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Induced pluripotent stem cells as a source for generating pancreatic islet endocrine cells represent a great research tool for deciphering the molecular mechanisms of lineage commitment, a layered multi-step process. Additionally, targeted gene silencing by using GapmeRs, short antisense oligonucleotides, proved instrumental in studying the role of different developmental genes. Here we describe our approach to induce mTOR silencing by using specific GapmeRs during the differentiation of induced pluripotent stem cells toward pancreatic progenitors. We will describe our current differentiation protocol, the transfection procedure, and the quality control steps required for a successful experiment., (© 2023. Springer Science+Business Media, LLC.)

The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5′ conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (−2139 to −1958 bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature β cells in vivo. Pdx1ΔAREAII/− mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon+ α relative to insulin+ β cells, associated with the transcriptional and epigenetic derepression of the α-cell-determining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and β cells that were incapable of maturation. Creating the Pdx1ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet β cells carrying abnormal chromatin marking in vital β-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and β-cell-specific Pdx1 functions. [ABSTRACT FROM AUTHOR]

Pancreatic islet endocrine cells generated from patient-derived induced pluripotent stem cells represent a great strategy for both disease modeling and regenerative medicine. Nevertheless, these cells inherently miss the effects of the intricate network of systemic signals characterizing the living organisms. Xenotransplantation of in vitro differentiating cells into murine hosts substantially compensates for this drawback.Here we describe our transplantation strategy of encapsulated differentiating pancreatic progenitors into diabetic immunosuppressed (NSG) overtly diabetic mice generated by the total ablation of insulin-producing cells following diphtheria toxin administration. We will detail the differentiation protocol employed, the alginate encapsulation procedure, and the xenotransplantation steps required for a successful and reproducible experiment., (© 2021. Springer Science+Business Media, LLC.)

The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing β-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is, thus, extremely valuable for managing the disease progression. Extracellular glucose is known to influence cell identity by impacting the redox balance. Here, we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex effect of exogenous glucose on differentiating pancreas progenitors characterized by a distinct proteome signature. [ABSTRACT FROM AUTHOR]

The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5' conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (-2139 to -1958 bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature β cells in vivo Pdx1 ΔAREAII/- mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon + α relative to insulin + β cells, associated with the transcriptional and epigenetic derepression of the α-cell-determining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and β cells that were incapable of maturation. Creating the Pdx1 ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet β cells carrying abnormal chromatin marking in vital β-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and β-cell-specific Pdx1 functions., (© 2017. Published by The Company of Biologists Ltd.)