NT2 neurons support network activity.

<p><b>A</b>. Centre image shows neuronal cell bodies and processes. Plots of fluorescence over time from the circled areas are displayed to the right and below. Black traces illustrate response to electrical stimulation in control conditions and grey traces following TTX application. <b>B</b>. Bargraphs summarise calcium elevation responses of neurons and astrocytes for a number of experiments. Scale bar in A, 5 mV, 2 ms. Cells were used within 14 days of differentiation.</p

Stem cell derived neurons and astrocytes display spontaneous activity.

<p><b>A</b>. Image on the left displays an NT2N neuronal aggregate with extending processes. The white dotted area is displayed expanded to the right at indicated time points during the experiment. White arrows indicate processes that become active during acquisition. Red numbered circles indicate exemplar active cells with corresponding fluorescence timecourse displayed below. Neurons can display synchronised activity (1,3). Propagating activity from distant processes can also be detected (4). <b>B</b>. Image on the left displays an NT2N-A coculture. Spontaneously active astrocytes are circled and numbered. White dotted area is displayed expanded to the right at indicated time points during the experiment. White arrows indicate active astrocytes. Fluorescence time courses from the astrocytes are illustrated below, indicating regular calcium oscillations. <b>C</b>. Autocorrelation analysis of neurons from experiment A and astrocytes from experiment B. Scale bars A-1-4: 2, 0.5, 10, 10%. B-1-4: 5, 5,10, 10%. Cells were used within 14 days of differentiation.</p

Functional annotational clustering of genes enriched in NT2.D1 neurospheres following treatment with LiCl.

<p>Metacore™ (GeneGo) was used for this analysis using genes that were statistically significant p<0.05. Pathways with a p-value <0.05 were considered significantly modulated.</p

Calcium wave propagation in astrocytic networks.

<p><b>A</b>. Images showing a field in a pure NT2 astrocyte culture loaded with Fluo-4. Following image at t = 0 s, a mechanical stimulation was applied at point indicated by orange triangle tip. Subsequent images display the progression of a calcium wave in surrounding astrocytes. <b>B</b>. The time course of fluorescence in the circled astrocytes at different distances from the origin. <b>C</b>. Plot of wave distance with time for a number of experiments fitted with a linear regression. <b>D</b>. Maximum projection images showing extent of wave propagation in control, with PPADS and following PPADS wash off. <b>E</b>. Bargraphs summarising a number of experiments where fluorescence was measured in astrocytes during wave propagation, and the effect of CBX and PPADS. Cells were used within 14 days of differentiation.</p

Functional annotational clustering of genes enriched in NT2.D1 neurospheres following treatment with VPA.

<p>Metacore™ (GeneGo) was used for this analysis using genes that were statistically significant p<0.05. Pathways with a p-value <0.05 were considered significantly modulated.</p

Proportion of cells expressing SSEA4, Oct4, GFAP and NFM following treatment with VPA+RA and LiCl+RA measured using flow cytometry.

<p>Number of cells expressed as a percentage of total number of cells A) Undifferentiated NT2.D1 cells, B) RA, C) LiCl (1 mM) and D) VPA (0.5 mM). Results are shown as ±S.E.M (n = 3). p<0.05(*), p<0.01(**), p<0.001(***). p values were calculated by One-way ANOVA and corrected for multiple comparisons by Bonferroni’s multiple comparison post-test in comparison to the RA only control.</p