We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3bright+-sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1+/ALK3bright+ populations (enriched in PDX1+/ALK3+/CAII− cells) differentiate into all pancreatic lineages, including functional β-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1+/ALK3+/CAII− progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.

MicroRNAs regulate gene expression by inhibiting translation or inducing target mRNA degradation. MicroRNAs regulate organ differentiation and embryonic development, including pancreatic specification and islet function. We showed previously that miR-7 is highly expressed in human pancreatic fetal and adult endocrine cells. Here we determined the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic days e10.5 and e11.5, then began to increase at e13.5 through e14.5, and eventually decreased by e18. In situ hybridization and immunostaining analysis showed that miR-7 colocalizes with endocrine marker Isl1, suggesting that miR-7 is expressed preferentially in endocrine cells. Whole-mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall downregulation of insulin production, decreased β-cell numbers, and glucose intolerance in the postnatal period. This phenomenon is specific for miR-7 and possibly due to a systemic effect on pancreatic development. On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds leads to β-cell death and generation of β-cells expressing less insulin than those in MO control. Therefore, in addition to the potential indirect effects on pancreatic differentiation derived from its systemic downregulation, the knockdown of miR-7 appears to have a β-cell-specific effect as well. These findings suggest that modulation of miR-7 expression could be utilized in the development of stem cell therapies to cure diabetes.

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas.

The exocrine pancreas can give rise to endocrine insulin-producing cells upon ectopic expression of key transcription factors. However, the need for genetic manipulation remains a translational hurdle for diabetes therapy. Here we report the conversion of adult human nonendocrine pancreatic tissue into endocrine cell types by exposure to bone morphogenetic protein 7. The use of this U.S. Food and Drug Administration–approved agent, without any genetic manipulation, results in the neogenesis of clusters that exhibit high insulin content and glucose responsiveness both in vitro and in vivo. In vitro lineage tracing confirmed that BMP-7–induced insulin-expressing cells arise mainly from extrainsular PDX-1+, carbonic anhydrase II− (mature ductal), elastase 3a (acinar)−, and insulin− subpopulations. The nongenetic conversion of human pancreatic exocrine cells to endocrine cells is novel and represents a safer and simpler alternative to genetic reprogramming.

SUMMARYTreatment of human pancreatic non-endocrine tissue with Bone Morphogenetic Protein 7 (BMP-7) leads to the formation of glucose-responsive β-like cells. Here, we show that BMP-7 acts on extrainsular cells expressing PDX1 and the BMP receptor activin-like kinase 3 (ALK3/BMPR1A). In vitro lineage tracing indicates that ALK3+ cell populations are multipotent. PDX1+/ALK3+ cells are absent from islets but prominently represented in the major pancreatic ducts and pancreatic duct glands. We identified the puri-nergic receptor P2Y1 (P2RY1) as a surrogate surface marker for PDX1. Sorted P2RY1+/ALK3bright+ cells form BMP-7-expandable colonies characterized by NKX6.1 and PDX1 expression. Unlike the negative fraction controls, these colonies can be differentiated into multiple pancreatic lineages upon BMP-7 withdrawal. RNA-seq further corroborates the progenitor-like nature of P2RY1+/ALK3bright+ cells and their multilineage differentiation potential. Our studies confirm the existence of progenitor cells in the adult human pancreas and suggest a specific anatomical location within the ductal and glandular networks.

Conventional culture vessels are not designed for physiological oxygen (O 2 ) delivery. Both hyperoxia and hypoxia-commonly observed when culturing cells in regular plasticware-have been linked to reduced cellular function and death. Pancreatic islets, used for the clinical treatment of diabetes, are especially sensitive to sub-and supraphysiological O 2 concentrations. A result of current culture standards is that a high percentage of islet preparations are never transplanted because of cell death and loss of function in the 24-48 h postisolation. Here, we describe a new culture system designed to provide quasiphysiological oxygenation to islets in culture. The use of dishes where islets rest atop a perfluorocarbon (PFC)-based membrane, coupled with a careful adjustment of environmental O 2 concentration to target the islet physiological pO 2 range, resulted in dramatic gains in viability and function. These observations underline the importance of approximating culture conditions as closely as possible to those of the native microenvironment, and fill a widely acknowledged gap in our ability to preserve islet functionality in vitro. As stem cell-derived insulin-producing cells are likely to suffer from the same limitations as those observed in real islets, our findings are especially timely in the context of current efforts to define renewable sources for transplantation.

The emerge of personalized medicine demands high-quality human biospecimens with appropriate clinical annotation, especially in complex diseases such as cancer, neurodegenerative, cardiovascular, and metabolic alterations in which specimen heterogeneity and individual responses often complicate the development of precision therapeutic programs. In the growing field of extracellular vesicles (EVs) research, exosomes (EXOs)—a particular type of EVs—have been proposed as an advantageous diagnostic tool, as effective delivery vehicles and as therapeutic targets. However, the lack of consensus on isolation methods and rigorous criteria to characterize them puts the term EXO into question at the time that might explain some of the controversial results found in the literature. A lack of response in the biobank network to warrant standard optimized procedures for the isolation, characterization, and storage of EXOs will undoubtedly lead to a waste of resources and failure. This review is aimed at highlighting the increasing importance of EXOs for the clinic, especially in the cancer field, and at summarizing the initiatives taken to improve current isolation procedures, classification criteria, and storage conditions of EXOs as an effort to identify technological demands that biobank platforms face for the incorporation of EXOs and other extracellular vesicle fractions as valuable biospecimens for research.

SummaryThe transplantation of human embryonic stem cell (hESC)-derived insulin-producing β cells for the treatment of diabetes is finally approaching the clinical stage. However, even with state-of-the-art differentiation protocols, a significant percentage of undefined non-endocrine cell types are still generated. Most importantly, there is the potential for carry-over of non-differentiated cell types that may produce teratomas. We sought to modify hESCs so that their differentiated progeny could be selectively devoid of tumorigenic cells and enriched for cells of the desired phenotype (in this case, β cells). Here we report the generation of a modified hESC line harboring two suicide gene cassettes, whose expression results in cell death in the presence of specific pro-drugs. We show the efficacy of this system at enriching for β cells and eliminating tumorigenic ones both in vitro and in vivo. Our approach is innovative inasmuch as it allows for the preservation of the desired cells while eliminating those with the potential to develop teratomas.