Rapid Ngn2-induction of excitatory neurons from hiPSC-derived neural progenitor cells

Abstract

Since the discovery of somatic reprogramming, human induced pluripotent stem cells (hiPSCs) have been exploited to model a variety of neurological and psychiatric disorders. Because hiPSCs represent an almost limitless source of patient-derived neurons that retain the genetic variations thought to contribute to disease etiology, they have been heralded as a patient-specific platform for high throughput drug screening. However, the utility of current protocols for generating neurons from hiPSCs remains limited by protracted differentiation timelines and heterogeneity of the neuronal phenotypes produced. Neuronal induction via the forced expression of exogenous transcription factors rapidly induces defined populations of functional neurons from fibroblasts and hiPSCs. Here, we describe an adapted protocol that accelerates maturation of functional excitatory neurons from hiPSC-derived neural progenitor cells (NPCs) via lentiviral transduction of Neurogenin 2 (using both mNgn2 and hNGN2). This methodology, relying upon a robust and scalable starting population of hiPSC NPCs, should be readily amenable to scaling for hiPSC-based high-throughput drug screening.

THC Treatment Alters Glutamate Receptor Gene Expression in Human Stem Cell-Derived Neurons

“…We and others have previously demonstrated that NGN2 -hiPSC neurons [35], NGN2 -NPC neurons [36], and directed differentiation forebrain neurons [37,40] have neuronal morphology (online suppl. Fig.…”

“…NGN2 -NPC neurons: NPCs were dissociated with Accutase, plated on Matrigel, spinfected with doxycycline-inducible NGN2 lentivirus, and selected with 0.2 μg/mL puromycin, as previously described [36]. Neurons were fed neural differentiation media every other day for 3 weeks after induction.…”

“…Lentivirus production was as previously described [36]: 12.2 μg lentiviral DNA, 8.1 μg MDL-gagpol, 3.1 μg Rev-RSV, and 4 μg CMV-VSVG were mixed together with 500 μL of Opti-MEM (Life Technologies) and 1 μg/μL polyethylenimine (Polysciences) 25kD linear and added per 15 cm plate of HEK 293T cells. Medium was changed 5 h later.…”

“…Because neural cells differentiated from human-induced pluripotent stem cells (hiPSCs) most resemble fetal brain tissue [31,32,33,34], they provide an unprecedented platform for studying the molecular, cellular, and functional results of fetal THC exposure to neural cells, across a variety of genetic backgrounds. In this study, we demonstrate that THC treatment of human excitatory neurons (whether generated by NGN2 induction from hiPSCs ( NGN2 - hiPSC neurons) [35] or hiPSC-derived neural progenitor cells ( NGN2 -NPC neurons) [36], or via directed differentiation from NPCs (forebrain neurons) [37,40] recapitulated several known molecular consequences of THC exposure, such as changes in glutamate receptor subunit expression, at least partially in a CB1R-dependent manner.…”

Personalized medicine in a dish: the growing possibility of neuropsychiatric disease drug discovery tailored to patient genetic variants using stem cells

“…They are straightforward to maintain in vitro, requiring less frequent feeding and passaging than their source hiPSCs (25). NPCs proliferate robustly, are cryopreservable, and easily differentiated or induced to mature neurons (25,26) and astrocytes (27). Overall, NPCs are an ideal cell type for mechanistic studies of disease biology as well as adaptation to high throughput drug screens.…”