In the first year the work was focused on establishing a new protocol to generate OPC from neural stem cells, and the generation of viral vectors encoding ChR2. We had anticipated that this task would require 3 months of work, but in reality the task was more complicated and beset by technical difficulties. Ultimately the Experienced Researcher (Dr Otsu) succeeded in establishing an effective protocol capable of producing OPC. However, the scale of work required for this optimization, and the necessary characterization that was then performed to guarantee the nature of the cells produced, proved greater than expected. Importantly, this process of optimization and characterization has formed the greater part of the work carried out in this project, and has lead to important new discoveries on OL biology (discussed later).
During the first year Dr Otsu also began production of lentiviral vectors encoding ChR2. Two vectors were generated via a commercial service and used successfully to produce a number of lines of stem-cell derived OPC (ChR2-OPC). The expression of ChR2 proteins was analysed in these lines using fluorescence microscopy (to detect YFP reporter tags) and immunofluorescent (IF) staining to detect ChR2 proteins. All lines exhibited appropriate expression of ChR2 protein, albeit at rather low levels. One ChR2-OPC line was investigated functionally using calcium imaging. Blue light stimulation was found to increase the concentration of intracellular Ca2+ suggesting that the ChR2 proteins are functional.
Work in the second year was mainly focused on further optimization of the stem-cell derived OPC protocol and a deep characterization of the cells properties including transcriptional profile, ability to generate OL and myelin, and their lineage plasticity (ability to generate other types of neural cells). This work has provided a number of interesting and unexpected discoveries that are the subject of a manuscript (now in preparation). For more details please see Progress beyond the state of the art.
Dissemination and exploitation
At the end of the project (24 months) work establishing and investigating the new stem-cell derived OPC lines was complete and a manuscript reporting this work is now under submission.
During the project Dr Otsu also developed expertise in the production of primary OPC since these were required to validate the new stem-cell derived OPC lines. His skills in primary OPC contributed to another project in the lab that has now been published:
Begum G, Otsu M, Ahmed U, Ahmed Z, Stevens A, Fulton D. (2018) NF-Y-dependent regulation of glutamate receptor 4 expression and cell survival in cells of the oligodendrocyte lineage. Glia. Epub ahead of print, DOI: 10.1002/glia.23446.
Dr Otsu also presented the work described above at two international conferences:
(1) The XIII European Meeting on Glial Cells in Health and Disease, July 8-11 2017, Edinburgh, UK; (2) British Neuroscience Association Festival of Neuroscience, 10-13 April 2017, Birmingham, UK.
