RESUMEN
The study of neurological disorders requires experimentation on human neurons throughout their development. Primary neurons can be difficult to obtain, and animal models may not fully recapitulate phenotypes observed in human neurons. Human neuronal culturing schemes which contain a balanced mixture of excitatory and inhibitory neurons that resemble physiological ratios seen in vivo will be useful to probe the neurological basis of excitation-inhibition (E-I) balance. Here, we describe a method for directly inducing a homogenous population of cortical excitatory neurons and cortical interneurons from human pluripotent stem cells, as well as the generation of mixed cultures using these induced neurons. The obtained cells display robust neuronal synchronous network activity as well as complex morphologies that are amenable to studies probing the molecular and cellular basis of disease mutations or other aspects of neuronal and synaptic development.
Asunto(s)
Neuronas GABAérgicas , Células Madre Pluripotentes , Animales , Humanos , Técnicas de Cocultivo , Células Cultivadas , InterneuronasRESUMEN
Synchronous firing of neurons, often referred to as "network activity" or "network bursting," is an indication of a mature and synaptically connected network of neurons. We previously reported this phenomenon in 2D human neuronal in vitro models (McSweeney et al. iScience 25:105187, 2022). Using induced neurons (iNs) differentiated from human pluripotent stem cells (hPSCs) coupled with high-density microelectrodes arrays (HD-MEAs), we probed the underlying patterns of neuronal activity and found irregularities in network signaling across mutant states (McSweeney et al. iScience 25:105187, 2022). Here, we describe methods for plating cortical excitatory iNs differentiated from hPSCs on top of HD-MEAs and culturing iNs to maturity, examples of representative human wild-type Ngn2-iN data, and troubleshooting tips and tricks for the experimenter interested in integrating HD-MEAs into one's research approach.