In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function

Wellcome Open Res. 2020 Feb 3;5:16. doi: 10.12688/wellcomeopenres.15626.1. eCollection 2020.

Abstract

The development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Developmental cues are transduced into cellular processes through the action of multiple intracellular second messengers including calcium. Calcium signals in cells are shaped by large number of proteins and mutations in several of these have been reported in human patients with brain disorders. However, the manner in which such mutations impact human brain development in vivo remains poorly understood. A key limitation in this regard is the need for a model system in which calcium signaling can be studied in neurons of patients with specific brain disorders. Here we describe a protocol to differentiate human neural stem cells into cortical neuronal networks that can be maintained as live cultures up to 120 days in a dish. Our protocol generates a 2D in vitro culture that exhibits molecular features of several layers of the human cerebral cortex. Using fluorescence imaging of intracellular calcium levels, we describe the development of neuronal activity as measured by intracellular calcium transients during development in vitro. These transients were dependent on the activity of voltage gated calcium channels and were abolished by blocking sodium channel activity. Using transcriptome analysis, we describe the full molecular composition of such cultures following differentiation in vitro thus offering an insight into the molecular basis of activity. Our approach will facilitate the understanding of calcium signaling defects during cortical neuron development in patients with specific brain disorders and a mechanistic analysis of these defects using genetic manipulations coupled with cell biological and physiological analysis.

Keywords: Stem cells; calcium imaging; disease in a dish; human brain disease; neuronal differentiation; transcriptomics.

Grant support

This work was supported by the Wellcome Trust through a Wellcome-DBT India Alliance Senior Fellowship to PR [IA/S/14/2/501540]. This work was also supported by the National Centre for Biological Sciences-TIFR, the Department of Biotechnology, Government of India through the Accelerator Program for Discovery in Brain Disorders (BT/PR17316/MED/31/326/2015). AJ was supported by a Research Associate Fellowship from the Department of Biotechnology, Government of India. We thank the NCBS Imaging and NGS facility for support.