Forward programming of human pluripotent stem cells to generate glutamatergic and GABAergic neurons in a tri-culture model with astrocytes

Over the past decade, forward programming of human pluripotent stem cells (hPSCs) using various transcription factor (TF) combinations has been widely applied in neuroscience research. Ectopic NGN2 expression in hPSCs has been widely used for rapidly generating in vitro models of induced neurons (iNs) that are predominantly composed of excitatory glutamatergic neurons. Achieving a more balanced synaptic communication between excitatory and inhibitory neurons is essential for physiologically relevant in vitro models. Additionally, incorporating hPSC-derived astrocytes into models, rather than commonly used primary astrocytes, would more closely mimic in vivo disease phenotypes, especially for those associated with astrocyte dysfunction. Inducible hPSC lines were generated by targeting the AAVS1 safe harbor site with TF transgene cassettes using CRISPR/Cas9. Forward programming was achieved through forced expression of NGN2 for glutamatergic neurons (iGlutNs), ASCL1/DLX2 for GABAergic neurons (iGABANs) and SOX9/NFIB for astrocytes (iAstros). Cell identity was validated by immunocytochemistry and bulk RNA sequencing. Functional properties were characterized on multielectrode arrays (MEAs). Bulk RNA sequencing confirmed lineage-specific differentiation while revealing distinct transcriptomic profiles between iAstros and human primary astrocytes. Functional assays demonstrated robust inhibitory control of network dynamics in co-culture with iGABANs on MEA, with enhanced responses to GABAA receptor-targeting drugs including picrotoxin, bicuculline and clonazepam. Neurons co-cultured with iAstros showed reduced spontaneous activity compared to those cultured with primary astrocytes. We successfully generated hPSC-derived excitatory and inhibitory neurons to establish an appropriate E/I balance in vitro, supported by primary astrocytes. Although astrocyte identity was confirmed in our hPSC-derived astrocytes, further optimization is required to achieve full functional maturation. This approach to developing an isogenic co-culture system derived from a single hPSC line may more faithfully replicate native neural network dynamics, offering a physiologically relevant platform for studying neurological disorders and screening therapeutic compounds.