Summary Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro . Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

It is currently believed that molecular agents that specifically bind to and neutralize the toxic proteins/peptides, amyloid β (Aβ42), tau, and the tau-derived peptide PHF6, hold the key to attenuating the progression of Alzheimer's disease (AD). We thus tested our previously developed nonaggregating Aβ42 double mutant (Aβ42 DM ) as a multispecific binder for three ADassociated molecules, wild-type Aβ42, the tau K174Q mutant, and a synthetic PHF6 peptide. Aβ42 DM acted as a functional inhibitor of these molecules in in vitro assays and in neuronal cell-based models of AD. The double mutant bound both cytotoxic tau K174Q and synthetic PHF6 and protected neuronal cells from the accumulation of tau in cell lysates and mitochondria. Aβ42 DM also reduced toxic intracellular levels of calcium and the overall cell toxicity induced by overexpressed tau, synthetic PHF6, Aβ42, or a combination of PHF6and Aβ42. Aβ42 DM inhibited PHF6-induced overall mitochondrial dysfunction: In particular, Aβ42 DM inhibited PHF6-induced damage to submitochondrial particles (SMPs) and suppressed PHF6-induced elevation of the ζ-potential of inverted SMPs (proxy for the inner mitochondrial membrane, IMM). PHF6 reduced the lipid fluidity of cardiolipin/DOPC vesicles (that mimic the IMM) but not DOPC (which mimics the outer mitochondrial membrane), and this effect was inhibited by Aβ42 DM . This inhibition may be explained by the conformational changes in PHF6 induced by Aβ42 DM in solution and in membrane mimetics. On this basis, the paper presents a mechanistic explanation for the inhibitory activity of Aβ42 DM against Aβ42-and tauinduced membrane permeability and cell toxicity and provides confirmatory evidence for its protective function in neuronal cells.