Figure 2.
Validation of neuronal sphere cultures as a readout for health and activity. (A) iNeurons expressing a genetically encoded calcium indicator (GCaMP6) were cocultured with Astrostim (10:1) for 9 d. After subsequent chronic CNO treatment (12–14 d), calcium imaging was performed to measure spontaneous oscillations. Scale bar: 50 µm; inset: 10 µm. (B) Representative calcium trace of a control (non–CNO treated) coculture (top), presented as a ratio of fluorescence intensity (F) to maximum intensity. Average frequency of intracellular calcium oscillations (bottom) of control and CNO-treated cocultures (n = 2–4 cells or ROIs from three to five videos or cocultures per group). (C) Timeline of differentiation of OptoNeurons from hPSCs (top). Large-scale formation of OptoNeuron sphere cultures (with YFP-fused ChR2) was achieved using microwell plates (bottom). Dissociated cells compacted over time to form spheres. Scale bars: 200 µm. (D) Neural spheres formed with microwell (µ-well) plates allowed for customizable cellular density and demonstrated more consistent shape (circularity, with a value of 1 representing a circle; left) and size (right) compared with cultures which were allowed to self-aggregate (n = 6–33 spheres each). (E) RNAseq of day-14 OptoNeuron sphere cultures confirmed significant upregulation of neuronal-restricted genes compared to day-12 iAstros (n = 3; for Padj ≤ 0.05, FPKM > 1); see Data S3. (F) OptoNeuron spheres were attached to microelectrode arrays (MEAs) for live electrophysiological recordings. Shown are two merged spheres on an MEA (left). OptoNeurons on MEAs elicited voltage spikes after acutely induced blue light optical stimulation (5 ms pulse width) at increasing frequencies (right; see Fig. S3). Scale bar: 200 µm. (G) Viability (n = 7; measured by a CellTiter-Glo 3D Assay and normalized to sphere area; left) and spontaneous (middle) and blue light–evoked (right) activity of day-21 OptoNeuron spheres (n = 7–41 electrodes each, after threshold) under different media conditions, including neural media NM or BP, with or without ascorbic acid and BDNF/GDNF growth factors (GF). Results are shown as mean ± SEM. In D and G, dots represent individual spheres or electrodes. For G, significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test, with *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.

Validation of neuronal sphere cultures as a readout for health and activity. (A) iNeurons expressing a genetically encoded calcium indicator (GCaMP6) were cocultured with Astrostim (10:1) for 9 d. After subsequent chronic CNO treatment (12–14 d), calcium imaging was performed to measure spontaneous oscillations. Scale bar: 50 µm; inset: 10 µm. (B) Representative calcium trace of a control (non–CNO treated) coculture (top), presented as a ratio of fluorescence intensity (F) to maximum intensity. Average frequency of intracellular calcium oscillations (bottom) of control and CNO-treated cocultures (n = 2–4 cells or ROIs from three to five videos or cocultures per group). (C) Timeline of differentiation of OptoNeurons from hPSCs (top). Large-scale formation of OptoNeuron sphere cultures (with YFP-fused ChR2) was achieved using microwell plates (bottom). Dissociated cells compacted over time to form spheres. Scale bars: 200 µm. (D) Neural spheres formed with microwell (µ-well) plates allowed for customizable cellular density and demonstrated more consistent shape (circularity, with a value of 1 representing a circle; left) and size (right) compared with cultures which were allowed to self-aggregate (n = 6–33 spheres each). (E) RNAseq of day-14 OptoNeuron sphere cultures confirmed significant upregulation of neuronal-restricted genes compared to day-12 iAstros (n = 3; for Padj ≤ 0.05, FPKM > 1); see Data S3. (F) OptoNeuron spheres were attached to microelectrode arrays (MEAs) for live electrophysiological recordings. Shown are two merged spheres on an MEA (left). OptoNeurons on MEAs elicited voltage spikes after acutely induced blue light optical stimulation (5 ms pulse width) at increasing frequencies (right; see Fig. S3). Scale bar: 200 µm. (G) Viability (n = 7; measured by a CellTiter-Glo 3D Assay and normalized to sphere area; left) and spontaneous (middle) and blue light–evoked (right) activity of day-21 OptoNeuron spheres (n = 7–41 electrodes each, after threshold) under different media conditions, including neural media NM or BP, with or without ascorbic acid and BDNF/GDNF growth factors (GF). Results are shown as mean ± SEM. In D and G, dots represent individual spheres or electrodes. For G, significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test, with *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.

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