A Double Fail-Safe Approach to Prevent Tumorigenesis and Select Pancreatic β Cells from Human Embryonic Stem Cells.

The 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.

Label-Free LC-MS/MS Strategy for Comprehensive Proteomic Profiling of Human Islets Collected Using Laser Capture Microdissection from Frozen Pancreata.

Diabetes mellitus is caused by either loss of pancreatic islets β-cells (Type 1 Diabetes, T1D), insufficient insulin release in the islet β-cells coupled with insulin resistance in target tissues (Type 2 Diabetes, T2D), or impaired insulin release (genetic forms of diabetes and, possibly, T1D subtypes). The investigation of the islet proteome could elucidate facets of the pathogenesis of diabetes. Enzymatically isolated and cultured (EIC) islets are frequently used to investigate biochemical signaling pathways that could trigger β-cell changes and death in diabetes.

P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics.

Treatment 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.

Proteomic profiling of human islets collected from frozen pancreata using laser capture microdissection.

Unlabelled: The etiology of Type 1 Diabetes (T1D) remains elusive. Enzymatically isolated and cultured (EIC) islets cannot fully reflect the natural protein composition and disease process of in vivo islets, because of the stress from isolation procedures. In order to study islet protein composition in conditions close to the natural environment, we performed proteomic analysis of EIC islets, and laser capture microdissected (LCM) human islets and acinar tissue from fresh-frozen pancreas sections of three cadaveric donors.

G-CSF and Exenatide Might Be Associated with Increased Long-Term Survival of Allogeneic Pancreatic Islet Grafts.

Background: Allogeneic human islet transplantation is an effective therapy for the treatment of patients with Type 1 Diabetes (T1D). The low number of islet transplants performed worldwide and the different transplantation protocols used limit the identification of the most effective therapeutic options to improve the efficacy of this approach.

Methods: We present a retrospective analysis on the data collected from 44 patients with T1D who underwent islet transplantation at our institute between 2000 and 2007.

Gene expression profiling of human fibrocytic myeloid-derived suppressor cells (f-MDSCs).

Myeloid-derived suppressor cells (MDSCs) have been shown to control self-reactive and anti-graft effector T-cells in autoimmunity and transplantation, but their therapeutic use is limited by their scarce availability in the peripheral blood of tumor-free donors. We isolated and characterized a novel population of myeloid suppressor cells, named fibrocytic MDSC (f-MDSC), which are differentiated from umbilical cord blood (UCB) precursors (Zoso et al., 2014).

Moderate Intensity Training Impact on the Inflammatory Status and Glycemic Profiles in NOD Mice.

The nonobese diabetic (NOD) mouse represents a well-established experimental model analogous to human type 1 diabetes mellitus (T1D) as it is characterized by progressive autoimmune destruction of pancreatic β-cells. Experiments were designed to investigate the impact of moderate-intensity training on T1D immunomodulation and inflammation. Under a chronic exercise regime, NOD mice were trained on a treadmill for 12 weeks (12 m/min for 30 min, 5 d/wk) while age-matched, control animals were left untrained.

BMP-7 Induces Adult Human Pancreatic Exocrine-to-Endocrine Conversion.

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.

Fibrin gels engineered with pro-angiogenic growth factors promote engraftment of pancreatic islets in extrahepatic sites in mice.

With a view toward reduction of graft loss, we explored pancreatic islet transplantation within fibrin matrices rendered pro-angiogenic by incorporation of minimal doses of vascular endothelial growth factor-A165 and platelet-derived growth factor-BB presented complexed to a fibrin-bound integrin-binding fibronectin domain. Engineered matrices allowed for extended release of pro-angiogenic factors and for their synergistic signaling with extracellular matrix-binding domains in the post-transplant period. Aprotinin addition delayed matrix degradation and prolonged pro-angiogenic factor availability within the graft.

Human fibrocytic myeloid-derived suppressor cells express IDO and promote tolerance via Treg-cell expansion.

By restraining T-cell activation and promoting Treg-cell expansion, myeloid-derived suppressor cells (MDSCs) and tolerogenic DCs can control self-reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4-day culture with FDA-approved cytokines (recombinant human-GM-CSF and recombinant human-G-CSF).

Influence of in vitro and in vivo oxygen modulation on β cell differentiation from human embryonic stem cells.

The possibility of using human embryonic stem (hES) cell-derived β cells as an alternative to cadaveric islets for the treatment of type 1 diabetes is now widely acknowledged. However, current differentiation methods consistently fail to generate meaningful numbers of mature, functional β cells. In order to address this issue, we set out to explore the role of oxygen modulation in the maturation of pancreatic progenitor (PP) cells differentiated from hES cells.

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.

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.

MicroRNA expression in alpha and beta cells of human pancreatic islets.

microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways.

From cellular therapies to tissue reprogramming and regenerative strategies in the treatment of diabetes.

Diabetes mellitus represents a global epidemic affecting over 350 million patients worldwide and projected by the WHO to surpass the 500 million patient mark within the next two decades. Besides Type 1 and Type 2 diabetes mellitus, the study of the endocrine compartment of the pancreas is of great translational interest, as strategies aimed at restoring its mass could become therapies for glycemic dysregulation, drug-related diabetes following diabetogenic therapies, or hyperglycemic disturbances following the treatment of cancer and nesidioblastosis. Such strategies generally fall under one of the 'three Rs': replacement (islet transplantation and stem cell differentiation); reprogramming (e.

Immuno-isolation of pancreatic islet allografts using pegylated nanotherapy leads to long-term normoglycemia in full MHC mismatch recipient mice.

Two major hurdles need to be surmounted for cell therapy for diabetes: (i) allo-immune rejection of grafted pancreatic islets, or stem/precursor cell-derived insulin-secreting cells; and (ii) continuing auto-immunity against the diabetogenic endogenous target antigen. Nanotherapeutics offer a novel approach to overcome these problems and here we ask if creation of "stealth" islets encapsulated within a thin cage of pegylated material of 100-200 nanometers thick provides a viable option for islet transplantation. The aims of this study were to test islet viability and functionality following encapsulation within the pegylated cage, and functional efficacy in vivo in terms of graft-derived control of normoglycemia in diabetic mice.

Concise review: mesenchymal stem cells for diabetes.

Mesenchymal stem cells (MSCs) have already made their mark in the young field of regenerative medicine. Easily derived from many adult tissues, their therapeutic worth has already been validated for a number of conditions. Unlike embryonic stem cells, neither their procurement nor their use is deemed controversial.

A physiological pattern of oxygenation using perfluorocarbon-based culture devices maximizes pancreatic islet viability and enhances β-cell function.

Conventional culture vessels are not designed for physiological oxygen (O2) 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 O2 concentrations.

Intracardial embryonic delivery of developmental modifiers in utero.

Our knowledge of organ ontogeny is largely based on loss-of-function (knockout) or gain-of-function (transgenesis) approaches. However, developmental modulators such as proteins, mRNAs, microRNAs(miRNAs), small interfering RNAs, and other small molecules may complement the above DNA-modifying technologies in a much more direct way. Unfortunately, their use is often limited by the ability of these compounds to cross the placenta and reach physiologically relevant concentrations when administered systemically to the mother.

Optimization of perfluoro nano-scale emulsions: the importance of particle size for enhanced oxygen transfer in biomedical applications.

Nano-scale emulsification has long been utilized by the food and cosmetics industry to maximize material delivery through increased surface area to volume ratios. More recently, these methods have been employed in the area of biomedical research to enhance and control the delivery of desired agents, as in perfluorocarbon emulsions for oxygen delivery. In this work, we evaluate critical factors for the optimization of PFC emulsions for use in cell-based applications.

Regeneration of pancreatic beta-cell mass for the treatment of diabetes.

Introduction: The study of the endocrine compartment of the pancreas (the islets of Langerhans) is of great translational interest, as strategies aimed at restoring its mass could become therapies for glycemic dysregulation in type 1 and 2 diabetes mellitus, drug-related diabetes following diabetogenic therapies or hyperglycemic disturbances following the treatment of cancer and nesidioblastosis. Such strategies generally fall under one of the 'three Rs,' namely, replacement (islet transplantation and stem cell differentiation), reprogramming (chiefly from the exocrine compartment of the pancreas) and regeneration (replication and induction of endogenous stem cells).

Areas Covered: This expert opinion focuses on the latter, as islets are known to regenerate under specific circumstances of physiological (e.

Aptamer-mediated blockade of IL4Rα triggers apoptosis of MDSCs and limits tumor progression.

In addition to promoting tumor progression and metastasis by enhancing angiogenesis and invasion, myeloid-derived suppressor cells (MDSC) and tumor-associated macrophage (TAM) also inhibit antitumor T-cell functions and limit the efficacy of immunotherapeutic interventions. Despite the importance of these leukocyte populations, a simple method for their specific depletion has not been developed. In this study, we generated an RNA aptamer that blocks the murine or human IL-4 receptor-α (IL4Rα or CD124) that is critical for MDSC suppression function.

Generation of glucose-responsive, insulin-producing cells from human umbilical cord blood-derived mesenchymal stem cells.

We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on stepwise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human C-peptide production in response to a glucose challenge.

Peptide-conjugated PAMAM dendrimer as a universal DNA vaccine platform to target antigen-presenting cells.

DNA-based vaccines hold promise to outperform conventional antigen-based vaccines by virtue of many unique features. However, DNA vaccines have thus far fallen short of expectations, due in part to poor targeting of professional antigen-presenting cells (APC) and low immunogenicity. In this study, we describe a new platform for effective and selective delivery of DNA to APCs in vivo that offers intrinsic immune-enhancing characteristics.