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Figure 3. Schematic representation of differentiation procedures for generation of GABAergic and glutamatergic human induced
pluripotent stem cell (hiPSC) neurons. Pluripotent hiPSCs can be induced via dual SMAD inhibition to produce telencephalic neural
progenitors cells (NPCs) which are positive for FOXG1 - a brain-specific transcriptional repressor essential for the early development
of the telencephalon - marking this population. Notably, FOXG1 gene is associated with the FOXG1 syndrome, which has previously
been described as a congenital variant of Rett syndrome. In the further differentiation steps, the absence of a SHH (sonic hedgehog)
agonist facilitates dorsal patterning of the NPCs, characterized by expression of the markers PAX8 (a human neuroectoderm cell fate
determinant) and EMX1/2 (transcription factors expressed primarily in dorsal telencephalon. On the contrary, activation of SHH signaling
via the addition of SSH agonists induces ventralization of NPCs, characterized by expression of NKX2-1 (a transcription factor that
specifies ventral lineages during development). Dorsal progenitors can be further differentiated into glutamatergic neurons, whilst ventral
precursors can be differentiated into GABAergic neurons

edited isogenic controls to model monogenic epilepsies. These are powerful models, especially if they are
used in combination with patient data and animal models, and they will expand our knowledge of underlying
cellular deficiencies observed in epilepsy. Nevertheless, several challenges remain and need to be addressed to
further improve these hiPSC models. In terms of 2D monocultures, which are very applicable for large drug
screens, the biggest challenge is heterogeneity of the generated neuronal populations. These heterogeneous
populations are problematic if the disease phenotype affects only specific neuronal subpopulations; therefore,
the effect of the drugs tested can be over- or under-interpreted. Another issue linked to heterogeneous
neuronal populations is the reproducibility among different populations. Varying subpopulations of
neurons with different cellular disease phenotypes is another challenge since such subpopulations can vary
significantly between different experiments and influence functional readouts. Another issue to take into
consideration is that epilepsy is a complex neurological disorder, where excitatory and inhibitory neuronal
communication is disturbed. The ideal case would be to generate a mixed population with both types of
neurons, ideally with glial contributions to investigate the activities of neurons in interaction with each other
[66]
or with glial cells, which is a more in vivo-like condition , but this would also be more challenging in terms
of the interpretation of results and drug efficacy. The most in vivo-like situation would be comparative studies
in vascularized cerebral organoids, but this line of research, which is described below, is still in its infancy
and not applicable for large-scale drug tests.

iPSC-derived brain organoids in neurodevelopment and epilepsy
Epilepsy is a complex brain disorder with excitatory and inhibitory neuron involvement, in which neural
network activity is further dependent on neurotransmitter recycling via astrocytes. Therefore, even though
2D models are useful in electrophysiological measurements and drug screening, they have limitations when
it comes to investigation of more complex interactions. To imitate the natural physiological conditions,
architecture and neurodevelopmental features of the brain, differentiated neurons can be organized into 3D
[67]
cerebral organoids .

A more sophisticated model is the 3D cerebral organoid generated using intrinsic patterning, which can
yield semi-organized organoids containing neurons from different parts of the brain [22,68] . For example
hiPSC-derived cerebral organoids show primitive cortical organization, dorsal forebrain-like progenitor
zones and expression of markers specific for the hippocampus and forebrain. Cerebral organoids are useful