A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells

Abstract

Progress in elucidating the molecular and cellular pathophysiology of neuropsychiatric disorders has been hindered by the limited availability of living human brain tissue. The emergence of induced pluripotent stem cells (iPSCs) has offered a unique alternative strategy using patient-derived functional neuronal networks. However, methods for reliably generating iPSC-derived neurons with mature electrophysiological characteristics have been difficult to develop. Here, we report a simplified differentiation protocol that yields electrophysiologically mature iPSC-derived cortical lineage neuronal networks without the need for astrocyte co-culture or specialized media. This protocol generates a consistent 60:40 ratio of neurons and astrocytes that arise from a common forebrain neural progenitor. Whole-cell patch-clamp recordings of 114 neurons derived from three independent 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) and AP amplitude (66.5±1.3 mV). Nearly 100% of neurons were capable of firing APs, of which 79% had sustained trains of mature APs with minimal 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). We expect this protocol to be of broad applicability for implementing iPSC-based neuronal network models of neuropsychiatric disorders.

The SAC1 domain in synaptojanin is required for autophagosome maturation at presynaptic terminals

“…Medium was changed daily and cells were passaged every 4-6 days either mechanically or enzymatically with collagenase type IV (100 U/ml) (Life Technologies), and addition of 10 lM ROCK inhibitor (Sigma) to the medium after passaging. Neural progenitor cells (NPCs) were created according to published protocols (de Esch et al, 2014;Gunhanlar et al, 2017) with minor modifications. Briefly, for embryoid body (EB) generation, iPSCs were dissociated and transferred to non-adherent plates in normal iPSC medium without bFGF and cultured on a shaker at 37°C/5% CO 2 .…”

Human pluripotent stem cell modeling of tuberous sclerosis complex reveals lineage-specific therapeutic vulnerabilities

“…Cells were collected from glass coverslips in 24-well culture plates. Before transfer to the recording chamber in an upright Nikon Eclipse E600FN microscope, culture medium was gradually changed to oxygenated artificial cerebral spinal fluid (ACSF) containing (mM): 124 NaCl, 5 KCl, 1.25 NaH2PO4, 1.2 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 glucose, in 30 mins at room temperature (Gunhanlar et al, 2018). Whole-cell patch clamp recordings were obtained from differentiated neurons at 4-6 weeks following the initiation of the differentiation procedure (Sun et al, 2018).…”

Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease

“…The density of synaptic junctions formed on cultured neurons also offers a strong indicator of the structural development of a given neuronal population as well as the degree of connectivity. Complex dendritic arborization and axonal distinction [Gouder et al, 2015;Kang et al, 2017] coupled with stable expression of pan-neuronal markers such as NeuN, MAP-2, and Tuj1 [Hu et al, 2010;Gunhanlar et al, 2018] should likewise be considered.…”