Single-cell resolution analysis of the human pancreatic ductal progenitor cell niche.

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 ß-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.

CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation.

Approximately 12% of all human cancers worldwide are caused by infections with oncogenic viruses. Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8) is one of the oncogenic viruses responsible for human cancers, including Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL), and the lymphoproliferative disorder multicentric Castleman's disease (MCD). Chronic inflammation mediated by KSHV infection plays a decisive role in the development and survival of these cancers. NF-κB, a family of transcription factors regulating inflammation, cell survival, and proliferation, is persistently activated in KSHV-infected cells. The KSHV latent and lytic expressing oncogenes involved in NF-κB activation are vFLIP/K13 and vGPCR, respectively. However, the mechanisms by which NF-κB is activated by vFLIP and vGPCR are poorly understood. In this study, we have found that a host molecule, Cell Adhesion Molecule 1 (CADM1), is robustly upregulated in KSHV-infected PBMCs and KSHV-associated PEL cells. Further investigation determined that both vFLIP and vGPCR interacted with CADM1. The PDZ binding motif localized at the carboxyl terminus of CADM1 is essential for both vGPCR and vFLIP to maintain chronic NF-κB activation. Membrane lipid raft associated CADM1 interaction with vFLIP is critical for the initiation of IKK kinase complex and NF-κB activation in the PEL cells. In addition, CADM1 played essential roles in the survival of KSHV-associated PEL cells. These data indicate that CADM1 plays key roles in the activation of NF-κB pathways during latent and lytic phases of the KSHV life cycle and the survival of KSHV-infected cells.

The Role of MicroRNAs in Diabetes-Related Oxidative Stress.

Cellular stress, combined with dysfunctional, inadequate mitochondrial phosphorylation, produces an excessive amount of reactive oxygen species (ROS) and an increased level of ROS in cells, which leads to oxidation and subsequent cellular damage. Because of its cell damaging action, an association between anomalous ROS production and disease such as Type 1 (T1D) and Type 2 (T2D) diabetes, as well as their complications, has been well established. However, there is a lack of understanding about genome-driven responses to ROS-mediated cellular stress. Over the last decade, multiple studies have suggested a link between oxidative stress and microRNAs (miRNAs). The miRNAs are small non-coding RNAs that mostly suppress expression of the target gene by interaction with its 3'untranslated region (3'UTR). In this paper, we review the recent progress in the field, focusing on the association between miRNAs and oxidative stress during the progression of diabetes.

Intra-Amniotic Soluble Endoglin Impairs Lung Development in Neonatal Rats.

Soluble endoglin (sENG) is increased in the amniotic fluid of women with preeclampsia and chorioamnionitis. Preterm infants born to women with these disorders have an increased risk of aberrant lung development. Whether this increased risk is secondary to elevated sENG levels is unclear. The objective of this study was to determine whether intrauterine exposure to an adenovirus overexpressing sENG impairs neonatal lung angiogenesis by modulating lung endothelial nitric oxide synthase (eNOS) signaling. Pregnant Sprague-Dawley rats were randomly assigned to receive ultrasound-guided intra-amniotic injections of adenovirus overexpressing sENG (Ad-sENG) or control adenovirus (Ad-control) on embryonic day 17. After this exposure, rat pups were maintained in normoxia and evaluated on postnatal day 14. Intra-amniotic Ad-sENG decreased lung vascular endothelial growth factor receptor 2 and eNOS expression in rat pups. This was accompanied by a marked decrease in lung angiogenesis and alveolarization. Ad-sENG-exposed pups also had an increase in right ventricular systolic pressure, weight ratio of right ventricle to left ventricle plus septum, and pulmonary vascular remodeling. In addition, exposure of human pulmonary artery endothelial cells to recombinant sENG reduced endothelial tube formation and protein levels of eNOS, phosphorylated eNOS, and phosphorylated Smad1/5. Together, our findings demonstrate that intrauterine exposure to an adenovirus overexpressing sENG disrupts lung development by impairing Smad1/5-eNOS signaling. We speculate that sENG-mediated dysregulation of Smad1/5-eNOS signaling contributes to impaired lung development and potentially primes the developing lung for further postnatal insults. Further studies exploring the relationship between amniotic fluid sENG levels and preterm respiratory outcomes will be necessary.

Concise review: clinical programs of stem cell therapies for liver and pancreas.

Regenerative medicine is transitioning into clinical programs using stem/progenitor cell therapies for repair of damaged organs. We summarize those for liver and pancreas, organs that share endodermal stem cell populations, biliary tree stem cells (hBTSCs), located in peribiliary glands. They are precursors to hepatic stem/progenitors in canals of Hering and to committed progenitors in pancreatic duct glands. They give rise to maturational lineages along a radial axis within bile duct walls and a proximal-to-distal axis starting at the duodenum and ending with mature cells in the liver or pancreas. Clinical trials have been ongoing for years assessing effects of determined stem cells (fetal-liver-derived hepatic stem/progenitors) transplanted into the hepatic artery of patients with various liver diseases. Immunosuppression was not required. Control subjects, those given standard of care for a given condition, all died within a year or deteriorated in their liver functions. Subjects transplanted with 100-150 million hepatic stem/progenitor cells had improved liver functions and survival extending for several years. Full evaluations of safety and efficacy of transplants are still in progress. Determined stem cell therapies for diabetes using hBTSCs remain to be explored but are likely to occur following ongoing preclinical studies. In addition, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being used for patients with chronic liver conditions or with diabetes. MSCs have demonstrated significant effects through paracrine signaling of trophic and immunomodulatory factors, and there is limited evidence for inefficient lineage restriction into mature parenchymal or islet cells. HSCs' effects are primarily via modulation of immune mechanisms.

Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis.

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Radial-axis lineages start in PBGs near the ducts' fibromuscular layers with stem cells and end at the ducts' lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota's Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas' committed progenitors. Both could be driven by three-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis.

Cell and organ bioengineering technology as applied to gastrointestinal diseases.

This review illustrates promising regenerative medicine technologies that are being developed for the treatment of gastrointestinal diseases. The main strategies under validation to bioengineer or regenerate liver, pancreas, or parts of the digestive tract are twofold: engineering of progenitor cells and seeding of cells on supporting scaffold material. In the first case, stem cells are initially expanded under standard tissue culture conditions. Thereafter, these cells may either be delivered directly to the tissue or organ of interest, or they may be loaded onto a synthetic or natural three-dimensional scaffold that is capable of enhancing cell viability and function. The new construct harbouring the cells usually undergoes a maturation phase within a bioreactor. Within the bioreactor, cells are conditioned to adopt a phenotype similar to that displayed in the native organ. The specific nature of the scaffold within the bioreactor is critical for the development of this high-function phenotype. Efforts to bioengineer or regenerate gastrointestinal tract, liver and pancreas have yielded promising results and have demonstrated the immense potential of regenerative medicine. However, a myriad of technical hurdles must be overcome before transplantable, engineered organs become a reality.

Engineered tools to study endocrine dysfunction of pancreas.

Pancreas, a vital organ with intricate endocrine and exocrine functions, is central to the regulation of the body's glucose levels and digestive processes. Disruptions in its endocrine functions, primarily regulated by islets of Langerhans, can lead to debilitating diseases such as diabetes mellitus. Murine models of pancreatic dysfunction have contributed significantly to the understanding of insulitis, islet-relevant immunological responses, and the optimization of cell therapies. However, genetic differences between mice and humans have severely limited their clinical translational relevance. Recent advancements in tissue engineering and microfabrication have ushered in a new era of in vitro models that offer a promising solution. This paper reviews the state-of-the-art engineered tools designed to study endocrine dysfunction of the pancreas. Islet on a chip devices that allow precise control of various culture conditions and noninvasive readouts of functional outcomes have led to the generation of physiomimetic niches for primary and stem cell derived islets. Live pancreatic slices are a new experimental tool that could more comprehensively recapitulate the complex cellular interplay between the endocrine and exocrine parts of the pancreas. Although a powerful tool, live pancreatic slices require more complex control over their culture parameters such as local oxygenation and continuous removal of digestive enzymes and cellular waste products for maintaining experimental functionality over long term. The combination of islet-immune and slice on chip strategies can guide the path toward the next generation of pancreatic tissue modeling for better understanding and treatment of endocrine pancreatic dysfunctions.

Immunometabolic biomarkers for partial remission in type 1 diabetes mellitus.

Shortly after diagnosis of type 1 diabetes mellitus (T1DM) and initiation of insulin therapy, many patients experience a transient partial remission (PR) phase, also known as the honeymoon phase. This phase presents a potential therapeutic opportunity due to its association with immunoregulatory and ß cell-protective mechanisms. However, the lack of biomarkers makes its characterization difficult. In this review, we cover the current literature addressing the discovery of new predictive and monitoring biomarkers that contribute to the understanding of the metabolic, epigenetic, and immunological mechanisms underlying PR. We further discuss how these peripheral biomarkers reflect attempts to arrest ß cell autoimmunity and how these can be applied in clinical practice.

SliceChip: a benchtop fluidic platform for organotypic culture and serial assessment of human and rodent pancreatic slices.

Enzymatically isolated pancreatic islets are the most commonly used ex vivo testbeds for diabetes research. Recently, precision-cut living slices of human pancreas are emerging as an exciting alternative because they maintain the complex architecture of the endocrine and exocrine tissues, and do not suffer from the mechanical and chemical stress of enzymatic isolation. We report a fluidic pancreatic SliceChip platform with dynamic environmental controls that generates a warm, oxygenated, and bubble-free fluidic pathway across singular immobilized slices with continuous deliver of fresh media and the ability to perform repeat serial perfusion assessments. A degasser ensures the system remains bubble-free while systemic pressurization with compressed oxygen ensures slice medium remains adequately oxygenated. Computational modeling of perfusion and oxygen dynamics within SliceChip guide the system's physiomimetic culture conditions. Maintenance of the physiological glucose dependent insulin secretion profile across repeat perfusion assessments of individual pancreatic slices kept under physiological oxygen levels demonstrated the culture capacity of our platform. Fluorescent images acquired every 4 hours of transgenic murine pancreatic slices were reliably stable and recoverable over a 5 day period due to the inclusion of a 3D-printed bioinert metallic anchor that maintained slice position within the SliceChip. Our slice on a chip platform has the potential to expand the useability of human pancreatic slices for diabetes pathogenesis and the development of new therapeutic approaches, while also enabling organotypic culture and assessment of other tissue slices such as brain and patient tumors.

Dynamic scRNA-seq of live human pancreatic slices reveals functional endocrine cell neogenesis through an intermediate ducto-acinar stage.

Human pancreatic plasticity is implied from multiple single-cell RNA sequencing (scRNA-seq) studies. However, these have been invariably based on static datasets from which fate trajectories can only be inferred using pseudotemporal estimations. Furthermore, the analysis of isolated islets has resulted in a drastic underrepresentation of other cell types, hindering our ability to interrogate exocrine-endocrine interactions. The long-term culture of human pancreatic slices (HPSs) has presented the field with an opportunity to dynamically track tissue plasticity at the single-cell level. Combining datasets from same-donor HPSs at different time points, with or without a known regenerative stimulus (BMP signaling), led to integrated single-cell datasets storing true temporal or treatment-dependent information. This integration revealed population shifts consistent with ductal progenitor activation, blurring of ductal/acinar boundaries, formation of ducto-acinar-endocrine differentiation axes, and detection of transitional insulin-producing cells. This study provides the first longitudinal scRNA-seq analysis of whole human pancreatic tissue, confirming its plasticity in a dynamic fashion.

A postnatal network of co-hepato/pancreatic stem/progenitors in the biliary trees of pigs and humans.

A network of co-hepato/pancreatic stem/progenitors exists in pigs and humans in Brunner's Glands in the submucosa of the duodenum, in peribiliary glands (PBGs) of intrahepatic and extrahepatic biliary trees, and in pancreatic duct glands (PDGs) of intrapancreatic biliary trees, collectively supporting hepatic and pancreatic regeneration postnatally. The network is found in humans postnatally throughout life and, so far, has been demonstrated in pigs postnatally at least through to young adulthood. These stem/progenitors in vivo in pigs are in highest numbers in Brunner's Glands and in PDGs nearest the duodenum, and in humans are in Brunner's Glands and in PBGs in the hepato/pancreatic common duct, a duct missing postnatally in pigs. Elsewhere in PDGs in pigs and in all PDGs in humans are only committed unipotent or bipotent progenitors. Stem/progenitors have genetic signatures in liver/pancreas-related RNA-seq data based on correlation, hierarchical clustering, differential gene expression and principal component analyses (PCA). Gene expression includes representative traits of pluripotency genes (SOX2, OCT4), endodermal transcription factors (e.g. SOX9, SOX17, PDX1), other stem cell traits (e.g. NCAM, CD44, sodium iodide symporter or NIS), and proliferation biomarkers (Ki67). Hepato/pancreatic multipotentiality was demonstrated by the stem/progenitors' responses under distinct ex vivo conditions or in vivo when patch grafted as organoids onto the liver versus the pancreas. Therefore, pigs are logical hosts for translational/preclinical studies for cell therapies with these stem/progenitors for hepatic and pancreatic dysfunctions.

c-Myc Drives inflammation of the maternal-fetal interface, and neonatal lung remodeling induced by intra-amniotic inflammation.

Background: Intra-amniotic inflammation (IAI) is associated with increased risk of preterm birth and bronchopulmonary dysplasia (BPD), but the mechanisms by which IAI leads to preterm birth and BPD are poorly understood, and there are no effective therapies for preterm birth and BPD. The transcription factor c-Myc regulates various biological processes like cell growth, apoptosis, and inflammation. We hypothesized that c-Myc modulates inflammation at the maternal-fetal interface, and neonatal lung remodeling. The objectives of our study were 1) to determine the kinetics of c-Myc in the placenta, fetal membranes and neonatal lungs exposed to IAI, and 2) to determine the role of c-Myc in modulating inflammation at the maternal-fetal interface, and neonatal lung remodeling induced by IAI. Methods: Pregnant Sprague-Dawley rats were randomized into three groups: 1) Intra-amniotic saline injections only (control), 2) Intra-amniotic lipopolysaccharide (LPS) injections only, and 3) Intra-amniotic LPS injections with c-Myc inhibitor 10058-F4. c-Myc expression, markers of inflammation, angiogenesis, immunohistochemistry, and transcriptomic analyses were performed on placenta and fetal membranes, and neonatal lungs to determine kinetics of c-Myc expression in response to IAI, and effects of prenatal systemic c-Myc inhibition on lung remodeling at postnatal day 14. Results: c-Myc was upregulated in the placenta, fetal membranes, and neonatal lungs exposed to IAI. IAI caused neutrophil infiltration and neutrophil extracellular trap (NET) formation in the placenta and fetal membranes, and neonatal lung remodeling with pulmonary hypertension consistent with a BPD phenotype. Prenatal inhibition of c-Myc with 10058-F4 in IAI decreased neutrophil infiltration and NET formation, and improved neonatal lung remodeling induced by LPS, with improved alveolarization, increased angiogenesis, and decreased pulmonary vascular remodeling. Discussion: In a rat model of IAI, c-Myc regulates neutrophil recruitment and NET formation in the placenta and fetal membranes. c-Myc also participates in neonatal lung remodeling induced by IAI. Further studies are needed to investigate c-Myc as a potential therapeutic target for IAI and IAI-associated BPD.

Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets.

UNLABELLED: Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato-pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum-free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2-3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger-2-AE2 and CFTR), or regulatable C-peptide secreting neoislet-like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin-induced diabetic mice, results in functional islets secreting glucose-regulatable human C-peptide. CONCLUSION: The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes.

Neonatal intermittent hypoxia persistently impairs lung vascular development and induces long-term lung mitochondrial DNA damage.

Adults born preterm have an increased risk of pulmonary vascular disease. Extreme preterm infants often require supplemental oxygen but they also exhibit frequent intermittent hypoxemic episodes (IH). Here, we test the hypothesis that neonatal IH induces lung endothelial cell mitochondrial DNA (mitDNA) damage and contributes to long-term pulmonary vascular disease and pulmonary hypertension (PH). Newborn C57BL/6J mice were assigned to the following groups: 1) normoxia, 2) hyperoxia (O2 65%), 3) normoxia cycling with IH (O2 21% + O2 10%), and 4) hyperoxia cycling with IH (O2 65% + O2 10%) for 3 wk. IH episodes were initiated on postnatal day 7. Lung angiogenesis, PH, and mitDNA lesions were assessed at 3 wk and 3 mo. In vitro, the effect of IH on tubule formation and mitDNA lesions was evaluated in human pulmonary microvascular endothelial cells (HPMECs). Data were analyzed by ANOVA. In vitro, IH exposure reduced tubule formation and increased mitDNA lesions in HPMECs. This was most marked in HPMECs exposed to hyperoxia cycling with IH. In vivo, neonatal IH increased lung mitDNA lesions, impaired angiogenesis, and induced PH in 3-wk-old mice. These findings were pronounced in mice exposed to hyperoxia cycling with IH. At 3 mo follow-up, mice exposed to neonatal IH had persistently increased lung mitDNA lesions and impaired lung angiogenesis, even without concomitant hyperoxia exposure. Neonatal IH induces lung endothelial cell mitDNA damage and causes persistent impairment in lung angiogenesis. These findings provide important mechanistic insight into the pathogenesis of pulmonary vascular disease in preterm survivors.NEW & NOTEWORTHY Our current study demonstrates that neonatal intermittent hypoxia (IH) alters lung endothelial cell function, induces mitochondrial DNA lesions, and impairs lung vascular growth into adulthood. Moreover, when superimposed on hyperoxia, neonatal IH induces a severe lung vascular phenotype that is seen in preterm infants with PH. These findings suggest that neonatal IH contributes to PH in adults born preterm and importantly, that mitochondrial protection strategies may mitigate these deleterious effects.

Patch grafting of organoids of stem/progenitors into solid organs can correct genetic-based disease states.

Patch grafting, a novel strategy for transplantation of stem/progenitor organoids into porcine livers, has been found successful also for organoid transplantation into other normal or diseased solid organs in pigs and mice. Each organoid contained ∼100 cells comprised of biliary tree stem cells (BTSCs), co-hepato/pancreatic stem/progenitors, and partnered with early lineage stage mesenchymal cells (ELSMCs), angioblasts and precursors to endothelia and stellate cells. Patch grafting enabled transplantation into livers or pancreases of ≥108th (pigs) or ≥106th-7th (mice) organoids/patch. Graft conditions fostered expression of multiple matrix-metalloproteinases (MMPs), especially secretory isoforms, resulting in transient loss of the organ's matrix-dictated histological features, including organ capsules, and correlated with rapid integration within a week of organoids throughout the organs and without emboli or ectopic cell distribution. Secondarily, within another week, there was clearance of graft biomaterials, followed by muted expression of MMPs, restoration of matrix-dictated histology, and maturation of donor cells to functional adult fates. The ability of patch grafts of organoids to rescue hosts from genetic-based disease states was demonstrated with grafts of BTSC/ELSMC organoids on livers, able to rescue NRG/FAH-KO mice from type I tyrosinemia, a disease caused by absence of fumaryl acetoacetate hydrolase. With the same grafts, if on pancreas, they were able to rescue NRG/Akita mice from type I diabetes, caused by a mutation in the insulin 2 gene. The potential of patch grafting for cell therapies for solid organs now requires translational studies to enable its adaptation and uses for clinical programs.

Stem cell-derived islet cells for transplantation.

PURPOSE OF REVIEW: The promise of islet transplantation for type 1 diabetes has been hampered by the lack of a renewable source of insulin-producing cells. However, steadfast advances in the field have set the stage for stem cell-based approaches to take over in the near future. This review focuses on the most intriguing findings reported in recent years, which include not only progress in adult and embryonic stem cell differentiation, but also the direct reprogramming of nonendocrine tissues into insulin-producing beta cells. RECENT FINDINGS: In spite of their potential for tumorigenesis, human embryonic stem (hES) cells are poised to be in clinical trials within the next decade. This situation is mainly due to the preclinical success of a differentiation method that recapitulates beta cell development. In contrast, adult stem cells still need one such gold standard of differentiation, and progress is somewhat impeded by the lack of consensus on the best source. A concerted effort is necessary to bring their potential to clinical fruition. In the meantime, reported success in reprogramming might offer a 'third way' towards the rescue of pancreatic endocrine function. SUMMARY: Here we discuss the important strategic decisions that need to be made in order to maximize the therapeutic chances of each of the presented approaches.

The Importance of Proper Oxygenation in 3D Culture.

Cell culture typically employs inexpensive, disposable plasticware, and standard humidified CO2/room air incubators (5% CO2, ∼20% oxygen). These methods have historically proven adequate for the maintenance of viability, function, and proliferation of many cell types, but with broad variation in culture practices. With technological advances it is becoming increasingly clear that cell culture is not a "one size fits all" procedure. Recently, there is a shift toward comprehension of the individual physiological niches of cultured cells. As scale-up production of single cell and 3D aggregates for therapeutic applications has expanded, researchers have focused on understanding the role of many environmental metabolites/forces on cell function and viability. Oxygen, due to its role in cell processes and the requirement for adequate supply to maintain critical energy generation, is one such metabolite gaining increased focus. With the advent of improved sensing technologies and computational predictive modeling, it is becoming evident that parameters such as cell seeding density, culture media height, cellular oxygen consumption rate, and aggregate dimensions should be considered for experimental reproducibility. In this review, we will examine the role of oxygen in 3D cell culture with particular emphasis on primary islets of Langerhans and stem cell-derived insulin-producing SC-ß cells, both known for their high metabolic demands. We will implement finite element modeling (FEM) to simulate historical and current culture methods in referenced manuscripts and innovations focusing on oxygen distribution. Our group and others have shown that oxygen plays a key role in proliferation, differentiation, and function of these 3D aggregates. Their culture in plastic consistently results in core regions of hypoxia/anoxia exacerbated by increased media height, aggregate dimensions, and oxygen consumption rates. Static gas permeable systems ameliorate this problem. The use of rotational culture and other dynamic culture systems also have advantages in terms of oxygen supply but come with the caveat that these endocrine aggregates are also exquisitely sensitive to mechanical perturbation. As recent work demonstrates, there is a strong rationale for the use of alternate in vitro systems to maintain physio-normal environments for cell growth and function for better phenotypic approximation of in vivo counterparts.

Temporal single-cell regeneration studies: the greatest thing since sliced pancreas?

The application of single-cell analytic techniques to the study of stem/progenitor cell niches supports the emerging view that pancreatic cell lineages are in a state of flux between differentiation stages. For all their value, however, such analyses merely offer a snapshot of the cellular palette of the tissue at any given time point. Conclusions about potential developmental/regeneration paths are solely based on bioinformatics inferences. In this context, the advent of new techniques for the long-term culture and lineage tracing of human pancreatic slices offers a virtual window into the native organ and presents the field with a unique opportunity to serially resolve pancreatic regeneration dynamics at the single-cell level.

Association between the Mediterranean Diet and Metabolic Syndrome with Serum Levels of miRNA in Morbid Obesity.

BACKGROUND: The Mediterranean diet (MD) could be involved in the regulation of different miRNAs related to metabolic syndrome (MS). METHODS: We analyzed the serum level of mir-let7a-5p, mir-21, mir-590, mir-107 and mir-192 in patients with morbid obesity and its association with the MD and MS. RESULTS: There is an association between the adherence to MD and higher serum levels of mir-590. Mir-590 was lower in those patients who consumed >2 commercial pastries/week. Mir-let7a was lower in those who consumed ≥1 sweetened drinks, in those who consumed ≥3 pieces of fruit/day and in those who consumed less red than white meat. A lower mir-590 and mir-let7a, and a higher mir-192 level, were found in patients who met the high-density lipoprotein cholesterol (HDL) criterion of MS. A higher mir-192 was found in those patients who met the triglyceride criterion of MS and in those with type 2 diabetes (T2DM). CONCLUSIONS: There is an association between specific serum levels of miRNAs and the amount and kind of food intake related to MD. Mir-590 was positively associated with a healthy metabolic profile and type of diet, while mir-192 was positively associated with a worse metabolic profile. These associations could be suggestive of a possible modulation of these miRNAs by food.