Generation of Functional Beta-Like Cells from Human Exocrine Pancreas.

Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells.

Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture.

A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells.

Cell therapy for type 1 diabetes.

Cell therapy in the form of human islet transplantation has been a successful form of treatment for patients with type 1 diabetes for over 10 years, but is significantly limited by lack of suitable donor material. A replenishable supply of insulin-producing cells has the potential to address this problem; however to date success has been limited to a few preclinical studies. Two of the most promising strategies include differentiation of embryonic stem cells and induced pluripotent stem cells towards insulin-producing cells and transdifferentiation of acinar or other closely related cell types towards β-cells.

Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing β-like cells.

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer.

Nanoscale perturbations of room temperature ionic liquid structure at charged and uncharged interfaces.

The nanoscale interactions of room temperature ionic liquids (RTILs) at uncharged (graphene) and charged (muscovite mica) solid surfaces were evaluated with high resolution X-ray interface scattering and fully atomistic molecular dynamics simulations. At uncharged graphene surfaces, the imidazolium-based RTIL ([bmim(+)][Tf(2)N(-)]) exhibits a mixed cation/anion layering with a strong interfacial densification of the first RTIL layer. The first layer density observed via experiment is larger than that predicted by simulation and the apparent discrepancy can be understood with the inclusion of, dominantly, image charge and π-stacking interactions between the RTIL and the graphene sheet.

Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells.

Aims/hypothesis: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) offer unique opportunities for regenerative medicine and for the study of mammalian development. However, developing methods to differentiate hESCs/hIPSCs into specific cell types following a natural pathway of development remains a major challenge.

Methods: We used defined culture media to identify signalling pathways controlling the differentiation of hESCs/hIPSCs into pancreatic or hepatic progenitors.

Pancreatic transcription factors containing protein transduction domains drive mouse embryonic stem cells towards endocrine pancreas.

Protein transduction domains (PTDs), such as the HIV1-TAT peptide, have been previously used to promote the uptake of proteins into a range of cell types, including stem cells. Here we generated pancreatic transcription factors containing PTD sequences and administered these to endoderm enriched mouse embryonic stem (ES) cells under conditions that were designed to mimic the pattern of expression of these factors in the developing pancreas. The ES cells were first cultured as embryoid bodies and treated with Activin A and Bone morphogenetic protein 4 (BMP4) to promote formation of definitive endoderm.

Efficient differentiation of AR42J cells towards insulin-producing cells using pancreatic transcription factors in combination with growth factors.

The AR42J-B13 rat pancreatic acinar cell line was used to identify pancreatic transcription factors and exogenous growth factors (GFs) that might facilitate the reprogramming of exocrine cells into islets. Adenoviruses were used to induce exogenous expression of the pancreatic transcription factors (TFs) Pdx1, MafA, Ngn3 and Pax4. Individually Pdx1, MafA and Pax4 had no effect on the expression of endocrine markers, whilst adeno-Ngn3 on its own increased the expression of Pax4, Ngn3 and NeuroD.

Resummed thermodynamic perturbation theory for central force associating potential. Multi-patch models.

A resummed thermodynamic perturbation theory for associating fluids with multiply bondable central force associating potential is extended for the fluid with multiple number of multiply bondable associating sites. We consider a multi-patch hard-sphere model for associating fluids. The model is represented by the hard-sphere fluid system with several spherical attractive patches on the surface of each hard sphere.

Resummed thermodynamic perturbation theory for central force associating potential: One-patch model.

A resummed thermodynamic perturbation theory for associating fluids with multiply bondable central force associating potential is proposed. We consider a simple one-patch model for associating fluids. The model is represented by the hard-sphere system with a circular attractive patch on the surface of each hard-sphere.

A factor(s) secreted from MIN-6 beta-cells stimulates differentiation of definitive endoderm enriched embryonic stem cells towards a pancreatic lineage.

In the mouse the developing pancreas is controlled by contact with, and signalling molecules secreted from, surrounding cells. These factors are best studied using explant cultures of embryonic tissue. The present study was undertaken to determine whether embryonic stem (ES) cells could be used as an alternative model in vitro system to investigate the role of cell-cell interactions in the developing pancreas.

An engineered zinc finger protein reveals a role for the insulin VNTR in the regulation of the insulin and adjacent IGF2 genes.

An engineered zinc finger protein (eZFP) was isolated from a library based on its ability to activate expression of the endogenous insulin gene in HEK-293 cells. Using a panel of insulin promoter constructs, the eZFP was shown to act through the variable number of tandem repeat (VNTR) region located 365 base pairs upstream of the transcription start site. The eZFP also activated expression of the IGF2 gene that lies close to INS on chromosome 11p15.

Biphasic induction of Pdx1 in mouse and human embryonic stem cells can mimic development of pancreatic beta-cells.

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes. Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development. Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse and human ES cell.

The importance of polarizability in the modeling of solubility: quantifying the effect of solute polarizability on the solubility of small nonpolar solutes in popular models of water.

In recent work by Paschek [J. Chem. Phys.

Investigation of the salting out of methane from aqueous electrolyte solutions using computer simulations.

We calculate the excess chemical potential of methane in aqueous electrolyte solutions of NaCl using Monte Carlo computer simulations. In a recent work [Docherty et al. J.

A potential model for methane in water describing correctly the solubility of the gas and the properties of the methane hydrate.

We have obtained the excess chemical potential of methane in water, over a broad range of temperatures, from computer simulation. The methane molecules are described as simple Lennard-Jones interaction sites, while water is modeled by the recently proposed TIP4P/2005 model. We have observed that the experimental values of the chemical potential are not reproduced when using the Lorentz-Berthelot combining rules.

Transcription factor cycling on the insulin promoter.

Using MIN6 beta-cells and chromatin immunoprecipitation (ChIP) assays, the chronological sequence of binding of MafA, E47/beta2 and PDX-1 to the insulin promoter in living beta-cells were investigated. All four factors were shown to bind to the mouse insulin 2 promoter in a cyclical manner with a periodicity of approximately 10-15 min. The cyclical binding of MafA, E47 and beta2 was largely unaffected by the glucose or insulin concentration in the media.

Relative contribution of PDX-1, MafA and E47/beta2 to the regulation of the human insulin promoter.

The insulin promoter binds a number of tissue-specific and ubiquitous transcription factors. Of these, the homoeodomain protein PDX-1 (pancreatic duodenal homeobox factor-1), the basic leucine zipper protein MafA and the basic helix-loop-helix heterodimer E47/BETA2 (beta-cell E box transactivator 2; referred to here as beta2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and beta2 to activate the rat insulin 1 promoter.

Secretion of bioactive human insulin following plasmid-mediated gene transfer to non-neuroendocrine cell lines, primary cultures and rat skeletal muscle in vivo.

The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats.

Engineering a glucose-responsive human insulin-secreting cell line from islets of Langerhans isolated from a patient with persistent hyperinsulinemic hypoglycemia of infancy.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a neonatal disease characterized by dysregulation of insulin secretion accompanied by profound hypoglycemia. We have discovered that islet cells, isolated from the pancreas of a PHHI patient, proliferate in culture while maintaining a beta cell-like phenotype. The PHHI-derived cell line (NES2Y) exhibits insulin secretory characteristics typical of islet cells derived from these patients, i.

Impaired expression of transcription factor IUF1 in a pancreatic beta-cell line derived from a patient with persistent hyperinsulinaemic hypoglycaemia of infancy (nesidioblastosis).

Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI), or nesidioblastosis, is a rare disorder which may be familial or sporadic, and which is characterized by unregulated secretion of insulin and profound hypoglycaemia in the neonate. The defect has been linked in some patients to mutations in the sulphonyl urea receptor gene (SUR). The present study investigated potential defects in the regulation of the insulin gene by glucose in a beta-cell line (NES 2Y) derived from a patient with PHHI.

Expression of parathyroid hormone-related protein in abnormal human parathyroids.

The expression of parathyroid hormone-related protein (PTHrP) in abnormal human parathyroids was investigated. Northern blot analysis of RNA extracted from human benign parathyroid adenomata (n = 4) revealed multiple PTHrP mRNA species ranging in size from 1.8 to 4 kb.

Monoclonal antibodies to ROS 17/2.8 cells recognize antigens, some of which are restricted to osteoblasts and chondrocytes.

We have raised a panel of 15 monoclonal antibodies (MAbs) recognizing cell surface antigens of the rat osteoblast-like cell line ROS 17/2.8. The MAbs were selected on the basis of preferential binding to ROS 17/2.

Parathyroid hormone-related proteins in cultured epithelial cells.

The distribution and molecular characteristics of parathyroid hormone-related protein (PTH-rP) in conditioned media and cell extracts of cultured human keratinocytes, and in media from a variety of both normal and transformed epithelial and non-epithelial cell cultures were studied. PTH-rP of Mr 20,000 was observed in keratinocyte-conditioned media, and a larger form, Mr 29,000, in the keratinocyte cell extract. PTH-like bioactivity was also detected in media from 12 out of 17 epithelial cell cultures, but was not present in media from 14 cell cultures of non-epithelial origin.

Multiple forms of parathyroid hormone-like proteins in a human tumour.

The extensive chromatographic characterization of four parathyroid hormone (PTH)-like proteins in a human bronchial carcinoid tumour associated with humoral hypercalcaemia and severe osteitis fibrosa is described. PTH-like bioactivity was detected in acetic acid extracts of the tumour using an in-vitro osteo-sarcoma cell bioassay. The active tumour proteins were positively charged at physiological pH and had apparent Mr of approximately 29,000, 16,000, 4000-9000 and less than 4000.