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  • Induced pluripotent stem cells from patients with focal cortical dysplasia and refractory epilepsy.

Induced pluripotent stem cells from patients with focal cortical dysplasia and refractory epilepsy.

Molecular medicine reports (2017-03-06)
Daniel Rodrigo Marinowic, Fernanda Majolo, Alessandra Deise Sebben, Vinicius Duval da Silva, Tiago Giuliani Lopes, Eliseu Paglioli, André Palmini, Denise Cantarelli Machado, Jaderson Costa da Costa
ABSTRACT

Focal cortical dysplasia (FCD) is caused by numerous alterations, which can be divided into abnormalities of the cortical architecture and cytological variations; however, the exact etiology of FCD remains unknown. The generation of induced pluripotent stem cells (iPSCs) from the cells of patients with neurological diseases, and their subsequent tissue‑specific differentiation, serves as an invaluable source for testing and studying the initial development and subsequent progression of diseases associated with the central nervous system. A total of 2 patients demonstrating seizures refractory to drug treatment, characterized as FCD Type IIb, were enrolled in the present study. Fibroblasts were isolated from residual skin fragments obtained from surgical treatment and from brain samples obtained during surgical resection. iPSCs were generated following exposure of fibroblasts to viral vectors containing POU class 5 homeobox 1 (OCT4), sex determining region Y‑box 2 (SOX2), Kruppel‑like factor 4 and c‑MYC genes, and were characterized by immunohistochemical staining for the pluripotent markers homeobox protein NANOG, SOX2, OCT4, TRA1‑60 and TRA1‑81. The brain samples were tested with antibodies against protein kinase B (AKT), phosphorylated‑AKT, mechanistic target of rapamycin (mTOR) and phosphorylated‑mTOR. Analysis of the AKT/mTOR pathway revealed a statistically significant difference between the cerebral tissues of the two patients, which were of different ages (45 and 12 years old). Clones with the morphological features of embryonic cells were detected on the 13th day and were characterized following three subcultures. The positive staining characteristics of the embryonic cells confirmed the successful generation of iPSCs derived from the patients' fibroblasts. Therefore, the present study presents a method to obtain a useful cellular source that may help to understand embryonic brain development associated with FCD.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-TRA-1-60 Antibody, clone TRA-1-60, Cy3 conjugate, clone TRA-1-60, from mouse, CY3 conjugate
Sigma-Aldrich
Anti-OCT-4 [POU5F1] Antibody, clone 7F9.2, Alexa Fluor 488 conjugate, clone 7F9.2, from mouse, ALEXA FLUOR 488
Sigma-Aldrich
Anti-TRA-1-81 Antibody, clone TRA-1-81, Cy3 conjugate, clone TRA-1-81, from mouse, CY3 conjugate
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Anti-NANOG Antibody, clone 7F7.1, Alexa Fluor 488 conjugate, clone 7F7.1, from mouse, ALEXA FLUOR 488
Sigma-Aldrich
Anti-NeuN Antibody, from chicken, purified by affinity chromatography