Figure 5.

Rapid Generation of Neurons from Human iPSCs by Endogenous Activation of Neuronal Lineage-Specific TFs with the PB-CRISPRa System

(A–C) Experiments were performed in NEUROG2-mCherry knockin human iPSC reporter line. Three days after transfection with PB-SAM-NEUROG1/2 all-in-one vector, iPSCs underwent morphological changes and showed a bipolar morphology, typical of neural progenitors. Importantly, these cells also expressed mCherry (red), the surrogate marker for NEUROG2. (D–F) Experiments were performed in Doublecortin (DCX)-ZsGreen human iPSC reporter line. Three days after transfection with PB-SAM-NEUROG1/2 all-in-one vector, iPSCs started to express ZsGreen (green), the surrogate marker for DCX, indicating that the activation of NEUROG1 and NEUROG2 had promoted the iPSCs to differentiate into DCX-expressing young neurons. (G–I) Seven days after transfection, the DCX-ZsGreen reporter cells presented longer neurites and extensive arborization, typical of more mature neurons. (J–O) Immunocytochemistry revealed that these cells also expressed pan neuronal marker β3 tubulin (J) and mature neuronal markers NeuN (K), NF160 (M), and MAP2 (N). (L) and (O) are overlapped images with DAPI. Scale bar, 50 μm.