Directed differentiation of human pluripotent stem cells (hPSCs) into telencephalic excitatory neurons and inhibitory neurons

An overview of the procedure is presented in Figure 2A. First, human ES/iPS cell colonies were detached from DR4 MEF feeders with Dispase and grown as suspension culture for five days to form embryoid bodies (EBs). The EB medium was the standard human ES medium supplemented with SMAD inhibitors Dorsomorphin (5 µM, Sigma Aldrich) and SB431542 (10 µM, Tocris) but without FGF2. Next, EBs were plated for the induction of neural rosettes over 10 days (Figure 2B). The neural induction media contained Neurobasal-A (Base medium, Gibco), B-27 without Vitamin-A (2%, Gibco), GlutaMax (1%, Gibco), and Penstrep (100 units/mL working concentration, Gibco). Human recombinant FGF2 (20 ng/ml for dorsal telencephalon induction, 10 ng/ml for ventral telencephalon-MGE induction; R&D systems), EGF (20 ng/ml, EMD Millipore), IGF1 (10 ng/ml, EMD Millipore) and small molecules SAG (10 nM and 100 nM, Enzo Life Sciences, Farmingdale, New York) and retinoic acid (RA) (10 µM, Sigma Aldrich) were added to the neural induction media in specific combinations (Figure 2A). Neural rosettes were isolated by mechanical picking and re-plated for neuronal differentiation. For rosettes harvested from the dorsal telencephalon patterning scheme, an extra expansion step was included, where the neural progenitors were allowed to proliferate in suspended neurospheres over seven days (Figure 2—figure supplement 1). The neuronal differentiation was divided into an initial stage (20 days) and a secondary, astrocyte- supported stage (~2 months), where the immature human neurons were co-cultured with a rat cortical astrocyte monolayer. The neuronal differentiation media contained Neurobasal-A (Base medium, Gibco), B-27 without Vitamin-A (2%, Gibco), GlutaMax (1%, Gibco), and Penstrep (100 units/mL working concentration, Gibco), recombinant human BDNF (10 ng/ml, PeproTech, Rocky Hill, New Jersey) and recombinant human NT-3 (10 ng/ml, PeproTech). Additional neurotrophic or signaling molecules were used for the initial stage of inhibitory neuron differentiation. Between Differentiation Day 15 and 25 (Figure 2—figure supplement 1), we used BDNF (10 ng/ml, extra, PeproTech) and NT-3 (10 ng/ml, extra, PeproTech), GDNF (10 ng/ml, R&D systems), cyclic AMP (0.5 mM, Sigma Aldrich) and BMP4 (20 ng/ml, R&D systems); between Differentiation Day 25 and 35, we used BDNF (10 ng/ml, extra, PeproTech) and NT-3 (10 ng/ml, extra, PeproTech), GDNF (10 ng/ml, R&D systems), cyclic AMP (0.5 mM, Sigma Aldrich) and IGF1 (10 ng/ml, EMD Millipore). The rat cortical astrocytes were prepared from newborn to P2 rat pups, using a procedure modified from a previous publication (McCarthy and de Vellis, 1980), according to a lab protocol approved by the administrative panel on laboratory animal care (APLAC) at Stanford University. At the transition to astrocyte-supported neuronal differentiation, human neurons were dissociated with Accutase and plated at the density of 10,000 cells/cm².