Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Kraków, Poland.
Department of Neurology, Jagiellonian University Medical College, Krakow, Poland.
Methods Mol Biol. 2024;2835:135-146. doi: 10.1007/978-1-0716-3995-5_12.
Disease modeling of neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), is hindered by limited accessibility of affected cells. This problem can be overcome by generation of human induced pluripotent stem cells (hiPSC), which can be then differentiated into required cells. Here, we describe the detailed protocol of hiPSC establishment from peripheral blood mononuclear cells (PBMC) of two ALS patients with detected expansion of G4C2 (GGGGCC) repeats in the first intron of C9ORF72 gene, known to be linked with the most common form of familial ALS.Successful PBMC reprogramming with non-integrating Sendai vectors was confirmed by expression of pluripotency markers: OCT4, NANOG, SSEA4, and TRA-1-60 in obtained hiPSC and their ability to differentiate into cells of three germ layers.The generated ALS-patient-specific hiPSC create a possibility for deciphering molecular basis of this devastating neuromuscular disease.
神经肌肉疾病（如肌萎缩侧索硬化症，ALS）的疾病建模受到受影响细胞有限可及性的阻碍。通过生成人诱导多能干细胞（hiPSC）可以克服这个问题，然后可以将其分化为所需的细胞。在这里，我们描述了从两位 ALS 患者的外周血单核细胞（PBMC）中建立 hiPSC 的详细方案，这些患者的 C9ORF72 基因第一内含子中检测到 G4C2（GGGGCC）重复扩增，已知与最常见的家族性 ALS 形式有关。通过表达多能性标志物证实了非整合性 Sendai 载体对 PBMC 的成功重编程：在获得的 hiPSC 中 OCT4、NANOG、SSEA4 和 TRA-1-60，以及它们分化为三个胚层细胞的能力。生成的 ALS 患者特异性 hiPSC 为破译这种毁灭性神经肌肉疾病的分子基础创造了可能性。
