Leveraging Human Induced Pluripotent Stem Cells (iPSCs) to Investigate the Pathophysiology of Mental Illness

Background and Aim: The interactions among neurons and astrocyteare very important during both early neurodevelopment and in the adultbrain. Accordingly, the presence of astrocytes significantly improves thefunctional maturation of human pluripotent stem cell-derived neurons.Presently, most experiments introduce non-human sources of astrocytesvia co-culture using human induced pluripotent stem cells (iPSCs)-derived neurons. Nevertheless, differentiation protocols, which arebased on common progenitor giving rise to both neurons and astrocytes,appear to proceed more similarly to in vivo neurodevelopment. Havingcapitalized on the latter approach, we now report on the developmentof a simplified differentiation protocol for deriving functionally matureneuronal networks from iPSCs.Methods: Human iPSC-derived NPCs (passages 5–11) were plated onsterile coverslips and coated with poly-L-ornithine. One fully confluent 10cm dish of NPCs was divided over a 12-well plate. A 100 μL drop of NPCcell suspension was placed on the laminin-coated spot for 1 h to allowfor attachment of NPCs on coverslips in neural differentiation medium(Neurobasal medium, 1% N2 supplement, 2% B27-RA supplement, 1%minimum essential medium/non-essential amino acid, 20 ng/ml brainderivedneurotrophic factor, 20 ng/mL glial cell-derived neurotrophicfactor, 1 μM dibutyryl cyclic adenosine monophosphate, 200 μMascorbic acid, 2 μg/ml laminin and 1% penicillin/streptomycin). Cellswere refreshed with medium 3 times per week. During weeks 1–4, the medium was fully refreshed. After 4 weeks of neural differentiation, onlyhalf of the volume of medium per well was refreshed. Electrophysiologyand confocal imaging were performed between 8 and 10 weeks afterplating of NPCs.Results: Our protocol produces a consistent 60:40 ratio of neurons andastrocytes, which arise from a common forebrain neural progenitor.Whole-cell patch-clamp recordings of 114 neurons derived from threeindependent iPSC lines confirmed their electrophysiological maturity,including resting membrane potential (−58.2 ± 1.0 mV), capacitance(49.1 ± 2.9 pF), action potential (AP) threshold (−50.9 ± 0.5 mV) andAP amplitude (66.5 ± 1.3 mV). Nearly 100% of neurons were capableof firing APs, of which 79% had sustained trains of mature APs withminimal accommodation (peak AP frequency: 11.9 ± 0.5 Hz) and 74%exhibited spontaneous synaptic activity (amplitude, 16.03 ± 0.82 pA;frequency, 1.09 ± 0.17 Hz).Conclusion: In the present study, we provide a simplified differentiationprotocol for the generation of electrophysiologically mature iPSC-derivedneuronal networks with no need for astrocyte co-culture or specializedmedia. Furthermore, these results provide a quantitative basis forconsidering the variability of distinct electrophysiological parametersfor the modeling of iPSC-based disease. This protocol is envisioned tobe a considerable utility for implementing cellular modeling approachestowards the study of human neuropsychiatric disease pathophysiology.

کلیدواژه ها:

Human pluripotent stem cells ، Neurons ، Astrocytes ، Synaptic transmission ، Electrophysiology