Robust Expression of Functional NMDA Receptors in Human Induced Pluripotent Stem Cell-Derived Neuronal Cultures Using an Accelerated Protocol.

Jacob B Ruden,Brad A Grueter,Mrinalini Dixit,José C Zepeda,Laura L Dugan

doi:10.3389/fnmol.2021.777049

Abstract

N-methyl-D-aspartate (NMDA) receptors are critical for higher-order nervous system function, but in previously published protocols to convert human induced pluripotent stem cells (iPSCs) to mature neurons, functional NMDA receptors (NMDARs) are often either not reported or take an extended time to develop. Here, we describe a protocol to convert human iPSC-derived neural progenitor cells (NPCs) to mature neurons in only 37 days. We demonstrate that the mature neurons express functional NMDARs exhibiting ligand-activated calcium flux, and we document the presence of NMDAR-mediated electrically evoked postsynaptic current. In addition to being more rapid than previous procedures, our protocol is straightforward, does not produce organoids which are difficult to image, and does not involve co-culture with rodent astrocytes. This could enhance our ability to study primate/human-specific aspects of NMDAR function and signaling in health and disease.

Highlights

Much of our understanding of NMDA receptors (NMDARs) function has come from studies on cell cultures from rodents and other nonprimate animal model systems, or from immortalized cell lines with induced expression of human NMDAR subunits
The ability to study human NMDARs in the context of all the regulatory and co-activating factors necessary for human neuronal function may be critical to understanding the roles of NMDARs in physiologic and pathophysiologic conditions
Immunohistochemistry of XCL-4 converted mature cell cultures exhibited the presence of β-tubulin III (Figures 1A,B), microtubule-associated protein 2 (Figures 1A,D), NMDAR1 (Figure 1B and Supplementary Figure 4), NMDAR2A (Supplementary Figure 4), NMDAR2B (Supplementary Figure 4), synaptotagmin 1 (Figure 1C), and postsynaptic density protein 95 (Figure 1C), which are markers indicative of mature neurons

Summary

Introduction

N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamatergic receptors which are critical for neurotransmission and higher-order function of the nervous system, including long-term potentiation (LTP), memory formation and consolidation, and maintenance of neuronal plasticity (Liu et al, 2007; Hunt and Castillo, 2012; Luscher and Malenka, 2012; Paoletti et al, 2013; Chakraborty et al, 2017). Differences between mice and humans in protein abundance of postsynaptic density factors found in the NMDAR complex have been reported (Bayes et al, 2012). These differences could have major implications for understanding the roles of NMDARs in health and disease and as drug targets. The ability to study human NMDARs in the context of all the regulatory and co-activating factors necessary for human neuronal function may be critical to understanding the roles of NMDARs in physiologic and pathophysiologic conditions

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Conclusion