Figure 3.
Cell-specific activation of astrocytes within all-inducible bioengineered organoids. (A) Differentiated iAstros or Astrostim cells were cocultured with OptoNeurons. (B) Neural spheres have genetically encoded capabilities for cell-selective optogenetic (neuronal) or chemogenetic (astrocyte-specific) activation. Cocultures exhibited cell-restricted markers including S100 (see Video 3). Scanning electron microscopy scale: 100 µm; inset: 10 µm. S100/ChR2/DAPI scale and inset: 200 µm. (C) Spheres containing day-22 OptoNeurons or cocultured with iAstros (1:1) exhibited significantly increased firing rates when exposed to 40 Hz blue light stimulation (denoted as +) on MEAs (n = 6–19 electrodes each, after threshold). OptoNeurons, Coculture, and iAstros data are shown left to right in the plots. (D) Astrostim-OptoNeuron coculture spheres (1:1,000), visualized with YFP-fused ChR2 and stained for S100, with (bottom) and without (top) 2-d CNO treatment. Shown are 1-µm slices (left) and maximum projections (133 µm, right). Scale: 50 µm. See Video 6. (E) Chronic CNO treatment of Astrostim cells for up to 18 d did not significantly affect viability of cocultured OptoNeurons (1:10) compared with controls, as measured by a CellTiter-Glo 3D Assay. Luminescence readings were normalized to sphere cross-sectional areas (n = 12–24 spheres each). (F) A significant increase in secreted THBS1 protein was observed in conditioned media from coculture spheres with 2-d CNO treatment compared with controls, as measured by ELISA (n = 3 each). (G) Spontaneous and light-induced response of OptoNeuron–Astrostim cocultures (10:1) on MEAs under basal conditions (NM). 11-d chronic treatment of cocultures resulted in significantly decreased activity compared with controls (n = 25–32 electrodes each, postthreshold). (H) Spontaneous and light-induced response of OptoNeuron–Astrostim cocultures (10:1) on MEAs under optimal conditions (BP+GF). Chronic 16-d CNO treatment did not significantly alter light-induced firing rates (n = 64–84 electrodes each, after threshold; left). Representative traces are shown (right). Results are shown as mean ± SEM. For F, significance was determined using a two-tailed unpaired t test. In C, E–H, dots represent individual electrodes or spheres. For C, G, and H, significance was determined using a two-tailed paired t test to compare spontaneous to light-induced firing rates within the same groups only. For G and H, significance was determined using a one-way ANOVA followed by a Šidák post hoc test for multiple comparisons between spontaneous or light-induced firing rates across groups, with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Cell-specific activation of astrocytes within all-inducible bioengineered organoids. (A) Differentiated iAstros or Astrostim cells were cocultured with OptoNeurons. (B) Neural spheres have genetically encoded capabilities for cell-selective optogenetic (neuronal) or chemogenetic (astrocyte-specific) activation. Cocultures exhibited cell-restricted markers including S100 (see Video 3). Scanning electron microscopy scale: 100 µm; inset: 10 µm. S100/ChR2/DAPI scale and inset: 200 µm. (C) Spheres containing day-22 OptoNeurons or cocultured with iAstros (1:1) exhibited significantly increased firing rates when exposed to 40 Hz blue light stimulation (denoted as +) on MEAs (n = 6–19 electrodes each, after threshold). OptoNeurons, Coculture, and iAstros data are shown left to right in the plots. (D) Astrostim-OptoNeuron coculture spheres (1:1,000), visualized with YFP-fused ChR2 and stained for S100, with (bottom) and without (top) 2-d CNO treatment. Shown are 1-µm slices (left) and maximum projections (133 µm, right). Scale: 50 µm. See Video 6. (E) Chronic CNO treatment of Astrostim cells for up to 18 d did not significantly affect viability of cocultured OptoNeurons (1:10) compared with controls, as measured by a CellTiter-Glo 3D Assay. Luminescence readings were normalized to sphere cross-sectional areas (n = 12–24 spheres each). (F) A significant increase in secreted THBS1 protein was observed in conditioned media from coculture spheres with 2-d CNO treatment compared with controls, as measured by ELISA (n = 3 each). (G) Spontaneous and light-induced response of OptoNeuron–Astrostim cocultures (10:1) on MEAs under basal conditions (NM). 11-d chronic treatment of cocultures resulted in significantly decreased activity compared with controls (n = 25–32 electrodes each, postthreshold). (H) Spontaneous and light-induced response of OptoNeuron–Astrostim cocultures (10:1) on MEAs under optimal conditions (BP+GF). Chronic 16-d CNO treatment did not significantly alter light-induced firing rates (n = 64–84 electrodes each, after threshold; left). Representative traces are shown (right). Results are shown as mean ± SEM. For F, significance was determined using a two-tailed unpaired t test. In C, E–H, dots represent individual electrodes or spheres. For C, G, and H, significance was determined using a two-tailed paired t test to compare spontaneous to light-induced firing rates within the same groups only. For G and H, significance was determined using a one-way ANOVA followed by a Šidák post hoc test for multiple comparisons between spontaneous or light-induced firing rates across groups, with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal