Human stem cell-derived GABAergic neurons functionally integrate into human neuronal networks

Ana Gonzalez-Ramos,Zaal Kokaia,My Andersson,Merab Kokaia,Eliška Waloschková,Marco Ledri,Johan Bengzon,Apostolos Mikroulis

doi:10.1038/s41598-021-01270-x

Abstract

Gamma-aminobutyric acid (GABA)-releasing interneurons modulate neuronal network activity in the brain by inhibiting other neurons. The alteration or absence of these cells disrupts the balance between excitatory and inhibitory processes, leading to neurological disorders such as epilepsy. In this regard, cell-based therapy may be an alternative therapeutic approach. We generated light-sensitive human embryonic stem cell (hESC)-derived GABAergic interneurons (hdIN) and tested their functionality. After 35 days in vitro (DIV), hdINs showed electrophysiological properties and spontaneous synaptic currents comparable to mature neurons. In co-culture with human cortical neurons and after transplantation (AT) into human brain tissue resected from patients with drug-resistant epilepsy, light-activated channelrhodopsin-2 (ChR2) expressing hdINs induced postsynaptic currents in human neurons, strongly suggesting functional efferent synapse formation. These results provide a proof-of-concept that hESC-derived neurons can integrate and modulate the activity of a human host neuronal network. Therefore, this study supports the possibility of precise temporal control of network excitability by transplantation of light-sensitive interneurons.

Highlights

Gamma-aminobutyric acid (GABA)-releasing interneurons comprise a highly abundant cell type in the central nervous system
The MAP2 gene expression was already detected at 4 days in vitro (DIV) and showed a tendency to increase its expression over time (Fig. 1C and K)
SYN1 displayed a gradual increase of expression from 7 to 35 DIV, at which time point the maximal expression was reached indicating the onset of synaptic maturation of differentiated neurons (Fig. 1K)

Summary

Introduction

GABA-releasing interneurons comprise a highly abundant cell type in the central nervous system. The possibility to differentiate patient-specific stem cells to mature regional- and transmitter- specific subtypes of particular human interneuron populations provides an exceptional platform for studying pathophysiology as well as a potential therapeutic approach for diseases. To this goal, several studies have focused on generating GABAergic neurons from human stem cells (hSC), both induced pluripotent stem cells and ESCs9–11. Despite the high value of these studies, translation to the clinic requires a human cell source, generating a robust and consistent yield of GABAergic neurons, and proof of functional cell integration In this regard, hSCs offer great potential as an unlimited source of derived neurons for cell-based therapeutic strategies. We further confirm integration of hdINs in human epileptic brain slices obtained from surgeries for drug-resistant epilepsy, and thereby provide opportunity to modulate disease-altered human neuronal networks

Methods

Results

Conclusion