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Efficient Derivation of Excitatory and Inhibitory Neurons from Human Pluripotent Stem Cells Stably Expressing Direct Reprogramming Factors

Song, S; Ashok, A; Williams, D; Kaufman, M; Duff, K; Sproul, A; (2021) Efficient Derivation of Excitatory and Inhibitory Neurons from Human Pluripotent Stem Cells Stably Expressing Direct Reprogramming Factors. Current Protocols , 1 (6) , Article e141. 10.1002/cpz1.141. Green open access

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Abstract

It is essential to generate isolated populations of human neuronal subtypes in order to understand cell-type-specific roles in brain function and susceptibility to disease pathology. Here we describe a protocol for in-parallel generation of cortical glutamatergic (excitatory) and GABAergic (inhibitory) neurons from human pluripotent stem cells (hPSCs) by using the neurogenic transcription factors Ngn2 and a combination of Ascl1 and Dlx2, respectively. In contrast to the majority of neural transdifferentiation protocols that use transient lentiviral infection, the use of stable hPSC lines carrying doxycycline-inducible transcription factors allows neuronal differentiation to be initiated by addition of doxycycline and neural medium. This article presents a method to generate lentivirus from cultured mammalian cells and establish stable transcription factor–expressing cell lines (Basic Protocol 1), followed by a method for monolayer excitatory and inhibitory neuronal differentiation from the established lines (Basic Protocol 2). The resulting neurons reproducibly exhibit properties consistent with human cortical neurons, including the expected morphologies, expression of glutamatergic and GABAergic genes, and functional properties. Our approach enables the scalable and rapid production of human neurons suitable for modeling human brain diseases in a subtype-specific manner and examination of differential cellular vulnerability.

Type: Article
Title: Efficient Derivation of Excitatory and Inhibitory Neurons from Human Pluripotent Stem Cells Stably Expressing Direct Reprogramming Factors
Open access status: An open access version is available from UCL Discovery
DOI: 10.1002/cpz1.141
Publisher version: https://doi.org/10.1002/cpz1.141
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: doxycycline inducible tet-O system, excitatory neuron, human pluripotent stem cell, inhibitory neuron, neurogenic transcription factors
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > UK Dementia Research Institute
URI: https://discovery.ucl.ac.uk/id/eprint/10130353
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