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Background and Objectives: Recent studies have identified variants in the kinesin family member 5A (KIF5A) gene that predispose to amyotrophic lateral sclerosis (ALS). These ALS-linked KIF5A variants lead to the exclusion of exon 27, resulting in the production of a mutated protein with an altered C-terminal region (KIF5A ΔExon27). Through whole genome sequencing, we identified a novel KIF5A intronic variant, rs1057522322 (c.2993-6C > A; chr12:57582596C > A, GRCh38.p14), in a family segregating ALS. Our goal is to investigate the effect of this variant on exon 27 splicing and to assess its functional consequences on KIF5A-mediated cargo transport. Methods: Induced pluripotent stem cells (iPSCs) were generated from siblings with and without the c.2993-6C > A variant. RT-PCR was performed on RNA extracted from iPSC-derived neurons to assess exon 27 splicing. Functional studies were conducted on iPSC-derived motor neurons (MNs). Results: RT-PCR confirmed that the c.2993-6C > A variant induced exon 27 skipping in KIF5A. Immunofluorescent staining showed that KIF5A ΔExon27 abolished the axonal interaction with splicing factor proline- and glutamine-rich, a cargo specifically transported by KIF5A. Under stress conditions, MNs carrying the c.2993-6C > A variant exhibited TDP-43 proteinopathy. Discussion: KIF5A intronic variant c.2993-6C > A could be a risk factor for ALS. KIF5A ΔExon27 impairs KIF5A-mediated cargo transport and contributes to ALS pathogenesis in a TDP-43–dependent manner.

Respiratory syncytial virus (RSV) is one of the major causes of lower respiratory tract infections, particularly in infants and older adults. However, the host factors mediating infection remain poorly defined. It has been suggested that four host surface proteins, namely intercellular adhesion molecule-1 (ICAM-1), epidermal growth factor receptor (EGFR), nucleolin (NCL), and insulin-like growth factor 1 receptor (IGF1R), may interact with the RSV fusion (F) protein. To investigate these roles under physiologically relevant conditions, we employed human induced pluripotent stem cell (iPSC)-derived respiratory organoids as a model for RSV infection. In this model, ICAM-1 and EGFR were genetically depleted using the CRISPR-Cas9 genome editing technique, while NCL and IGF1R were inhibited with neutralizing antibodies. Suppression of ICAM-1 or NCL significantly reduced RSV nucleoprotein gene expression, whereas inhibition of EGFR or IGF1R had no observable effect on viral gene expression. Notably, simultaneous suppression of ICAM-1 and NCL resulted in a more substantial reduction in infectious viral titers and RSV F protein expression than inhibition of either protein alone. Our results suggest that both ICAM-1 and NCL may play important roles during RSV infection in human iPSC-derived respiratory organoids. Graphical abstract Takayama and colleagues used human induced pluripotent stem cell-derived respiratory organoids to investigate host factors that facilitate respiratory syncytial virus (RSV) entry. They identified intercellular adhesion molecule-1 (ICAM-1) and nucleolin (NCL) as important host factors, demonstrating that simultaneous suppression of both markedly reduces infection efficiency.

Long-term imaging formats are ideal for capturing dynamic neuronal network formation in vitro, yet fluorescent techniques are often constrained by the impact of phototoxicity on cell survival. Here we present a live-imaging protocol that was optimised via quantitative analysis of 3 target culturing conditions on neuromorphological health: extracellular matrix (human- versus murine-derived laminin), culture media (Neurobasal™ versus Brainphys™ Imaging media), and seeding density (1 × 105 versus 2 × 105 cells/cm2). A cortical neuron reporter line was differentiated from human embryonic stem cells by transduction of Neurogenin-2 and green fluorescent protein, then fluorescently imaged in 8 different microenvironments daily for 33 days. Alongside viability analysis by PrestoBlue assay and gene quantification by digital polymerase chain reaction, an automated image analysis pipeline was developed to characterise network morphology and organisation over time. Brainphys™ Imaging medium was observed to support neuron viability, outgrowth, and self-organisation to a greater extent than Neurobasal™ medium with either laminin type, while the combination of Neurobasal™ medium and human laminin reduced cell survival. Further, a higher seeding density fostered somata clustering, but did not significantly extend viability compared to low density. These findings suggest a synergistic relationship between species-specific laminin and culture media in phototoxic environments, which is positively mediated by light-protective compounds found in Brainphys™ Imaging medium.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04591-0.