Optogenetic control of iPS cell‐derived neurons in 2D and 3D culture systems using channelrhodopsin‐2 expression driven by the synapsin‐1 and calcium‐calmodulin kinase II promoters

Abstract

Development of an optogenetically controllable human neural network model in three‐dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light‐sensitive neurons were generated by transducing channelrhodopsin‐2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell‐type specific promoters. A mixed population of human iPSC‐derived cortical neurons, astrocytes and progenitor cells were obtained (Axol‐ChR2) upon neural differentiation. Pan‐neuronal promoter synapsin‐1 (SYN1) and excitatory neuron‐specific promoter calcium‐calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol‐ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine‐glycine‐aspartate (RGD) and small molecules (Y‐27632). Improved RGD‐alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol‐ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2‐ by both the CaMKII and SYN1 promoters. The RGD‐alginate hydrogel system supports the growth of differentiated Axol‐ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non‐invasive control of human neural networks in 3D.

Electrophysiological Analysis of Brain Organoids: Current Approaches and Advancements

“…Despite relatively few applications in hiPSC-derived cells thus far, the potential for optogenetics to improve hiPSC and brain organoid models by allowing for deeper mechanistic analysis has been recognized ( Chin and Goh, 2015 ; Su et al, 2015 ; Trujillo and Muotri, 2018 ). Relatively low transfection efficiency in hiPSC-derived cells compared to somatic cells may be partially responsible for the slow adoption of optogenetics in brain organoids and other hiPSC-derived cells; however, recent advances and comparisons of transfection techniques may help increase these studies moving forward ( Chin and Goh, 2015 ; Rapti et al, 2015 ; Lee et al, 2019 ). Indeed, a neuromuscular junction (NMJ) model implementing hiPSC-derived neurospheres and muscle tissue was recently used to assess functional deficits in amyotrophic lateral sclerosis (ALS) ( Osaki et al, 2018 ).…”

Advances in 3D neuronal microphysiological systems: towards a functional nervous system on a chip

“…Microscopy utilizing ratiometric fluorescent dyes, genetically encoded calcium reporters and two-photon imaging techniques has allowed for the real-time assessment of synchronous activity in many tissue types, including neurons (Grienberger and Konnerth 2012;Mitani and Komiyama 2018). Similarly, the utilization of optogenetic technology has been used in 3D neuronal models to induce action potentials selectively via specific genetic promoters and to activate organ systems through synapses (Renault et al 2015;Lee et al 2019), such as the neuromuscular junction (Osaki et al 2020). Multiplanar imaging of complex neural circuits has also improved and allows for the study of neurons both in vitro and in vivo (Yang et al 2016).…”

3D Cell Culture—Can It Be As Popular as 2D Cell Culture?

“…[ 108 ] Compared with cells subjected to static culture, mechanical stretching, or compression of 3D cell‐hydrogel arrays, such cells show more mineralization. [ 109 ] In addition, light, [ 110 ] electricity, [ 111,112 ] oxygen concentration, [ 113,114 ] and magnetism [ 115 ] also affect cell functions.…”