Chunk #23 — Results — Induction of neural stem cells (NSCs) from iPSCs and generation of human 2D neuronal culture and 3D neuro-spheroids (3DSs) from NSCs
We have further grown these neuronal stem cells in parallel into neuronal culture in two different environments, 2D neuronal culture (2D1-2D5, Fig 3) and 3D neuronal spheroids (3DS1-5, Figs 4 and 5) using a modified protocol [23]. To characterize these 2D and 3D neurons, we performed ICC to detect epitopes associated with immature and mature neurons. Confocal images from 2D neurons in five iPSC differentiated neuronal culture demonstrated protein marker expression of NeuN, GFAP, β tubulin III (BT3) and MAP2 (Fig 3). Serial sections from 3D neuronal spheroids (3DS1-3DS5; Figs 4 and 5, bright field (BF)) were stained with following antibodies: NeuN (green) and GFAP (red) (Fig 4), and MAP2 (green) and PAX6 (red; Fig 5). All of the sections from 3D neuronal spheroids exhibited staining patterns similar to that of the 2D cells (Fig 3). The presence of NeuN staining clearly indicated the withdrawal of the neurons from the cell cycle and the initiation of terminal differentiation of the neurons [30]. Almost all of neuronal stem cells were differentiated into neurons, as class III β-tubulin is a microtubule element
