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. 2013 Jul 23;4:2196. doi: 10.1038/ncomms3196

Figure 3. Characterization of Olig2PC-Astros and NPC-Astros.

Figure 3

(a) Left panels, representative Olig2PC-Astros and NPC-Astros cultured in adhesion. Notice the different morphology of the two types of astroglial cells. Middle panels, representative Olig2PC-Astros and NPC-Astros cultured in suspension. Right panels, representatives showing that both Olig2PC-Astros and NPC-Astros are supportive for the growth of hESC-derived neurons. Scale bars represent 250 μm. (b–d) The expression of HOXB4, OTX2 and NKX2.1 in S100β+ Olig2-Astros and NPC-Astros. Blue, DAPI-stained nuclei. Scale bars represent 50 μm. (e) Quantification of HOXB4-, OTX2- and NKX2.1-expressing cells in Olig2-Astros and NPC-Astros (n=4). (f) Quantitative analysis of the proliferation rate of Olig2PC-Astro and NPC-Astros relative to HEK 293 cells. Notice that NPC-Astros proliferate more robustly than Olig2PC-Astros when cultured with either BMP4 or FBS (n=4). (g) Quantitative PCR analysis of GLAST and GLT-1 mRNA expression in Olig2PC-Astros and NPC-Astros (n=3). (h) Glutamate uptake analysis showing that both types of astroglia were capable of glutamate uptake. Notice that NPC-Astros showing glutamate uptake at a higher rate at the 30-min time point than Olig2PC-Astros (n=4). This uptake capability is Na+-dependent and can be abolished by the Na+-free solution. (i) Voltage steps (clamped at −70 mV and stepped from −130 to +60 mV at 10 mV increments for 300 ms) induced outward currents in both Olig2PC-Astros and NPC-Astros. A small inward current is detected in some Olig2PC-Astros (6 out of 10) but not in NPC-Astros shown by the traces highlighted in red and enlarged in the insets. The traces are presented after leak subtraction to reveal the voltage-gated currents. (j) Current-voltage relationship of the outward currents. (k) Using Cs+ internal solution, the outward current is eliminated. The inward currents in Olig2PC-Astros are then recorded with the cells held at −70 mV. To maximally activate the inward currents, a 100-ms preconditioning pulse at −100 mV is delivered to the recorded cells before voltage steps (stepped from −70 to +50 mV at 10 mV increments for 50 ms). The representative traces show that the inward currents are reversibly blocked by TTX. The traces are presented after leak subtraction. Student’s t-test, *P<0.05, **P<0.01 and ***P<0.001. NS represents no significance. Data are presented as mean±s.e.m.