Functional Neural Development from Human Embryonic Stem Cells: Accelerated Synaptic Activity via Astrocyte Coculture

AP maturation occurs from 3 to 10 weeks. A, Representative voltage responses to a 30 pA current injection are shown for neurons grown for 4, 6, 7, and 10 weeks (W). APs were determined by clear evidence of threshold (arrow). Repetitive trains of APs were observed in some cells at 10 weeks (10Wi). B, Summarized data from these neurons demonstrate that the number of APs fired in response to a 30 pA current pulse increased significantly by 7 weeks of culture. C, D, AP amplitude (C) increased significantly between 6 and 7 weeks of differentiation and was mirrored by a significant decrease in half-width (D). E, When neurons were held between −55 and −60 mV, no significant difference in AP amplitude was observed between 6 and 7 weeks. F, The significant difference between 6 and 7 weeks in AP half-width remained when cells were held at negative potentials. *p < 0.05; **p < 0.01. NS, Nonsignificant. Error bars indicate SEM.

Peak I_Na increases during development. Ai, Rapidly inactivating voltage-gated inward currents were elicited by step depolarizations from −50 to 50 mV from a holding potential of −70 mV. Aii, These inward currents were completely blocked by TTX (1 μm). B, Pooled data demonstrate that peak I_Na increases during differentiation, reaching significance after 7 weeks (*p < 0.05). Error bars indicate SEM. C, I–V relationship for peak inward current is shown for neurons after 4 weeks (filled diamonds) and 7 weeks (open squares) of differentiation. Seven-week-old neurons display larger peak I_Na and a distinct (∼6 mV) leftward shift in half-maximal current, whereas maximal current was always elicited at 0 mV.

I_A increases parallel AP maturation, whereas I_K decreases throughout maturation. Ai, A representative trace showing a fast inactivating and a sustained-outward current elicited by voltage steps from a holding potential of −80 mV. Aii, 4-AP (1 mm) eliminated the fast inactivating K⁺ current, leaving a sustained K⁺ current. Aiii, Subtraction of the sustained current from total K⁺ current isolated the transient K⁺ current. Aiv, A concentration of 12 μm TEA further reduced the sustained K⁺ current. B, Summarized data demonstrate that transient K⁺ current density significantly increased between 6 and 7 weeks and remained elevated until 10 weeks of differentiation. C, Sustained K⁺ current density declined steadily over time in culture and was significantly reduced by 10 weeks of differentiation (*p < 0.05). Error bars indicate SEM.

Endogenous and exogenous astrocytes induce synapsin localization and promote synaptic activity. Ai, In neurons plated without exogenous astrocytes, synapsin-1 (green) was diffusely distributed in the cytoplasm of MAP2-positive neurons (red) after 6 weeks of differentiation (arrowhead). Representative voltage-clamp recordings from cells held at −70 mV showed no spontaneous PSCs in these cells (below). Aii, In contrast, neurons grown on a monolayer of astrocytes displayed punctate localization of synapsin-1 (arrows) and prominent PSCs (below) at 6 weeks of differentiation. Aiii, Aiv, After 9 weeks, both groups displayed punctuate localization of synapsin-1 protein (arrows) and noticeable synaptic activity. Bi, Bii, Synaptic activity was composed of AMPA receptor-mediated currents that were blocked with CNQX (100 μm), as well as GABA receptor-mediated currents that could be blocked with bicuculline (bic.) (50 μm). C, Summarized data show that significantly more neurons displayed synaptic activity from 1 to 3 weeks when grown on astrocytes (open bars) than those grown on laminin (filled bars; *p < 0.05). At later time points, there was no significant difference between the number of cells displaying synaptic activity when neurons were plated with or without astrocytes. Scale bars, 20 μm.