Stem-Cell-Derived ß-Like Cells with a Functional PTPN2 Knockout Display Increased Immunogenicity.

Type 1 diabetes is a polygenic disease that results in an autoimmune response directed against insulin-producing beta cells. PTPN2 is a known high-risk type 1 diabetes associated gene expressed in both immune- and pancreatic beta cells, but how genes affect the development of autoimmune diabetes is largely unknown. We employed CRISPR/Cas9 technology to generate a functional knockout of PTPN2 in human pluripotent stem cells (hPSC) followed by differentiating stem-cell-derived beta-like cells (sBC) and detailed phenotypical analyses. The differentiation efficiency of PTPN2 knockout (PTPN2 KO) sBC is comparable to wild-type (WT) control sBC. Global transcriptomics and protein assays revealed the increased expression of HLA Class I molecules in PTPN2 KO sBC at a steady state and upon exposure to proinflammatory culture conditions, indicating a potential for the increased immune recognition of human beta cells upon differential PTPN2 expression. sBC co-culture with autoreactive preproinsulin-reactive T cell transductants confirmed increased immune stimulations by PTPN2 KO sBC compared to WT sBC. Taken together, our results suggest that the dysregulation of PTPN2 expression in human beta cell may prime autoimmune T cell reactivity and thereby contribute to the development of type 1 diabetes.

Dietary bovine milk miRNAs transported in extracellular vesicles are partially stable during GI digestion, are bioavailable and reach target tissues but need a minimum dose to impact on gene expression.

PURPOSE: Extracellular RNAs are unstable and rapidly degraded unless protected. Bovine-milk extracellular vesicles (EVs) confer protection to dietary miRNAs, although it remains unclear whether this importantly improves their chances of reaching host target cells to exert biological effects. METHODS: Caco-2, HT-29, Hep-G2 and FHs-74 cell lines were exposed to natural/labelled milk EVs to evaluate cellular uptake. Five frequently reported human milk miRNAs (miR-146b-5p, miR-148a-3p, miR-30a-5p, miR-26a-5p, and miR-22-3p) were loaded into EVs. The intracellular concentration of each miRNA in cells was determined. In addition, an animal study giving an oral dose of loaded EVs in C57BL6/ mice were performed. Gene expression regulation was assessed by microarray analysis. RESULTS: Digestive stability analysis showed high overall degradation of exogenous miRNAs, although EV-protected miRNAs better resisted gastrointestinal digestion compared to free miRNAs (tenfold higher levels). Importantly, orally delivered EV-loaded miRNAs reached host organs, including brain, in mice. However, no biological effect has been identified. CONCLUSION: Milk EVs protect miRNAs from degradation and facilitate cellular uptake. miRNA concentration in EVs from bovine milk might be insufficient to produce gene modulation. Nevertheless, sizable amounts of exogenous miRNAs may be loaded into EVs, and orally delivered EV-loaded miRNAs can reach tissues in vivo, increasing the possibility of exerting biological effects. Further investigation is justified as this could have an impact in the field of nutrition and health (i.e., infant formulas elaboration).

ENTPD3 Marks Mature Stem Cell-Derived ß-Cells Formed by Self-Aggregation In Vitro.

Stem cell-derived ß-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult ß-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human ß-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

Specific and Non-Invasive Fluorescent Labelling of Extracellular Vesicles for Evaluation of Intracellular Processing by Intestinal Epithelial Cells.

The presence of extracellular vesicles (EVs) in milk has gained interest due to their capacity to modulate the infant's intestinal and immune system. Studies suggest that milk EVs are enriched in immune-modulating proteins and miRNA, highlighting their possible health benefits to infants. To assess uptake of milk EVs by intestinal epithelial cells, a method was developed using labelling of isolated EVs with fluorophore-conjugated lactadherin. Lactadherin is a generic and validated EV marker, which enables an effective labelling of phosphatidylserine (PS) exposing EVs. Labelled EVs could effectively be used to describe a dose- and time-dependent uptake into the intestinal epithelial Caco-2 cell line. Additionally, fluorescence microscopy was employed to show that EVs colocalize with endosomal markers and lysosomes, indicating that EVs are taken up via general endocytotic mechanisms. Collectively, a method to specifically label isolated EVs is presented and employed to study the uptake of milk EVs by intestinal epithelial cells.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.