Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.729989
Title: Culture of human pluripotent stem cells and neural networks in 3D using an optogenetic approach and a hydrogel model
Author: Lee, Si Yuen
ISNI:       0000 0004 6499 6046
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Abstract:
Development of optogenetically controllable human neural network models can provide an investigative system that is relevant to the human brain. Conventional cultures of neural networks in two-dimensions (2D) have major limitations of scale. For instance, the soma of neurons in 2D is unrealistically flattened and both axon and dendrite outgrowth is restricted. Using a combination of tissue engineering techniques and the inclusion of optogenetically modified human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs), the development of a three-dimensional (3D) human neural culture model within a defined 3D microenvironment is investigated in this study. Light-sensitive neurons were successfully generated by transducing Channelrhodopsin-2 (ChR2) into human iPSC-derived NPCs and neuroblastoma cells (SH-SY5Y) using lentiviral transduction. The use of neuron specific promoters for synapsin-1 (SYN1) and calcium-calmodulin kinase II (CaMKII) in driving the expression of ChR2-Yellow Florescent Protein (YFP) within the mixed neuronal populations from hiPSC-derived neurons (Axol cells) were compared. Viability of the cells at 7 day-post-infection was 80% - 97% in all conditions tested. In line with published literature, transduction efficiency of neurons at day 14 was found to be 3% - 7% for plasmids containing the SYN1 promoter and 2% - 5% for plasmids containing the CaMKII promoter. An increase in promoter driven ChR2-YFP expression was evaluated over 28 days as the neural subpopulations matured. Stably ChR2 expression continued through-out higher passages (≥ P10) and possibly for periods up to several months. Both SYN1 and CaMKII promoters were found to drive the expression of ChR2 in Axol cells targeting inhibitory and excitatory neurons, respectively. 3D culture systems to support cell growth and optogenetic application were developed and characterised. Alginate hydrogel functionalised with short peptide sequence arginine-glycine-aspartate (RGD), and small molecules such as Rho Kinase inhibitor (ROCKi) and ZVAD were incorporated to increase the viability of human pluripotent stem cells (hPSCs). Investigation of cell response reveals that a flow rate of 3 ml/min and an alginate concentration of 1.8% (w/v) are optimal and that stem cell survival is significantly improved through incorporation of RGD and ROCKi. Interestingly, ChR2-YFP expression of Axol and SY5Y cells was detectable when transferred to the 3D culture system. The optogenetically modified neurons were found responsive to light stimulation, showing firing patterns and calcium events typical of early developing neurons (e.g. mixed and burst waves; single and multipeak spikes). Neuronal activities were assessed using calcium imaging. Higher numbers of calcium events were associated with CaMKII driven ChR2-YFP expression than with SYN1 in Axol cells. However, calcium activity in SH-SY5Y cells was most noticeable in neurons expressing ChR2-YFP driven by the SYN1 promoter. In primary rodent neuronal cultures, synchronous calcium firing with repetitive action potentials (APs) resulted from ChR2-YFP expression was driven by both SYN1 and CaMKII promoter upon light stimulation. By combining multi-approaches, we report for the first time on the generation of an in vitro hiPSC-derived neural network model in 3D using functionalised alginate hydrogel and involving optogenetic targeting. Expression of ChR2-YFP was found driven by both SYN1 and CaMKII promoter in the RGD-alginate bead system that cultured with Axol cells.
Supervisor: Seymour, Leonard ; Ye, Hua Sponsor: Ministry of Higher Education Malaysia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.729989  DOI: Not available
Keywords: Neural tissue engineering ; Human pluripotent stem cells ; Optogenetics ; 3D culture ; Hydrogels
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