Induction of neuronal differentiation by transient transfection with <i>Ngn2</i> in complete stem cell growth medium with LIF.

<p>(A-D) Wide field scans of Tuj1 staining 5 days post transfection (dpt). Dark field view (A, B) showing Tuj1 positive cells in <i>Ngn2</i>-transfected cells (A), but not in mock-transfected cells (B). Corresponding phase contrast images (C,D) reveal the presence of stem cell like colonies (arrows). Scale bars: 200 µm. (E-H) Close-up views of developing neurons 5dpt expressing Tuj1 (E,F) and Map2ab (G,H). Scale bars: 20 µm. (I-N) Loss of Nanog expression (arrowheads) 3dpt in <i>Ngn2</i>-transfected (I,K,M), but not in mock-transfected cells (J,L,N). Transfected cells are visualized by expression of cotransfected GFP (I,J). Scale bars: 20 µm. (O) Gene expression pattern of untreated (ut), <i>Ngn2</i>-transfected (d5+,d7+), and mock-transfected (d5-,d7-) mESCs 5 and 7dpt. b: Brain cDNA. Dashed line indicates grouping of different parts from the same gel. A representative result from three independent experiments is shown. (P) Tuj1 positive cells in <i>Ngn2</i>-transfected and mock-transfected cells 5 and 7dpt. Absolute numbers are shown as non-differentiating cells continue proliferating. Therefore, the relative number would not really reflect the increase of neurons upon <i>Ngn2</i> compared to mock transfection. Mean numbers +/− SD of three independent experiments are shown.