Aβ assemblies plays a crucial role in AD pathology. We aimed at characterizing pathological Aβ assemblies produced by cells and investigate their seeding properties and their contribution to amyloid pathology in a FAD mouse model.To that end, coiled-coil constructs were designed to study precise APP orientations controlling Aβ production lines. Immunoprecipitation combined to mass spectrometry was used for the characterization of Aβ assemblies produced by cells, and knock-down were performed to identify the type of γ-secretase involved in their production. Nucleation properties of cell-derived Aβ assemblies were studied in vitro, and they were further injected in FAD mice to study their pathological properties.Aβ hexamers (Aβ42) were detected in cell extracts and culture media of cells expressing APP amyloidogenic fragments. They were generated by a specific dimeric orientation of APP, and predominantly associated to PS2-dependent γ secretases. Hexameric Aβ42 produced by cells displayed nucleating properties. Similar Aβ assemblies were detected both in FAD mice brains and in the cerebrospinal fluid of AD patients. Finally, cell-derived hexameric Aβ was able to seed other human Aβ forms, resulting in the aggravation of amyloid deposition in FAD mice.Aβ42 assemblies produced by cells have intrinsic nucleation features underlying their pathological properties. The PS2-dependent γ-secretase is critical for their production. They are present in FAD mice brains and human fluids, and their seeding properties aggravate the amyloid pathology in FAD mice.