Researchers induced human induced Pluripotent Stem Cells (iPSCs) to differentiate into hippocampal NPCs. The study revealed that during the long-term culture of NPCs, calcium signals were highly variable, and ligand-induced calcium transients could predict their ability to generate mature neurons. To investigate calcium signal transduction without the need for dye loading, the researchers utilized the Sleeping Beauty transposon plasmid (reported to provide stable transgenic expression in human embryonic stem cells) combined with the transposase and performed repeated screening with puromycin (1.6μg/mL, 48h) to transgenic expressing cells. Ultimately, NPCs expressing the calcium indicator protein GCaMP6f were obtained. The authors used BDNF (Brain-Derived Neurotrophic Factor), cAMP (Cyclic Adenosine Monophosphate), Laminin, Wnt3A, and ascorbic acid to induce the differentiation of parental NPCs expressing GCaMP6, enriching for granule neurons of the dentate gyrus (DG). The results showed that the NPCs expressing GCaMP6 (P6 generation) were Nestin and Sox2 positive, indicating that they still retained their stem-like characteristic, and their differentiation potential was unaffected. They could be directionally induced to differentiate into mature neurons expressing MAP2 and hippocampal granule cell marker proteins PROX1 and CALB1.

Figure 1: NPCs expressing GCaMP6 can be induced to differentiate into mature neurons. The “Parental” group represents the control group without GCaMP6 transfection.