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

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.

A human stem cell resource to decipher the biochemical and cellular basis of neurodevelopmental defects in Lowe syndrome

“…The neurons so generated showed the characteristic morphology and molecular markers of neuronal development including MAP2, DCX and Synapsin-1 ( Fig. 2 B) as previously reported ( Sharma et al, 2020 ). Western blot analysis of protein extracts from these lysates revealed the band corresponding to OCRL in wild-type neurons and absent in LSPH004 ( Fig.…”

“…Calcium imaging was performed in 30 DIV D149 and LSPH004 neurons, according to our previously published protocol with minor modifications ( Sharma et al, 2020 ). Briefly, neurons were washed with Tyrode's buffer solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 30 mM glucose and 25 mM HEPES, pH 7.4) for 10 min and later incubated with 4 uM fluo-4/AM (1 mM, Molecular probes, #F14201) and 0.002% pluronic F-127 (Sigma-Aldrich, #P2443) in the Tyrode's buffer solution in dark for 30–45 min at room temperature.…”

A human stem cell resource to decipher the biochemical and cellular basis of neurodevelopmental defects in Lowe Syndrome

“…The neurons so generated showed the characteristic morphology and molecular markers of neuronal development including MAP2, DCX and Synapsin-1 (Fig. 2B) as previously reported (Sharma et al, 2020). Western blot analysis of protein extracts from these lysates revealed the band corresponding to OCRL in wild type neurons and absent in LSPH004 (Fig.…”

“…Calcium imaging was performed in 30 DIV D149 and LSPH004 neurons, according to our previously published protocol with minor modifications (Sharma et al, 2020). Briefly, neurons were washed with Tyrode's buffer solution (5 mM KCl, 129 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose and 25 mM HEPES, pH 7.4) for 10mins and later incubated with 4 uM fluo-4/AM (1 mM, Molecular probes, #F14201) and 0.002% pluronic F-127 (Sigma, #P2443) in the Tyrode's buffer solution in dark for 30-45 min at room temperature.…”

IMPA1 dependent regulation of plasma membrane phosphatidylinositol 4,5-bisphosphate turnover and calcium signalling by lithium

“…Calcium imaging for measuring transients: For studying the effect of Li on the excitability of cortical neurons, spontaneous calcium events (calcium transients) were measured in D149 cortical neuronal cultures at 45 DIV for both untreated and Li treated neurons, according to previously published lab protocol [44]. The neuronal cultures were washed twice for 10 minutes with HBSS and then loaded with 4 μM of Fluo-4 AM (Molecular probes, F14201) in HBSS having 0.002% Pluronic F-127 for 30 min at room temperature.…”