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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.

SummaryKnowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete, yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress, we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNA-seq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens, aggrecan, perlecan, proteoglycans, and elastic fibers. We identified two populations of chondroprogenitor cells, mesenchyme cells and nascent chondrocytes, and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage, but also play a pivotal autocrine cell signaling role in determining chondrocyte fate. Graphical abstract Highlights•Craniofacial cartilage organoids were grown from human neural crest stem cells•These organoids exhibited elastic cartilage architecture and characteristic markers•Paracrine signaling drove chondrogenesis in mesenchyme cells and nascent chondrocytes•ECM components cemented chondrocyte cell fate through autocrine signaling Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Specialized functions of cells

SummaryHere, we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling.For complete details on the use and execution of this protocol, please refer to Foltz et al.1 Graphical abstract Highlights•Protocol for inducing hESCs or iPSCs to form neural crest stem cells (NCSCs)•Steps for differentiating NCSCs into craniofacial cartilage organoids•Instructions for preparing appropriate media and conditions for differentiation•Guidance for assessing changes in cell and organoid morphology during differentiation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling.