), associated with unresponsive multiple hand warts, immune thrombocytopenia, and an impaired immunological profile (CD4+ lymphocytopenia, memory B-cell deficiency, reduction in regulatory T-cells, and B-cell- and T-cell-activated profiles). In our case, ITK-related immune dysregulation and prior exposure to oncological treatments seem to have simultaneously intervened in the same individual, leading to the development of a unique clinical profile. It is essential to raise awareness of the two-way association between immune dysregulation disorders and multiple tumors.

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel defective in cystic fibrosis (CF). Several CFTR mutations are causative of CF, among which G542X is a nonsense mutation introducing a premature stop codon which prevents CFTR protein synthesis. We generated a new iPSC line from nasal cells carrying G542X homozygous mutation for CFTR: IGGi002A. This cell line has normal female karyotype, express pluripotency markers and could differentiate into three germ layers in vitro. This iPSC line may be used for disease modeling (cell differentiation and organoid formation) and development of personalized treatments by genome editing or pharmacological screening.

Sotos syndrome (SoS) is a neurodevelopmental disorder caused by haploinsufficiency of the NSD1 gene located on chromosome 5 region q35.3. In order to understand the pathogenesis of Sotos syndrome and in view of future therapeutic approaches for its efficient treatment, we generated two human induced pluripotent stem cells (iPSCs) lines from one SoS patient carrying a 5q35 microdeletion. The established iPSCs expressed pluripotency markers, showing the capacity to differentiate into the three germ layers.