<p>Significant advances over the last two decades in the study of in vitro prion formation and propagation have revealed that distinct cofactors can facilitate or induce spontaneous prion misfolding. This, in turn, has raised important questions about the role of cofactors and their potential significance in vivo in prion diseases. Key questions include whether cofactors are necessary for prion infectivity or whether they might play a crucial role in determining strain features without being essential for infection. Grounded in previous work that showed that polyanions such as RNA or dextran sulfate facilitate spontaneous prion misfolding in vitro, we have addressed whether other chemically similar cofactors could expand the diversity of PrP^Sc conformers and whether these would exhibit distinctive strain features. Using the Protein Misfolding Shaking Amplification (PMSA) and three different polyanionic cofactors (heparin, chondroitin sulfate and pentosan polysulfate), we obtained and characterized a total of nine conformers and compared them to previously generated strains obtained with dextran sulfate. All nine conformers proved infectious in transgenic mice, generating distinct prion strains and suggesting that different cofactors can indeed drive the formation of distinct conformers. However, the observed variations within conformers generated with the same cofactor indicate a degree of structural flexibility, likely resulting in related but distinguishable groups of conformers. Our study demonstrates that sulfated glycans not only facilitate in vitro spontaneous PrP^Sc generation but also enable the emergence of multiple distinct prion strains, providing insights into the molecular mechanisms underlying strain diversity and their potential relevance to spontaneous prion diseases.</p>