Study of neurodevelopment in focal cortical dysplasia using Induced. Pluripotent Stem Cells (iPSC). Marinowic, D.R.1; Majolo, F.2; Sebben, A.D.1; Silva, V. D.3; ...
Study of neurodevelopment in focal cortical dysplasia using Induced Pluripotent Stem Cells (iPSC) Marinowic, D.R.1; Majolo, F.2; Sebben, A.D.1; Silva, V. D.3; Plentz, I.1; Pallamolla, G. P.1; Paglioli, E.4; Pamini, A.4; Machado, D.C.1,2; Da Costa, J.C.1 1Brain
Institute of PUCRS; 2Biomedical Research Institute of PUCRS; 3Pathological Anatomy Laboratory of PUCRS; 4Epilepsy Surgery Program of PUCRS
INTRODUCTION The focal cortical dysplasia (FCD) is one of the most frequent forms of cortical development malformation that encompasses multiple types of changes both in the cortical architecture and cytological abnormalities. It is an underlying pathology of a significant proportion of partial epilepsy refractory to drug treatment. Especially the limited number of cases and the lack of suitable experimental models rarely document the mechanisms involved with the genesis of FCD. The generation of iPSC and the differentiation into specific cells and tissues will provide important testing and an unique ability to study the development and progress of CNS diseases.
OBJECTIVE The aim of this study was to evaluate neurodevelopmental alterations on iPSC from skin fibroblasts from patients with focal cortical dysplasia.
METHODS PRIMARY CULTURE
REPROGRAMMING
DIFFERENTIATION
iPSC
Obtaintion of fibroblast
Cell migration assay
Characterization
Histology (anatomy + pathways)
RNA extraction
RNA extraction
Cell migration assay
Vector exposure
Passage to matrigel
Molecular analysis
Data analysis
REPROGRAMMING PROTOCOL (iPSC)
Clones selection an picking
ANALYSIS
NEURODIFFERENTIATION PROTOCOL
Clones maintenance
Culture in neuronal induction medium
RNA extraction
RNA extraction
RESULTS
Imunofluorescence
RNA extraction
Both patients were diagnosed with FCD type IIb (Fig. 1,2 and 3). Clones with morphological features of embryonic cells could be detected on the 13th day after viral transfection (Fig. 4). The features of embryonic cells were further confirmed after three subcultures of the clones over Matrigel with antibodies against the pluripotency markers Nanog, SOX2, OCT4, TRA1-60, and TRA1-81 (Fig. 5). The AKT/mTOR phosphorylated and non-phosphorylated was higher in brain sections from patient #1 (Fig. 6). After neurodifferentiation, iPSC from patients and controls showed polarization and morphology similar to nerve cells (Fig. 7 and 8). In the cell migration assay, fibroblasts derived from FCD migrate with greater intensity within 24 hours (p
