�����ƽ��

Craniosynostosis is a multigenic congenital condition in which one or more calvarial sutures have prematurely fused during the development of the fetus. Pathogenic variants in FGFR2 are associated with the development of syndromic craniosynostosis, such as Crouzon, Apert and Pfeifer syndromes. Investigation of FGFR2 -linked craniosynostosis is hindered by the lack of appropriate in vitro models. Patient-derived human induced pluripotent stem cell (hiPSC) in vitro disease models provide the opportunity to investigate the disease, identify molecular targets for pharmaceutical treatments, and enable the generation of autologous pluripotent stem cell catalogues. Here, we report three patient-derived hiPSC lines carrying the C342Y, S252W or E565G FGFR2 pathogenic variant. The patient hiPSC lines express characteristic pluripotency markers and display distinct phosphorylation profiles under unstimulated conditions. FGFR2 C342Y showed autophosphorylation in the absence of bFGF ligand, although downstream docking proteins PLCγ and FRS2α were not phosphorylated. FGFR2 S252W and FGFR2 E565G hiPSCs showed increased phosphorylation of docking proteins PLCγ and FRS2α, whereas FGFR2 was not phosphorylated. These patient hiPSC lines provide molecular and cellular options to investigate FGFR2 -linked craniosynostosis in the patient-specific genomic context and develop therapeutic modalities.

Neurological damage caused by peripheral nerve injury can be devastating and is a common neurological disorder that, along with muscle disorders, reduces the quality of life. Neural crest cells (NCCs) are a transient cell population that occurs during embryogenesis, can differentiate into various cells upon transplantation, and has potential therapeutic effects on neurological diseases. However, there are limitations to cell therapy, such as uncontrolled differentiation and tumor formation. Extracellular vesicles (EVs), which are non-cellular potential therapeutic candidates, are nanosized membrane-bound vesicles. Studies have been reported using starch cells derived from neural cells, such as neural stem cells, because they are involved in cell-to-cell communication and carry a variety of bioactive molecules. We investigated the effects of EVs isolated from NCCs on neuronal cell death and inflammation. Additionally, we overexpressed the nerve growth factor (NGF), which is involved in neural cell growth and proliferation, in NCCs to further investigate the effects of EVs containing NGF. NCCoe-NGF-EVs showed neuroprotective and regenerative properties by modulating inflammatory pathway, promoting Schwann cell activation, and enhancing remyelination. In vitro studies on NCCoe-NGF-EVs suppressed pro-inflammatory cytokines and reduced oxidative stress-induced neuronal apoptosis through NF-?B pathway inhibition and ERK, AKT signal activation. We also evaluated the effect of EVs on neuropathy by performing in vivo study. Our results suggest that NCCoe-NGF-EV had neuroprotective effects by reducing neuronal apoptosis and promoting neuronal proliferation based on neurite outgrowth and anti-inflammation effects treated with NCCoe-NGF-EVs. In addition, NCCoe-NGF-EVs were protected muscle loss caused by peripheral nerve injury. NCCoe-NGF-EV induced regeneration of damaged nerves and inhibited cell death through anti-inflammatory effects. This study suggests the potential of NGF-enriched EVs as non-cellular therapeutic platform for peripheral neuropathies and other neuroinflammatory disorders.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-025-02033-9.

The neuromuscular junction (NMJ) is essential for transmitting signals from motor neurons (MNs) to skeletal muscles (SKMs), and its dysfunction can lead to severe motor disorders. However, our understanding of the NMJ is limited by the absence of accurate human models. Although human induced pluripotent stem cell (iPSC)-derived models have advanced NMJ research, their application is constrained by challenges such as limited differentiation efficiency, lengthy generation times, and cryopreservation difficulties. To overcome these limitations, we developed a rapid human NMJ model using cryopreserved MNs and SKMs derived from iPSCs. Within 12 days of coculture, we successfully recreated NMJ-specific connectivity that closely mirrors in vivo synapse formation. Using this model, we investigated amyotrophic lateral sclerosis (ALS) and replicated ALS-specific NMJ cytopathies with SOD1 mutant and corrected isogenic iPSC lines. Quantitative analysis of 3D confocal microscopy images revealed a critical role of MNs in initiating ALS-related NMJ cytopathies, characterized by alterations in the volume, number, intensity, and distribution of acetylcholine receptors, ultimately leading to impaired muscle contractions. Our rapid and precise in vitro NMJ model offers significant potential for advancing research on NMJ physiology and pathology, as well as for developing treatments for NMJ-related diseases.