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The study of fallopian tube (FT) function in health and disease has been hampered by limited knowledge of FT stem cells and lack of in vitromodels of stem cell renewal and differentiation. Using optimized organoid culture conditions to address these limitations, we find that FT stem cell renewal is highly dependent on WNT/β-catenin signaling and engineer endogenous WNT/β-catenin signaling reporter organoids to biomark, isolate, and characterize these cells. Using functional approaches, as well as bulk and single-cell transcriptomics analyses, we show that an endogenous hormonally regulated WNT7A-FZD5 signaling axis is critical for stem cell renewal and that WNT/β-catenin pathway-activated cells form a distinct transcriptomic cluster of FT cells enriched in extracellular matrix (ECM) remodeling and integrin signaling pathways. Overall, we provide a deep characterization of FT stem cells and their molecular requirements for self-renewal, paving the way for mechanistic work investigating the role of stem cells in FT health and disease.

Despite a wealth of potential applications inside target cells, protein-based therapeutics are largely limited to extracellular targets due to the inability of proteins to readily cross biological membranes and enter the cytosol. Bacterial toxins, which deliver a cytotoxic enzyme into cells as part of their intoxication mechanism, hold great potential as platforms for delivering therapeutic protein cargo into cells. Diphtheria toxin (DT) has been shown to be capable of delivering an array of model proteins of varying sizes, structures, and stabilities into mammalian cells as amino-terminal fusions. Here, seeking to expand the utility of DT as a delivery vector, we asked whether an active human enzyme, purine nucleoside phosphorylase (PNP), could be delivered by DT into cells to rescue PNP deficiency. Using a series of biochemical and cellular readouts, we demonstrate that PNP is efficiently delivered into target cells in a receptor- and translocation-dependent manner. In patient-derived PNP-deficient lymphocytes and pluripotent stem cell-differentiated neurons, we show that human PNP is efficiently translocated into target cells by DT, where it is able to restore intracellular hypoxanthine levels. Further, through replacement of the native receptor-binding moiety of DT with single-chain variable fragments that were selected to bind mouse HBEGF, we show that PNP can be retargeted into mouse splenocytes from PNP-deficient mice, resulting in restoration of the proliferative capacity of T-cells. These findings highlight the versatility of the DT delivery platform and provide an attractive approach for the delivery of PNP as well as other cytosolic enzymes implicated in disease.

The conclusive identity of Wnts regulating liver zonation (LZ) and regeneration (LR) remains unclear despite an undisputed role of β-catenin. Using single-cell analysis, we identified a conserved Wnt2 and Wnt9b expression in endothelial cells (ECs) in zone 3. EC-elimination of Wnt2 and Wnt9b led to both loss of β-catenin targets in zone 3, and re-appearance of zone 1 genes in zone 3, unraveling dynamicity in the LZ process. Impaired LR observed in the knockouts phenocopied models of defective hepatic Wnt signaling. Administration of a tetravalent antibody to activate Wnt signaling rescued LZ and LR in the knockouts and induced zone 3 gene expression and LR in controls. Administration of the agonist also promoted LR in acetaminophen overdose acute liver failure (ALF) fulfilling an unmet clinical need. Overall, we report an unequivocal role of EC-Wnt2 and Wnt9b in LZ and LR and show the role of Wnt activators as regenerative therapy for ALF.