Stroke is the second single most common cause of death and the largest cause of disability worldwide, highlighting the urgent need for new treatments. Mesenchymal stem cell transplantation aids regeneration and repair in animal models of ischaemia, possibly, at least in part, via interaction with microglia, modulating their inflammatory response into an anti-inflammatory phenotype favouring regeneration. Evidence shows that the beneficial effects of cell therapy are due to the release of soluble factors, raising the possibility of cell-free therapeutic strategy. Our collaborators on the current project, Pischuitta et al., (2016) recently provided evidence of the comparable effects of human amniotic stem cells (hAMSC) and hAMSC-conditioned media. They found that both hAMSC and hAMSC-CM treatments induced upregulation of the microglia marker, CD11b. In order to investigate the potential for hAMSC-CM to modulate microglia, several in vitro and in vivo approaches were utilised. The aims of this thesis were to characterise the signalling and polarisation of SIM-A9cells; establish a consistent protocol for the isolation of highly pure microglia from the mouse brain; establish and optimise an in vitro microglia migration model; and to examine the effects of hAMSC-CM on SIM-A9 cells microglial cells on signalling, polarisation and migration in vitro and on polarisation in vivo. The results revealed the ability of SIM-A9 microglial cells to be polarised in to a pro-inflammatory phenotype (induced with LPS), with the expression of pro-inflammatory genes II1B and Nos2 significantly increasing; as well as an anti-inflammatory phenotype (induced with IL-4), which led to the expression of the anti-inflammatory gene, Arg1 to significantly increase. Protocols used to extract primary microglia from the neonatal and adult mouse did not produce purity, reproducibility or good survival in culture.We showed that the addition of hAMSC-CM significantly reduced mRNA expression of II1B Tnfa and CcI2 in LPS-induced microglia. Using a multi-disciplinary approach, an in vitro microglia migration model that is a better representation of the stroke environment was established, optimised and characterised. This work showed that LPS significantly inhibited glutamate-induced migration of microglial cells, while IL-4 treatment enhanced migration. Interestingly, addition of hAMSC-CM reversed the inhibitory effect of LPS on glutamate-induced migration. Translating our in vitro findings in vivo, we then investigated the effect of hAMSC-CM after stroke, using the mouse stroke model. It was concluded that hAMSC-CM, injected 3 hours after stroke, had no effect on inflammatory markers after 24 hours of recovery. In conclusion, we have shown that hAMSC-CM can modulate microglial cells from apro-inflammatory phenotype into an anti-inflammatory phenotype. Therefore, hAMSC-CM has an anti-inflammatory effect that may be beneficial after stroke.
|Date of Award||20 Apr 2018|
- University Of Strathclyde
|Sponsors||University of Strathclyde & BBSRC (Biotech & Biological Sciences Research Council)|
|Supervisor||Hilary Carswell (Supervisor) & Robin Plevin (Supervisor)|