Figure 1.

Engineering of i³N iPSCs and Generation of Homogeneous Functional Glutamatergic Neurons by a Simplified Two-Step Procedure

(A) Schematic of the targeting of the AAVS1 locus with pUCM.Puro-CAG.rtTA3G-TRE3G.Ngn2 donor vector by TALEN-mediated integration. The third-generation doxycycline-inducible reverse transcriptional activator (rtTA3G) is driven by the CAG promoter and followed by rbGlob polyA tail. Mouse Ngn2 is driven by the tet response element (TRE3G) and followed by SV40 polyA tail. It is oriented tail-to-tail with rtTA3G. Orange boxes are exons of the PPP1R12C gene; gray boxes are regions of homology. PCR1 and PCR2 primers are used for 5′ and 3′ junction PCR screening and generate 1.1-kb and 1.5-kb PCR products, respectively. PCR3 primers (product size, 248 base pairs) are used to detect the nonintegrated allele at the AAVS1 locus.

(B) Flow diagram of the two-step procedure for generating i³Neurons.

(C) Representative phase-contrast images during the differentiation of i³Neurons. The timeline is the same as shown in (B). Scale bar, 50 μm.

(D) Representative images showing immunocytochemical staining for the pan-neuronal marker MAP2, βIII tubulin (TUJ1 antibody), and NeuN in i³Neurons after 4 weeks of differentiation. Nuclei were labeled by Hoechst. Scale bar, 25 μm.

(E) Representative images of immunocytochemical staining of mature 8-week-old i³Neurons show tau enrichment (detected with HT7) in an axon identified by the axon initiation segment marker ankyrin G (AnkG). Nuclei were labeled by Hoechst. Scale bar, 25 μm.

(F) Representative confocal images of i³Neurons showing immunolabeling of postsynaptic GluR2/3 containing AMPA-type glutamate receptors (red) and the presynaptic vesicular glutamate transporter VGlut1 (green). The colocalization of GluR2/3 and VGlut1 puncta marks glutamatergic synapses formed between i³Neurons. Scale bar, 5 μm.

(G) Representative traces of action potentials evoked by 500-ms current step injections at just above the firing threshold (green trace) and at a higher firing frequency (black trace).

(H) Spontaneous excitatory postsynaptic currents recorded from an i³N neuron (top) were blocked by CNQX, an AMPA receptor antagonist (bottom).

See also Figures S1 and S2.