Santos John Lenon de Souza, Paredes Bruno Diaz, Adanho Corynne Stephanie Ahouefa, Nonaka Carolina Kymie Vasques, da Silva Katia Nunes, Santos Ian Marinho, Loiola Erick Correia, Silva Viviane Aline Oliveira, Rocha Clarissa Araújo Gurgel, Souza Bruno Solano de Freitas
Gonçalo Moniz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Salvador, Brazil.
Center for Biotechnology and Cell Therapy, São Rafael Hospital, Salvador, Brazil.
Hum Cell. 2025 Mar 20;38(3):74. doi: 10.1007/s13577-025-01199-7.
Autism spectrum disorders (ASD) comprise a group of complex neurodevelopmental disorders that affect communication and social interactions. Over a thousand genes have been associated with ASD, with SCN2A standing out due to its critical role in neuronal function and development. Induced pluripotent stem cells (iPSCs) derived from individuals with ASD have become invaluable in vitro models for investigating the cellular and molecular mechanisms underlying the disorder. In this study, we generated and characterized four iPSC clones from peripheral blood mononuclear cells (PBMCs) of two ASD patients carrying loss-of-function variants in the SCN2A gene. These iPSC lines underwent comprehensive characterization through multiple assays. Reverse transcription polymerase chain reaction (RT-PCR), flow cytometry, and immunofluorescence analyses confirmed the presence of pluripotency markers. An embryoid body formation assay demonstrated their potential to differentiate into the three germ layers. Sequencing analysis confirmed the SCN2A variants, while short tandem repeat (STR) analysis authenticated the cell lines, and karyotype analysis ensured chromosomal integrity. The iPSCs exhibited typical morphologic characteristics, including large nuclei with prominent nucleoli, a high nucleus-to-cytoplasm ratio, densely packed cells, and well-defined borders. These cells maintained pluripotency markers, demonstrated the ability to differentiate into the three germ layers, and showed a normal karyotype. Furthermore, we successfully generated cerebral organoids from these cells. Our study establishes a robust platform for further exploration of the pathophysiological mechanisms of ASD, particularly those involving SCN2A.
自闭症谱系障碍（ASD）是一组影响沟通和社交互动的复杂神经发育障碍。已有一千多种基因与ASD相关，其中SCN2A因其在神经元功能和发育中的关键作用而备受关注。源自ASD患者的诱导多能干细胞（iPSC）已成为研究该疾病潜在细胞和分子机制的宝贵体外模型。在本研究中，我们从两名携带SCN2A基因功能丧失变异的ASD患者的外周血单个核细胞（PBMC）中生成并鉴定了四个iPSC克隆。这些iPSC系通过多种检测方法进行了全面表征。逆转录聚合酶链反应（RT-PCR）、流式细胞术和免疫荧光分析证实了多能性标志物的存在。胚状体形成试验证明了它们分化为三个胚层的潜力。测序分析证实了SCN2A变异，短串联重复序列（STR）分析验证了细胞系，核型分析确保了染色体完整性。这些iPSC表现出典型的形态学特征，包括核大、核仁突出、核质比高、细胞密集且边界清晰。这些细胞维持多能性标志物，证明了分化为三个胚层的能力，并显示出正常的核型。此外，我们成功地从这些细胞中生成了脑类器官。我们的研究建立了一个强大的平台，用于进一步探索ASD的病理生理机制，特别是那些涉及SCN2A的机制。
