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.

CASK loss of function differentially regulates neuronal maturation and synaptic function in human induced cortical excitatory neurons.

Loss-of-function (LOF) mutations in CASK cause severe developmental phenotypes, including microcephaly with pontine and cerebellar hypoplasia, X-linked intellectual disability, and autism. Unraveling the pathological mechanisms of CASK-related disorders has been challenging owing to limited human cellular models to study the dynamic roles of this molecule during neuronal maturation and synapse development. Here, we investigate cell-autonomous functions of CASK in cortical excitatory induced neurons (iNs) generated from CASK knockout (KO) isogenic human embryonic stem cells (hESCs) using gene expression, morphometrics, and electrophysiology. While immature CASK KO iNs show robust neuronal outgrowth, mature CASK KO iNs display severe defects in synaptic transmission and synchronized network activity without compromising neuronal morphology and synapse numbers. In the developing human cortical excitatory neurons, CASK functions to promote both structural integrity and establishment of cortical excitatory neuronal networks. These results lay the foundation for future studies identifying suppressors of such phenotypes relevant to human patients.

STEM Pride: Perspectives from transgender, nonbinary, and genderqueer scientists.

In celebration of Pride Month, we asked transgender, genderqueer, and nonbinary scientists to tell us about what fascinates them, their ambitions and achievements, and how their gender identities have shaped their experiences in STEM. We owe a special thanks to 500 Queer Scientists (https://500queerscientists.com/), whose network and efforts at increasing LGBTQ+ scientists' visibility made this article possible.

Bisphenol S alters development of the male mouse mammary gland and sensitizes it to a peripubertal estrogen challenge.

Humans are exposed to estrogenic chemicals in food and food packaging, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS; 4,4'-sulfonyldiphenol), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. Here, we quantified the effects of perinatal exposures to BPS on the male mouse mammary gland. In our first study, pregnant CD-1 mice were orally exposed to BPS (2 or 200 µg/kg/day) starting on pregnancy day 9 through lactation day 20, and male mammary glands were evaluated on embryonic day 16, prior to puberty, and in early adulthood. We observed modest changes in tissue organization in the fetal gland, and significant increases in growth of the gland induced by developmental BPS exposure in adulthood. In our second study, pregnant CD-1 mice were orally exposed to BPS (2, 200 or 2000 µg/kg/day) starting on pregnancy day 9 through lactational day 2. After weaning, the male pups were administered either oil (vehicle) or an estrogen challenge (1 µg ethinyl estradiol/kg/day) for ten days starting prior to puberty. After the 10-day estrogen challenge, we examined hormone-sensitive outcomes including anogenital index (AGI), weight of the seminal vesicles, and morphological parameters of the mammary gland. Although AGI and seminal vesicle weight were not affected by BPS, we observed dose-specific effects on the response of male mammary glands to the peripubertal estrogen challenge. Because male mammary glands are structurally less developed compared to females, they may provide a simple model tissue to evaluate the effects of putative xenoestrogens.