Duration of culture and sonic hedgehog signaling differentially specify PV versus SST cortical interneuron fates from embryonic stem cells.

Medial ganglionic eminence (MGE)-derived GABAergic cortical interneurons (cINs) consist of multiple subtypes that are involved in many cortical functions. They also have a remarkable capacity to migrate, survive and integrate into cortical circuitry after transplantation into postnatal cortex. These features have engendered considerable interest in generating distinct subgroups of interneurons from pluripotent stem cells (PSCs) for the study of interneuron fate and function, and for the development of cell-based therapies. Although advances have been made, the capacity to generate highly enriched pools of subgroup fate-committed interneuron progenitors from PSCs has remained elusive. Previous studies have suggested that the two main MGE-derived interneuron subgroups--those expressing somatostatin (SST) and those expressing parvalbumin (PV)--are specified in the MGE from Nkx2.1-expressing progenitors at higher or lower levels of sonic hedgehog (Shh) signaling, respectively. To further explore the role of Shh and other factors in cIN fate determination, we generated a reporter line such that Nkx2.1-expressing progenitors express mCherry and postmitotic Lhx6-expressing MGE-derived interneurons express GFP. Manipulations of Shh exposure and time in culture influenced the subgroup fates of ESC-derived interneurons. Exposure to higher Shh levels, and collecting GFP-expressing precursors at 12 days in culture, resulted in the strongest enrichment for SST interneurons over those expressing PV, whereas the strongest enrichment for PV interneurons was produced by lower Shh and by collecting mCherry-expressing cells after 17 days in culture. These findings confirm that fate determination of cIN subgroups is crucially influenced by Shh signaling, and provide a system for the further study of interneuron fate and function.

GABAergic interneuron transplants to study development and treat disease.

Advances in stem cell technology have engendered keen interest in cell-based therapies for neurological disorders. Postnatal engraftments of most neuronal precursors result in little cellular migration, a crucial prerequisite for transplants to integrate within the host circuitry. This may occur because most neurons migrate along substrates, such as radial glial processes, that are not abundant in adults. However, cortical GABAergic interneurons migrate tangentially from the subcortical forebrain into the cerebral cortex. Accordingly, transplants of cortical interneuron precursors migrate extensively after engraftment into a variety of CNS tissues, where they can become synaptically connected with host circuitry. We review how this remarkable ability to integrate post-transplant is being applied to the development of cell-based therapies for a variety of CNS disorders.

Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells.

Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.

Considerations for importing live genetically modified mice from academic laboratories.

Genetically modified mice have been an invaluable tool for the study of gene function in an intact biological system. Researchers frequently obtain genetically modified mice from other academic institutions. This form of collaboration between laboratories comes with a unique set of challenges, and a clear set of guidelines for navigating the process has yet to be defined. The author provides suggestions for how to initiate an exchange of animal resources and steps for ensuring a successful collaboration. Both parties should be clear about their expectations. The importing lab should prepare in advance for potential animal health considerations and breeding and colony management strategies prior to importation. The number, gender, age and genotype of the imported animals should be confirmed as soon as possible by the importing lab. It is in the best interest of all parties to be courteous, forthright and thorough when sharing animal resources so that everyone can benefit from the resulting research.

Pluripotent stem cells for the study of CNS development.

The mammalian central nervous system is a complex neuronal network consisting of a diverse array of cellular subtypes generated in a precise spatial and temporal pattern throughout development. Achieving a greater understanding of the molecular and genetic mechanisms that direct a relatively uniform population of neuroepithelial progenitors into diverse neuronal subtypes remains a significant challenge. The advent of pluripotent stem cell (PSC) technology allows researchers to generate diverse neural populations in vitro. Although the primary focus of PSC-derived neural cells has been their therapeutic potential, utilizing PSCs to study neurodevelopment is another frequently overlooked and equally important application. In this review, we explore the potential for utilizing PSCs to study neural development. We introduce the types of neurodevelopmental questions that PSCs can help to address, and we discuss the different strategies and technologies that researchers use to generate diverse subtypes of PSC-derived neurons. Additionally, we highlight the derivation of several thoroughly characterized neural subtypes; spinal motoneurons, midbrain dopaminergic neurons and cortical neurons. We hope that this review encourages researchers to develop innovative strategies for using PSCs for the study of mammalian, and specifically human, neurodevelopment.

Assessment of general anaesthetic cytotoxicity in murine cortical neurones in dissociated culture.

General anaesthetics are proposed to cause unconsciousness by modulating neuronal excitability in the mammalian brain through mechanisms that include enhancement of inhibitory GABA(A) receptor currents and suppression of excitatory glutamate receptor responses. Both intravenous and volatile agents may produce neurotoxic effects during early postnatal rodent brain development through similar mechanisms. In the following study, we investigated anaesthetic cytotoxicity in primary cortical neurones and glia from postnatal day 2-8 mice. Cultures at 4-20 days in vitro were exposed to combinations of ketamine (100 µM to 3 mM), nitrous oxide (75%, v/v) and/or isoflurane (1.5-5%, v/v) for 6-12 h. Neuronal survival and cell death were measured via microtubule associated protein 2 immunoassay and lactate dehydrogenase release assays, respectively. Clinically relevant anaesthetic concentrations of ketamine, nitrous oxide and isoflurane had no significant neurotoxic effects individually or when given as anaesthetic cocktails, even with up to 12 h exposure. This lack of neurotoxicity was observed regardless of whether cultures were prepared from postnatal day 0-2 or day 8 mice, and was also unaffected by number of days in vitro (DIV 4-20). Significant neurotoxic effects were only observed at supraclinical concentrations (e.g. 1-3 mM ketamine). Our study suggests that neurotoxicity previously reported in vivo is not due to direct cytotoxicity of anaesthetic agents, but results from other impacts of the anaesthetised state during early brain development.