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Optogenetic control of iPS cell‐derived neurons in 2D and 3D culture systems using channelrhodopsin‐2 expressed by the SYN1 and CaMKII promoters
摘要： Development 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.
关键词： channelrhodopsin-2 (ChR2),Optogenetics,induced pluripotent stem cell (iPSC),3D culture,calcium-calmodulin kinase II (CaMKII),synapsin-1 (SYN1),alginate hydrogel,neural tissue engineering
更新于2025-09-23 15:19:57
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Fluorescent Metal-Doped Carbon Dots for Neuronal Manipulations
摘要： There is a growing need for biocompatible nanocomposites that may efficiently interact with biological tissues through multiple modalities. Carbon dots (CDs) could serve as biocompatible fluorescence nanomaterials for targeted tissue/cell imaging. Important goals toward this end are to enhance the fluorescence quantum yields of the CDs and to increase their targetability to cells. Here, sonochemistry was used to develop a one-pot synthesis of CDs, including metal-doped CDs (M@CDs), demonstrating how various experimental parameters, such as sonication time, temperature, and power of sonication affect the size of the CDs (2–10 nm) and their fluorescence properties. The highest measured quantum yield of emission was ~16%. Similarly, we synthesized CDs doped with different metals (M@CDs) including Ga, Sn, Zn, Ag, and Au. The interaction of M@CDs with neuron-like cells was examined and showed efficient uptake and low cytotoxicity. Moreover, the influence of the M@CDs on the improvement of neurites during initiation and elongation growth phases were compared with pristine CDs. Our research demonstrates the use of M@CDs for imaging and for neuronal interactions. The M@CD nanocomposites are promising due to their biocompatibility, photo-stability and potential selective affinity, paving the way for multifunctional biomedical applications.
关键词： sonication,neurite outgrowth,biocompatibility,metal-doped carbon dots,neural tissue engineering,fluorescence
更新于2025-09-09 09:28:46