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. 2019 May 2;12(5):934–949. doi: 10.1016/j.stemcr.2019.04.007

Figure 7.

Figure 7

Expansion of Functional MGE Progenitors via Chemical Activation of WNT/CTNNB1 Signaling

(A) Schematic of the procedures for long-term expansion of MGE progenitors and generation of functional INs was shown. CHIR99021 (2 μM) was maintained from day 17 to 55 or day 115, and MGE progenitors were passaged every 7 days in NIM medium. At day 55, the maintained MGE progenitors were plated on polyornithine-treated coverslips with 20 μg/mL laminin supplied with brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor in NDM. INs were obtained at 1 month after plating.

(B and C) At day 55, much more sphere-shaped MGE progenitors were shown in the CHIR99021-maintained culture conditions, and CHIR99021-maintained MGE progenitors showed increased populations of proliferative cells immunolabeled with Ki67 and PH3 compared with the DMSO-treated control (B). Scale bars, 500 μm (for bright field) and 50 μm (for fluorescence staining). Statistical results were shown in (C). n = 171–397 cells in at least three independent experiments; unpaired t test, ∗∗∗p < 0.001.

(D and E) At day 115, CHIR99021-maintained MGE progenitors showed increased populations of proliferative cells immunolabeled with Ki67 and PH3 compared with the DMSO-treated control (D). Scale bar, 50 μm. Statistical results are shown (E). n = 89–283 cells in at least three independent experiments; unpaired t test, ∗∗p < 0.01, ∗∗∗p < 0.001.

(F) One day after plating on laminin-coated culture surface, day 55-CHIR99021-maintained MGE progenitors generated neurons robustly, whereas the remaining spheres in the DMSO-treated group showed lower potentiality to generate neurons even when plated with equal cell numbers, as observed via TUJ1 immunostaining. Scale bars, 50 μm.

(G) Day 55-CHIR99021-maintained MGE progenitors generated INs at day 85 after plating, but DMSO-treated MGE progenitors showed minimized capability to generate these neurons. Scale bar, 50 μm.

(H and I) Whole-cell patch-clamp study was performed in the 120-day-old neurons differentiated from CHIR99021-treated MGE progenitors (H). Scale bar, 50 μm. Differentiated neurons showed functional action potentials, which were induced by injection of currents as low as 30 pA into neurons (I).

(J) Active spontaneous synaptic currents were captured in 120-day-old neurons differentiated from CHIR99021-treated MGE progenitors. These synaptic currents were completely blocked by bicuculline, an inhibitor of GABAa receptor.