Investigation of the expression pattern and function of intracellular complement factor C3 in hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells (HSPCs) maintain lifelong blood production owing to a tight regulation of self-renewal and differentiation. Recent studies have revealed that the complement system also operates intracellularly, where it modulates key physiological processes. In particular, the complement factor C3 has been found in HSPCs and has emerged as a novel regulator of hematopoiesis. This thesis aimes to investigate the expression pattern and functional role of C3 in the hematopoietic system, focusing on HSPCs. Using Western blot, cell fractionation and immunofluorescence, C3-derived fragments and their subcellular localizations were characterized in hematopoietic cells. Higher intracellular C3 levels, particularly iC3b, were observed in less differentiated cells. Moreover, C3 was detected across all subcellular compartments, and for the first time, its presence was demonstared in the nucleus of LSK cells. Collectively, these findings suggest that C3 may play a role in regulating HSPC function, possibly through modulation of gene expression. To investigate intracellular C3 function, a knockdown model was established in NIH/3T3 and LSK cells. Although C3-shRNA efficiently reduced C3 mRNA levels, protein expression remained largely unchanged. This inverse correlation between C3 mRNA and protein levels was consistently observed across several cell types and conditions. In addition, the potential uptake of C3 from the extracellular environment suggests that de novo synthesis and external sources cooperate to maintain stable intracellular C3 levels. Despite this discrepancy, these experiments indicate that changes in intracellular C3 levels are associated with changes in glycolytic activity and cell proliferation, independent of cell type. Overall, these results highlight the complex regulation and role of the complement system in hematopoiesis and suggest a link between intracellular C3 and HSPC homeostasis. Although many aspects of intracellular C3 dynamics and modulation remain to be clarified, this work provides initial insights into C3 expression and function in HSPCs. Further investigations may uncover important mechanisms underlying the elaborate interplay of intracellular and niche-derived factors that regulate HSPC fate.