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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.

GABA B receptor (GABA B R) activation is known to alleviate pain by reducing neuronal excitability, primarily through inhibition of high voltage‐activated (HVA) calcium (Ca V 2.2) channels and potentiating G protein–coupled inwardly rectifying potassium (GIRK) channels. Although the analgesic properties of small molecules and peptides have been primarily tested on isolated murine dorsal root ganglion (DRG) neurons, emerging strategies to develop, study, and characterise human pluripotent stem cell (hPSC)‐derived sensory neurons present a promising alternative. In this study, hPSCs were efficiently differentiated into peripheral DRG‐induced sensory neurons (iSNs) using a combined chemical and transcription factor‐driven approach via a neural crest cell intermediate. Molecular characterisation and transcriptomic analysis confirmed the expression of key DRG markers such as BRN3A, ISLET1, and PRPH, in addition to GABA B R and ion channels including Ca V 2.2 and GIRK1 in iSNs. Functional characterisation of GABA B R was conducted using whole‐cell patch clamp electrophysiology, assessing neuronal excitability under current‐clamp conditions in the absence and presence of GABA B R agonists baclofen and α‐conotoxin Vc1.1. Both baclofen (100 μM) and Vc1.1 (1 μM) significantly reduced membrane excitability by hyperpolarising the resting membrane potential and increasing the rheobase for action potential firing. In voltage‐clamp mode, baclofen and Vc1.1 inhibited HVA Ca 2+ channel currents, which were attenuated by the selective GABA B R antagonist CGP 55845. However, modulation of GIRK channels by GABA B Rs was not observed in the presence of baclofen or Vc1.1, suggesting that functional GIRK1/2 channels were not coupled to GABA B Rs in hPSC‐derived iSNs. This study is the first to report GABA B R modulation of membrane excitability in iSNs by baclofen and Vc1.1, highlighting their potential as a future model for studying analgesic compounds.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired mucociliary clearance due to defects in motile cilia. This study investigates the impact of loss-of-function mutations in the CFAP300 gene on the ciliary structure and function in three PCD patients. Using a multimodal approach, we integrated molecular genetic testing, transmission electron microscopy, the high-speed video microscopy assay and immunofluorescence staining to analyze ciliary motility and protein expression in both ex vivo and in vitro-obtained ciliary cells. Our results revealed that the pathogenic variant c.198_200delinsCC (p.Phe67ProfsTer10) in CFAP300 led to the absence of the functional CFAP300 protein, the complete loss of outer and inner dynein arms and immotile cilia. Air–liquid interface (ALI)-cultured cells from patients exhibited no ciliary beating, contrasting with healthy controls. Immunostaining confirmed the absence of CFAP300 in patient-derived cilia, underscoring its critical role in dynein arm assembly. These findings highlight the diagnostic utility of ALI cultures combined with functional and protein analyses for PCD, offering a clinically actionable framework that can be readily incorporated into standard diagnostic workflows.