Fig 1.
GSK3β inhibition by CHIR 99021 during neuronal differentiation affects neurodevelopmental signaling pathways and organoid growth.
A. Schematic of organoid production protocol. Dual SMAD inhibition was performed during the neural induction step. GSK3β inhibitor CHIR 99021 (1μM, 10μM or 50μM), or vehicle DMSO, was added to the culture media throughout neuronal differentiation (day 14 and onward). B. Representative organoids at day 35 of organoid development; organoids treated with 50μM CHIR 99021 failed to grow. C. Quantification of organoid size. CHIR 99021 treatment had a dose-dependent effect on organoid size (DMSO: 5.856 ± 0.7229, n = 12; 1μM: 9.472 ± 0.8797, n = 12; 10μM: 3.294 ± 0.3062, n = 11; DMSO vs 1μM, **: p-value = 0.0044; DMSO vs 10μM, **: p-value = 0.0047; 1μM vs 10μM, p-value<0.0001; unpaired t-tests). Bar graphs represent mean ± SEM.
Fig 2.
GSK3β inhibition by CHIR 99021 decreases apoptosis, and at high dose, but not low dose, decreases proliferation.
A. Representative images of immunostained organoids. The apoptosis marker cleaved caspase 3 (c-Cas3) and proliferation marker KI67 were immunolabeled. B. Quantification of the c-Cas3 or KI67 positive areas normalized by the DAPI positive area for each organoid (c-Cas3: DMSO: 1 ± 0.1972, n = 27; 1μM: 0.7968 ± 0.1147, n = 32; 10μM: 0.3282 ± 0.04979, n = 15; DMSO vs 1μM, p-value = 0.3592, DMSO vs 10μM, *: p-value = 0.0185. KI67: DMSO: 0.8857 ± 0.1606, n = 21; 1μM: 1.508 ± 0.2367, n = 27; 10μM: 0.2625 ± 0.05119, n = 15; DMSO vs 1μM, *: p-value = 0.0463, t-test; DMSO vs μM, **: p-value = 0.0031; unpaired t-test). Bar graphs represent mean ± SEM.
Fig 3.
GSK3β inhibition by CHIR 99021 affects neuronal differentiation of neuroepithelium in a dose dependent manner-Western Blots.
A. Western Blot bands and B. quantification of neuroepithelium marker E-cadherin (E-Cad), neural progenitor cells (NPCs) marker SOX2, radial glia marker BLBP, intermediate progenitor marker TBR2, and neuronal markers TUJ1 and DCX, relative to DMSO control (n = 5 batches of organoid production for the three groups. E-cadherin: DMSO: 1 ± 0.0891; 1μM: 0.9285 ± 0.2585, p-value = 0.2881; 10μM: 1.819 ± 0.5351, *: p-value = 0.0286; SOX2: DMSO: 1 ± 0.09242; 1μM: 1.472 ± 0.1668, *:p-value = 0.0125; 10μM: 0.5228 ± 0.05321, *:p-value = 0.0405; BLBP: DMSO: 1 ± 0.01525; 1μM: 2.069 ± 0.7181, *:p-value = 0.0286; 10μM: 0.6944 ± 0.1048, p-value = 0.8825; TBR2: DMSO: 1 ± 0.1263; 1μM: 1.141 ± 0.09633, p-value = 0.4000; 10μM: 2.884 ± 0.3168, ***: p-value = 0.0006; TUJ1: DMSO: 1 ± 0.1075; 1μM: 0.9262 ± 0.04413, p-value = 0.5429; 10μM: 0.7731 ± 0.0886, p-value = 0.0823; DCX: DMSO: 1 ± 0.2530; 1μM: 1.864 ± 0.4717, p-value = 0.1449; 10μM: -1.516 ± 0.0886, p-value = 0.0044, unpaired t-tests).
Fig 4.
GSK3β inhibition by CHIR 99021 affects neuronal differentiation of neuroepithelium in a dose dependent manner-IHC.
A. Representative images of immune-stained organoids for detection of NPC marker PAX6, neuronal markers DCX and TUJ1, intermediate progenitor marker TBR2, and B. neuroepithelial marker E-cadherin. C. Quantification of the cell type marker positive areas normalized by the DAPI positive area for each organoid, and relative to DMSO organoids (E-Cad: DMSO: 1 ± 0.5046, n = 3; 1μM: 0.8878 ± 0.3554, n = 3; 10μM: 92.47± 11.34, n = 2; DMSO vs 1μM, **: p-value = 0.8646; DMSO vs 10μM, **: p-value = 0.0040. PAX6: DMSO: 1 ± 0.2189, n = 5; 1μm: 1.834 ± 0.1585, n = 7; 10μM: 0.5 ± 0.09146, n = 2; DMSO vs 1μM, **: p-value = 0.0099; DMSO vs 10μM, p-value = 0.2339. TBR2: DMSO: 1 ± 0.2185, n = 6; 1μM: 2.2 ± 0.7904, n = 5; 10μM: 5.5 ± 1.471, n = 5; DMSO vs 1μM, p-value = 0.1456; DMSO vs 10μM, **: p-value = 0.0087; DCX: DMSO: 1 ± 0.3027, n = 5; 1μM: 1.078 ± 0.1477, n = 7; 10μM: 0.1283± 0.2005, n = 2; DMSO vs 1μM, p-value = 0.8046; DMSO vs 10μM, *: p-value = 0.0449; TUJ1: DMSO: 1 ± 0.1175, n = 6; 1μM: 1.17 ± 0.3217, n = 5; 10μM: 1.512 ± 0.3407, n = 5; DMSO vs 1μM, p-value = 0.6026; DMSO vs 10μM, p-value = 0.1583; unpaired t-test.). Bar graphs represent mean ± SEM.
Fig 5.
GSK3β inhibition by CHIR 99021 increased neuronal migration.
A. Representative images of GFP expressing cells 7 days after injection and electroporation of the GFP expression plasmid, with immunostaining of proliferative cell marker KI67 and neuronal marker DCX. B. Quantification of the percentage of neurons (DCX+, KI67- cells) (DMSO: 91.33% ± 8.083, n = 3; 1μM: 93.33% ± 5.508, n = 3; p-value = 0.7411, unpaired t-test) C. GFP positive area, distance and maximum distance (in gray) from injection ventricle were measured. D. Quantification of cell distance from injected ventricle (DMSO: 127.2±6.189, n = 135; 1μM: 184.9±8.041, n = 157; ****: p-value<0.0001, Welch’s t-test). E. Quantification of maximum distance from injected ventricle (DMSO: 165.4±22.58, n = 11; 1μM: 295.5±21.81, n = 26; ***: p-value = 0.0003, Welch’s t-test). F. Quantification of area occupied by GFP positive cells (DMSO: 2435±616.5, n = 14; 1μM: 7379±1285, n = 33; **: p-value = 0.0011, Welch’s t-test). Bar graphs represent mean ± SEM.