Generation and molecular characterization of human pluripotent stem cell-derived pharyngeal foregut endoderm.

Approaches to study human pharyngeal foregut endoderm-a developmental intermediate that is linked to various human syndromes involving pharynx development and organogenesis of tissues such as thymus, parathyroid, and thyroid-have been hampered by scarcity of tissue access and cellular models. We present an efficient stepwise differentiation method to generate human pharyngeal foregut endoderm from pluripotent stem cells. We determine dose and temporal requirements of signaling pathway engagement for optimized differentiation and characterize the differentiation products on cellular and integrated molecular level. We present a computational classification tool, "CellMatch," and transcriptomic classification of differentiation products on an integrated mouse scRNA-seq developmental roadmap confirms cellular maturation. Integrated transcriptomic and chromatin analyses infer differentiation stage-specific gene regulatory networks. Our work provides the method and integrated multiomic resource for the investigation of disease-relevant loci and gene regulatory networks and their role in developmental defects affecting the pharyngeal endoderm and its derivatives.

Exploring the Substrate Specificity of a Sugar Transporter with Biosensors and Cheminformatics.

Sugars will eventually be exported transporters (SWEETs) are conserved sugar transporters that play crucial roles in plant physiology and biotechnology. The genomes of flowering plants typically encode about 20 SWEET paralogs that can be classified into four clades. Clades I, II, and IV have been reported to favor hexoses, while clade III SWEETs prefer sucrose. However, the molecular features of substrates required for recognition by members of this family have not been investigated in detail. Here, we show that SweetTrac1, a previously reported biosensor constructed from the Clade I Arabidopsis thaliana SWEET1, can provide insight into the structural requirements for substrate recognition. The biosensor translates substrate binding to the transporter into a change in fluorescence, and its application in a small-molecule screen combined with cheminformatics uncovered 12 new sugars and their derivatives capable of eliciting a response. Furthermore, we confirmed that the wild-type transporter mediates cellular uptake of three of these species, including the diabetes drugs 1-deoxynojirimycin and voglibose. Our results show that SWEETs can recognize different furanoses, pyranoses, and acyclic sugars, illustrating the potential of combining biosensors and computational techniques to uncover the basis of substrate specificity.

Single-cell analysis reveals effective siRNA delivery in brain tumors with microbubble-enhanced ultrasound and cationic nanoparticles.

RNA-based therapies offer unique advantages for treating brain tumors. However, tumor penetrance and uptake are hampered by RNA therapeutic size, charge, and need to be "packaged" in large carriers to improve bioavailability. Here, we have examined delivery of siRNA, packaged in 50-nm cationic lipid-polymer hybrid nanoparticles (LPHs:siRNA), combined with microbubble-enhanced focused ultrasound (MB-FUS) in pediatric and adult preclinical brain tumor models. Using single-cell image analysis, we show that MB-FUS in combination with LPHs:siRNA leads to more than 10-fold improvement in siRNA delivery into brain tumor microenvironments of the two models. MB-FUS delivery of Smoothened (SMO) targeting siRNAs reduces SMO protein production and markedly increases tumor cell death in the SMO-activated medulloblastoma model. Moreover, our analysis reveals that MB-FUS and nanoparticle properties can be optimized to maximize delivery in the brain tumor microenvironment, thereby serving as a platform for developing next-generation tunable delivery systems for RNA-based therapy in brain tumors.