Optogenetic control of iPS cell-derived neurons in 2D and 3D culture systems using channelrhodopsin-2 expressed by the SYN1 and CaMKII promoters
AffiliationDepartment of Oncology, Old Road Campus Research Building, University of Oxford, UK
MetadataShow full item record
AbstractDevelopment of optogenetically controllable human neural network model in three-dimensional (3D) can provide an investigative system that is physiologically relevant or mimic to the human brain. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cells (hiPSCs) 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 was 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 sub-populations 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 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.
CitationLee SY, George JH, Nagel DA, Ye H, Kueberuwa G, Seymour LW. Optogenetic control of iPS cell-derived neurons in 2D and 3D culture systems using channelrhodopsin-2 expressed by the SYN1 and CaMKII promoters. J Tissue Eng Regen Med. 2018 Dec 14.
JournalJournal of Tissue Engineering and Regenerative Medicine
- 3D culture of human pluripotent stem cells in RGD-alginate hydrogel improves retinal tissue development.
- Authors: Hunt NC, Hallam D, Karimi A, Mellough CB, Chen J, Steel DHW, Lako M
- Issue date: 2017 Feb
- Arrays of microLEDs and astrocytes: biological amplifiers to optogenetically modulate neuronal networks reducing light requirement.
- Authors: Berlinguer-Palmini R, Narducci R, Merhan K, Dilaghi A, Moroni F, Masi A, Scartabelli T, Landucci E, Sili M, Schettini A, McGovern B, Maskaant P, Degenaar P, Mannaioni G
- Issue date: 2014
- Optogenetic approaches to characterize the long-range synaptic pathways from the hypothalamus to brain stem autonomic nuclei.
- Authors: Piñol RA, Bateman R, Mendelowitz D
- Issue date: 2012 Sep 30
- Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.
- Authors: Avaliani N, Sørensen AT, Ledri M, Bengzon J, Koch P, Brüstle O, Deisseroth K, Andersson M, Kokaia M
- Issue date: 2014 Dec
- Functional control of transplantable human ESC-derived neurons via optogenetic targeting.
- Authors: Weick JP, Johnson MA, Skroch SP, Williams JC, Deisseroth K, Zhang SC
- Issue date: 2010 Nov