Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.
Phosphoinositide 3-kinase (PI3K)-dependent signaling has been implicated in the regulation of embryonic stem (ES) cell fate. To gain further insight into the mechanisms regulated by PI3Ks in murine ES cells, we have performed expression profiling using Affymetrix GeneChips to characterize the transcriptional changes that arise as a result of inhibition of PI3K-dependent signaling. Using filtering of greater than 1.5-fold change in expression and an analysis of variance significance level of p < .05, we have defined a dataset comprising 646 probe sets that detect changes in transcript expression (469 down and 177 up) on inhibition of PI3Ks. Changes in expression of selected genes have been validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analyses reveal significant over-representation of transcriptional regulators within our dataset. In addition, several known regulators of ES cell pluripotency, for example, Nanog, Esrrb, Tbx3, and Tcl-1, are among the downregulated genes. To evaluate the functional involvement of selected genes in regulation of ES cell self-renewal, we have used short interfering RNA-mediated knockdown. These studies identify genes not previously associated with control of ES cell fate that are involved in regulating ES cell pluripotency, including the protein tyrosine phosphatase Shp-1 and the Zscan4 family of zinc finger proteins. Further gain-of-function analyses demonstrate the importance of Zscan4c in regulation of ES cell pluripotency. STEM CELLS 2009;27:764-775 Disclosure of potential conflicts of interest is found at the end of this article.
NMDA receptor agonists have been used for many years to generate animal models of polymicrogyria, a malformation of cortical development. Fry et al. identify de novo GRIN1 mutations in eleven patients with severe bilateral polymicrogyria. Polymicrogyria-associated GRIN1 mutations cluster in specific protein domains and significantly alter NMDA receptor function.
The growth of a pluripotent embryonic stem (ES) cell population is dependent on cell survival, proliferation and self-renewal. The nucleotide ATP represents an important extracellular signalling molecule that regulates the survival of differentiated cells, however, its role is largely undefined in embryonic stem cells. Here we report a role for ATP-gated P2X7 receptors in ES cell survival. The functional expression of P2X7 receptors in undifferentiated mouse ES cells is demonstrated using a selective P2X7 antagonist and small interfering RNA knockdown of these receptors. Our data illustrate a key role for the P2X7 receptor as an essential pro-survival signal required for optimal ES cell colony growth in the presence of leukemia inhibitor factor (LIF). However, chronic exposure to exogenous ATP leads to rapid P2X7-dependent cell death via necrosis. Together, these data demonstrate a novel role for P2X7 receptors in regulation of ES cell behaviour where they can mediate either a pro-survival or pro-death signal depending on the mode of activation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.