Generation of neuronal/glial mixed cultures from human induced pluripotent stem cells (hiPSCs).

For a long time, the understanding of neurological diseases has been limited by the lack of representative experimental models able to recapitulate essential features of the human pathologies. Human induced pluripotent stem cells (hiPSCs) have emerged as a powerful tool for disease modeling, drug screening, and the development of novel cell and gene therapies. A critical issue for the prospective use of hiPSCs in basic and translational research for central nervous system (CNS) disorders is to validate robust protocols able to efficiently differentiate pluripotent cells into neurons and glial cells of interest, specifically those that are most affected in pathological conditions. We describe here a three-step differentiation protocol optimized for feeder-free hiPSCs. The protocol includes a first step of neural induction mediated by dual SMAD inhibition to generate homogeneous populations of neural progenitor cells (NPCs), a second step of NPCs expansion, and a third phase of NPCs differentiation into a mixed culture of neurons, oligodendrocytes, and astrocytes. This experimental platform is relevant to recapitulate the neural induction of hiPSCs and to monitor NPC lineage specification and neuronal/glial differentiation in physiological conditions as well as in the context of CNS diseases. The protocol allows monitoring early pathological hallmarks in the different CNS cell types, also offering a simplified in vitro model to study the neuronal-glial crosstalk.