Exploring the role of C5a-C5aR1 signalling in development through pluripotent stem cell modelling

Abstract

The complement system has been traditionally described as a powerful controller of innate immunity.With activation through the recognition of pathogenic surfaces, spontaneous hydrolysis, or extrinsic cleavage, a cleavage cascade is initiated culminating in the formation of the active complement fragments C3a, C3b, C5a, and C5b. These fragments direct immune cells to sites of inflammation, as well as tagging pathogens for destruction and directly lysing foreign cells. It is now clear however, that this complex family of proteins possesses a wide range of functions outside of immune regulation. From fertilisation and morphogenesis, to the control of foetal and adult stem cell populations, studies have described novel actions of complement factors. This thesis is focussed on the central complement receptor, C5aR1. Traditionally described as a potent activating and chemotactic receptor for immune cells, C5aR1 has been shown to function in a number of nonimmune cell populations. Of note, our laboratory has previously described a role for C5aR1 in neural tube closure, with loss of C5aR1 signalling associated with increased neural tube defects under folate deficient conditions. However, a mechanistic role of C5aR1 at this stage of development was not described.In this thesis, pluripotent stem cells were utilised as an in vitro model of human development to interrogate the expression and function of C5aR1 at a number of developmental stages. Specifically, in pluripotent stem cells representative of the blastocyst inner cell mass, in neural rosettes representative of the developing ventricular zone, and in matured post-mitotic cortical neurons. This builds on previous work by our laboratory, which had identified C5aR1 actions in the mouse ventricular zone, analogous to late neural rosette cultures, and had shown a neurotoxic effect of C5aR1 in post-mitotic mouse neurons.C5aR1 was found to be expressed in pluripotent stem cells, with a role in promoting maintenance of pluripotency in the absence of FGF2 signalling. Additionally, C5aR1 was found to be apically expressed in human neural rosettes, with signalling promoting proliferation and maintenance of cell polarity. This work correlated well with mouse studies performed outside of this thesis, to show that in vivo, loss of C5aR1 in this neural progenitor population resulted in behavioural deficits and microstructural brain changes. Lastly, we determined the mRNA expression of C5aR1 in human postmitotic cortical neurons. However, in contrast to previous mouse studies, exogenous C5a, alone or in the presence of secondary stressors, had little effect on the survival of these cells.Overall, the results presented in this thesis have further expanded our knowledge of C5a-C5aR1 signalling in development, describing novel roles for this signalling pathway. The use of pluripotent stem cells allowed for the exploration of C5aR1 actions in human development that would otherwise be limited to animal models. Additionally, it allowed for the correlation of previous animal resu...