Figure 5. Optogenetic modulation of ESC neural differentiation.
(a-c) 3D image stacks of ChR2-ESCs after 5 days with (b, c) or without (a) optical stimulation. (a, b) Cells were incubated with 2.5 μM retinoic acid; as a negative control, in (c) no neural lineage inducing agents were added. Cells were stained for cellular nuclei (DAPI; blue fluorescence) and the neural marker nestin (red fluorescence). 3D reconstruction consists of 10 x-y sections, z-step size is 0.98 μm. Scale bars: 50 μm. (d - g) Summary of blinded experiments comparing response to optical stimulation of native ESCs (d, e) and ChR2-ESCs (f, g). (d, f) Histograms of nestin fluorescence intensities of optically-stimulated versus non-stimulated cells. All individual x-y sections of all embryoid body-containing ROIs in each condition were used to calculate the intensity histograms (8-bit images gave rise to 256 intensity bins). (d) Optical stimulation of native ESCs did not significantly affect nestin expression. (f) Increased nestin expression in the optically-driven ES cells; histograms were normalized revealing an optically-driven increased fraction of nestin-positive voxels, as evaluated by non-parametric statistical testing. DAPI histograms reflecting the number of cells did not reveal any significant changes upon light stimulation, no proliferative effect could be observed. (e, g) Summary data of mean nestin-positive voxels per embryoid body-containing ROI (+/- s.e.m., n = 10 ROIs, two independent experiments). (e) Optical stimulation of native ESCs did not significantly affect nestin expression. (g) Optically-stimulated ChR2-ESCs gave rise to significantly increased nestin expression in the presence of 2.5 μM RA (two tailed t-test, p=0.03). (h-i) Confocal images of ChR2-ESCs after 5 days with (i) or without (h) optical stimulation. Cells were incubated with 2.5 μM retinoic acid. Cells were stained for cellular nuclei (DAPI; blue fluorescence) and the neuronal marker β-3-tubulin (red fluorescence). Scale bars: 30 μm. (j-k) Normalized percentage of β-3-tubulin expressing cells incubated with 2.5 μM RA, two independent experiments per condition, mean +/- s.e.m. (j) No significant change in cell numbers upon optically stimulated native ESCs can be detected. (k) In contrast, stimulation of ChR2-ESCs results in a significant increase (P=0.01, two tailed t-test) of the number of β-3-tubulin expressing cells of about 25%.