Neuropharmacology of human TERA2.cl.SP12 stem cell-derived neurons in ultra-long-term culture for antiseizure drug discovery

Abstract

Modeling the complex and prolonged development of the mammalian central nervous system in vitro remains a profound challenge. Most studies of human stem cell derived neurons are conducted over days to weeks and may or may not include glia. Here we have utilized a single human pluripotent stem cell line, TERA2.cl.SP12 to derive both neurons and glial cells and determined their differentiation and functional maturation over 1 year in culture together with their ability to display epileptiform activity in response to pro-convulsant agents and to detect antiseizure drug actions. Our experiments show that these human stem cells differentiate in vitro into mature neurons and glia cells and form inhibitory and excitatory synapses and integrated neural circuits over 6–8 months, paralleling early human neurogenesis in vivo; these neuroglia cultures display complex electrochemical signaling including high frequency trains of action potentials from single neurons, neural network bursts and highly synchronized, rhythmical firing patterns. Neural activity in our 2D neuron–glia circuits is modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and these actions were consistent in both young and highly mature neuron cultures. We also show for the first time that spontaneous and epileptiform activity is modulated by first, second and third generation antiseizure agents consistent with animal and human studies. Together, our observations strongly support the value of long-term human stem cell-derived neuroglial cultures in disease modeling and neuropsychiatric drug discovery.

Antiseizure properties of fenamate NSAIDs determined in mature human stem-cell derived neuroglial circuits

“…Human [TERA2.cl.SP12] stem cell derived neurons and glial cells were successfully differentiated and maintained for up to 88 weeks in cell culture. As previously detailed, neurons and glia differentiated and matured over the course of 4-6 months with increased axodendritic networks and synapse formation continuing through 12 months (Salmanzadeh et al, 2023). Figure 1 shows a snapshot of neurons and glia in phase contrast (Figures 1A, D) and immunolabeled (Figures 1B, C) over the course of year 1.…”

“…In the present study, therefore, we have addressed the hypothesis that fenamate NSAIDs have antiseizure properties by utilizing human stem cellderived neuron-glial cells maintained in long-term culture. We have recently reported that this 2D in vitro model forms complex excitatory (glutamatergic) and inhibitory (GABAergic) neural circuits over many weeks, paralleling in vivo development, with synaptogenesis and electrophysiological activity facilitated by the secondary development of astrocytes in the cultures; significantly, we have also shown that these human neuronal cell networks generate epileptiform activity in response to diverse convulsant agents and to be highly sensitive to the anticonvulsant actions of first, second and third generation antiepileptic drugs (Salmanzadeh et al, 2023).…”

“…Pluripotent human (TERA2. cl.SP12) stem cells were maintained and differentiated as previously described (e.g., Salmanzadeh et al, 2023). Briefly, non-differentiated stem cells were cultured in media consisting of Dulbecco's Modified Eagles Medium (DMEM, Sigma-Aldrich), 10% Fetal Bovine Serum (FBS, Gibco), 2 mM L-glutamine (Gibco) and penicillin-streptomycin (100 U/mL, 100 μg/mL; Invitrogen), and maintained in an incubator at 37 °C, 5% CO 2 , 100% relative humidity in 75 mL tissue culture flasks until 80% confluent.…”

“…The cells were later viewed and imaged using an inverted EVOS FL auto microscope (Thermo Fisher Scientific) and the βIII tubulin + neurons or GFAP + glial cells calculated as a percentage of the total number of DAPI + cells in 5 separate fields (0.23 mm 2 per field) for each slide. (Salmanzadeh et al, 2023). In the current study, three 6-well MEA plates were utilized.…”

“…4-AP is a well-established convulsant agent both in vivo and in vitro (Ventura-Mejía et al, 2023) and we have previously shown that it is highly effective in provoking epileptiform activity in our stem cell-derived neuroglial cultures (Salmanzadeh et al, 2023). Control spontaneous firing was therefore recorded for a minimum of 10 min, 4-AP (100 μM) then added to the cells and activity was recorded for a further 60 min.…”