Project description:The goal of this study was to compare the endogenous tRNA expression levels from cells transduced with a lentiviral vector encopding Cas9 and an sgRNA from a U6 or a Glutamine tRNA promoter. Using high-throughput sequencing methods (Illumina Hi-Seq 2000) we show that endogenous tRNA expression levels are not perturbed by expression of an sgRNA from a human tRNA. tRNA expression profiles were generated from 2 different transduced polyconal cell lines.
Project description:The goal of this study was to compare the endogenous tRNA expression levels from cells transduced with a lentiviral vector encopding Cas9 and an sgRNA from a U6 or a Glutamine tRNA promoter. Using high-throughput sequencing methods (Illumina Hi-Seq 2000) we show that endogenous tRNA expression levels are not perturbed by expression of an sgRNA from a human tRNA.
Project description:The generation of pancreatic organoids from human pluripotent stem cells represents a major breakthrough for regenerative medicine and the modeling of diseases such as diabetes. However, current approaches remain inefficient due to lengthy multi-step differentiation protocols and limited functional maturity in the organoids. In this study, we overcome these challenges using multi-phase optimization screens to achieve rapid generation of functionally mature pancreatic organoids from a stable endocrine progenitor culture. We conducted stepwise culture condition screens that enabled the stable culture of multiple pancreatic progenitor cell states, including the unprecedented stable propagation of NEUROD1-expressing endocrine progenitor-like cells (EpSCs). Further transcriptomic profiling of EpSC confirmed similarity of that to previously reported endocrine progenitor populations. Using EpSCs, we significantly reduced the number of steps and timing required to generate pancreatic organoids, enabling rapid testing of conditions for organoid maturation. Utilizing this optimized protocol, we further tested conditions to promote pancreatic organoid maturation. We identified that exosome-delivered WNT5B, in combination with RSPO1 (exoW/R), could strongly induce non-canonical WNT/JNK signaling, promoting pancreatic organoid maturation. This combinatorial exosome treatment enhances epithelial organization, reduces immature cell states, and significantly improves glucose responsiveness and insulin secretion. Collectively, our work establishes a robust pancreatic differentiation platform that integrates long-term progenitor expansion with optimized organoid maturation. This system provides a reproducible experimental framework for studying pancreatic development, investigating disease mechanisms, and facilitating future translational applications involving pancreatic organoids.
Project description:Protein truncating variants caused by stop codons are the most prevalent class of rare variant mutations in neurodevelopmental diseases, with UGA codons being most common. Suppressor transfer RNA (sup-tRNA) have therapeutic potential for premature termination codon (PTC) rescue, but have thus far underperformed by traditional AAV delivery platforms and progress has been hampered by the lack of methods to non-invasively assess in vivo activity in mammalian brain. To fill this material gap, we utilize transcranial in vivo bioluminescence imaging data from a luciferase-UGA mouse model to optimize viral payloads with sup-tRNA genes. These data demonstrate that U6 promoter-driven and single-stranded AAV2/9 constructs show variable and dose-dependent activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100bp genomic context provides broad and efficacious PTC rescue. Further, payload tRNA multiplexing and use of tRNA introns enable efficacy of low viral titers and sustained rescue. tRNA sequencing of scAAV delivered ArgUGA sup-tRNA in brain demonstrate no effects on endogenous tRNA levels, their acylation or processing, and these features are also maintained in scAAV delivered ArgUGA sup-tRNA. Collectively, this work defines a scalable strategy for precision UGA PTC stop codon suppression, supporting development of durable genetic rescue therapies for neurodevelopmental disorders in the mammalian brain.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.