Generation and Co-culture of Cortical Glutamatergic and GABAergic-Induced Neuronal Cells.

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.

Measuring Neuronal Network Activity Using Human Induced Neuronal Cells.

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.

oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids.

Cerebral organoids are comprised of diverse cell types found in the developing human brain, and can be leveraged in the identification of critical cell types perturbed by genetic risk variants in common, neuropsychiatric disorders. There is great interest in developing high-throughput technologies to associate genetic variants with cell types. Here, we describe a high-throughput, quantitative approach (oFlowSeq) by utilizing CRISPR-Cas9, FACS sorting, and next-generation sequencing. Using oFlowSeq, we found that deleterious mutations in autism-associated gene KCTD13 resulted in increased proportions of Nestin+ cells and decreased proportions of TRA-1-60+ cells within mosaic cerebral organoids. We further identified that a locus-wide CRISPR-Cas9 survey of another 18 genes in the 16p11.2 locus resulted in most genes with > 2% maximum editing efficiencies for short and long indels, suggesting a high feasibility for an unbiased, locus-wide experiment using oFlowSeq. Our approach presents a novel method to identify genotype-to-cell type imbalances in an unbiased, high-throughput, quantitative manner.