Direct Reprogramming of Mice Skin Fibroblasts into Insulin-Producing Cells In Vitro.

Transdifferentiation means mature cell conversion into other mature cells. Ethical issues, epigenetic failure, or teratoma development are found in cellular reprogramming strategies. Thus, new methods are needed. This study aimed to develop a new novel formula of chemical molecules and growth factors that differentiate skin fibroblasts into insulin-producing cells (IPCs). Newborn mice fibroblasts differentiated using four induction methods into IPCs to search for the best method. Fibroblasts, stem cells, and pancreatic markers were identified using an immunocytochemistry (ICC) assay. Insulin was measured using ELISA and dithizone (DTZ) assays. The skin fibroblasts were induced successfully into IPCs. The best method to obtain IPCs was indicated by measuring insulin concentration in differentiated cell supernatant from all induced cells by the four methods. The protein expression of the pancreatic markers of induced cells increased with time, as indicated by the ICC assay. OCT3/4 increased on day 9, after which the expression tended to decrease. DTZ-positive clusters were observed on day 16. Secreted insulin of differentiated cells was injected in streptozotocin-induced diabetic mice, which decreased blood glucose levels after injection. This study indicated an efficient new chemical method for transdifferentiating skin fibroblasts into functional IPCs, which is a promising method for diabetes mellitus therapy.

Cysteine Prevents the Development of Experimental Diabetes Induced by Zinc-Binding Substances.

In experimental rabbits, cysteine injected intravenously in a dose of 1000 mg/kg temporarily bound zinc in ß cells and prevented the formation of chelate zinc complexes in response to subsequent injection of diabetogenic zinc-binding substances that induce cell destruction. Injection of cysteine to animals was associated with a sharply negative reaction to zinc in ß cells, which attests to blockade of zinc ions. Injection of cysteine few minutes after dithizone and formation of zinc-dithizone complex was followed by displacement of dithizone from the complex and prevented the development of diabetes in most animals. The most plausible mechanism of preventive effect of cysteine is the formation of 2:1 zinc-cysteine complex in ß cells with possible fixation of Zn atom between sulfur atoms from SH groups of two cysteine molecules.

Zinc-Chelating Small Molecules Preferentially Accumulate and Function within Pancreatic ß Cells.

Diabetes is a hyperglycemic condition characterized by pancreatic ß-cell dysfunction and depletion. Whereas methods for monitoring ß-cell function in vivo exist, methods to deliver therapeutics to ß cells are lacking. We leveraged the rare ability of ß cells to concentrate zinc to preferentially trap zinc-binding molecules within ß cells, resulting in ß-cell-targeted compound delivery. We determined that zinc-rich ß cells and islets preferentially accumulated TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline) in a zinc-dependent manner compared with exocrine pancreas. Next, we asked whether appending a zinc-chelating moiety onto a ß-cell replication-inducing compound was sufficient to confer preferential ß-cell accumulation and activity. Indeed, the hybrid compound preferentially accumulated within rodent and human islets in a zinc-dependent manner and increased the selectivity of replication-promoting activity toward ß cells. These data resolve the fundamental question of whether intracellular accumulation of zinc-chelating compounds is influenced by zinc content. Furthermore, application of this principle yielded a proof-of-concept method for ß-cell-targeted drug delivery and bioactivity.

Differentiation of chicken umbilical cord mesenchymal stem cells into beta-like pancreatic islet cells.

In this study, we explored the possibility of using in vitro differentiation to create functional beta-like islet cells from chicken umbilical cord mesenchymal stem cells (UCMSCs). Passaged UCMSCs were induced to differentiate into pancreatic beta-like islet cells. Differentiated cells were observed through dithizone staining, and Pdx1 and insulin expressed in differentiated cells were detected with immunofluorescence. Insulin and C-peptide production from differentiated cells were analyzed using ELISA and western blotting. Differentiated cells were found to not only express Pdx1, insulin, and C-peptide, but also to display a glucose-responsive secretion of these hormones.

Differentiation of pancreatic stem and progenitor ß-cells into insulin secreting cells in mice with diabetes mellitus.

We studied in vitro differentiation of pancreatic stem and progenitor cells into insulin secreting cells in the model of streptozotocin-induced diabetes in C57Bl/6 mice. Streptozotocin was shown to increase the population of pancreatic oligopotent ß-cell precursors (CD45(-), TER119(-), CD133(+), and CD49f(low)) and did not affect multipotent (stem) progenitor cells (CD45(-), TER119(-), CD17(-), CD309(-)). During long-term culturing, diabetic multipotent progenitor cells showed high capacity for self-renewal. A population of dithizone-positive (insulin secreting cells) mononuclear cells was obtained releasing insulin after prolonged culturing in suspension enriched with diabetic CD45(-), TER119(-), CD17(-), and CD309(-) cells. The rate of generation of "new" insulin-producing cells and insulin release in the samples of experimental group considerably exceeded activity of the corresponding processes in the control group.

Dithizone staining of intracellular zinc: an unexpected and versatile counterscreen for auxotrophic marker genes in Saccharomyces cerevisiae.

Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle.

A preliminary spectroscopic investigation on the molecular interaction of metal-diphenylthiocarbazone complex with cellulose biopolymer and its application.

Biopolymer adsorbents are versatile in their application for removal of heavy metals. The present work is focused towards the preliminary study of the interaction of diphenylthiocarbazone (DTZ) complex of chromium(VI) in acidic medium with cellulose biopolymer. Chromium-DTZ complex could be quantitatively adsorbed on a cellulose column in the pH range 1.0-2.5 and the effect of various experimental parameters such as stability of the column and the complex, column breakthrough volume, and interfering ions have been studied in detail. The probable mechanism of adsorption of complex on the cellulose biopolymer was corroborated using Fourier transform infra-red spectroscopy (FT-IR), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) and solid state 13C nuclear magnetic resonance techniques (CP-MAS). The pores formed due to the hydrogen bond between the cellulose layers and then the ensuing occupation of the complex between these layers and on the surface of the biopolymer layer through electrostatic attractive force and Π interaction of aromatic ring with cellulose are expected to play a vital role in the interaction. The cellulose column could be regenerated using environmentally benign polyethylene glycol-400 (PEG-400) in acidic medium. The cellulose biosorbent has been successfully tested to study the removal of chromium as its dithizone complex from synthetic and real waste water samples.

Violets of the section Melanium, their colonization by arbuscular mycorrhizal fungi and their occurrence on heavy metal heaps.

Violets of the sections Melanium were examined for their colonization by arbuscular mycorrhizal fungi (AMF). Heartsease (Viola tricolor) from several heavy metal soils was AMF-positive at many sites but not at extreme biomes. The zinc violets Viola lutea ssp. westfalica (blue zinc violet) and ssp. calaminaria (yellow zinc violet) were always AMF-positive on heavy metal soils as their natural habitats. As shown for the blue form, zinc violets germinate independently of AMF and can be grown in non-polluted garden soils. Thus the zinc violets are obligatorily neither mycotrophs nor metalophytes. The alpine V. lutea, likely ancestor of the zinc violets, was at best poorly colonized by AMF. As determined by atomic absorption spectrometry, the contents of Zn and Pb were lower in AMF colonized plants than in the heavy metal soils from where the samples had been taken. AMF might prevent the uptake of toxic levels of heavy metals into the plant organs. Dithizone staining indicated a differential deposition of heavy metals in tissues of heartsease. Leaf hairs were particularly rich in heavy metals, indicating that part of the excess of heavy metals is sequestered into these cells.

Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells.

The maintenance of viable and functional islets is critical in successful pancreatic islet transplantation from cadaveric sources. During the isolation procedure, islets are exposed to a number of insults including ischemia, oxidative stress and cytokine injury that cause a reduction in the recovered viable islet mass. A novel approach was designed in which streptozotocin (STZ)-damaged rat pancreatic islets (rPIs) were indirectly cocultured with rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) to maintain survival of the cultured rPIs. The results indicated that islets cocultured with rBM-MSCs secreted an increased level of insulin after 14 days, whereas non-cocultured islets gradually deteriorated and cell death occurred. The cocultivation of rBM-MSCs with islets and STZ-damaged islets showed the expression of IL6 and transforming growth factor-ß1 in the culture medium, besides the expression of the antiapoptotic genes (Mapkapk2, Tnip1 and Bcl3), implying the cytoprotective, anti-inflammatory and antiapoptotic effects of rBM-SCs through paracrine actions.

Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro.

It was recently reported that pluripotent mesenchymal stem cells (MSCs) in rodent bone marrow (BM) have the capacity to generate insulin-producing cells (IPCs) in vitro. However, little is known about this capacity in human BM-MSCs. We developed a nongenetic method to induce human BM-MSCs to transdifferentiate into IPCs both phenotypically and functionally. BM-MSCs from 12 human donors were sequentially cultured in specially defined conditions. Their differentiation extent toward beta-cell phenotype was evaluated systemically. Specifically, after induction human BM-MSCs formed spheroid islet-like clusters containing IPCs, which was further confirmed by dithizone (DTZ) staining and electron microscopy. These IPCs expressed multiple genes related to the development or function of pancreatic beta cells (including NKX6.1, ISL-1, Beta2/Neurod, Glut2, Pax6, nestin, PDX-1, ngn3, insulin and glucagon). The coexpression of insulin and c-peptide was observed in IPCs by immunofluorescence. Moreover, they were able to release insulin in a glucose-dependent manner and ameliorate the diabetic conditions of streptozotocin (STZ)-treated nude mice. These results indicate that human BM-MSCs might be an available candidate to overcome limitations of islet transplantation.

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells.

Although several studies have suggested that insulin-secreting cells can be generated in vitro from cells residing in adult exocrine pancreas, neither the origin of these cells nor their precise insulin secretory properties was obtained. We show here that insulin-secreting cells can be derived from adult mouse pancreatic exocrine cells by suspension culture in the presence of EGF and nicotinamide. The frequency of insulin-positive cells was only 0.01% in the initial preparation and increased to approximately 5% in the culture conditions. Analysis by the Cre/loxP-based direct cell lineage tracing system indicates that these newly made cells originate from amylase/elastase-expressing pancreatic acinar cells. Insulin secretion is stimulated by glucose, sulfonylurea, and carbachol, and potentiation by glucagon-like peptide-1 also occurs. Insulin-containing secretory granules are present in these cells. In addition, we found that the enzymatic dissociation of pancreatic acini itself leads to activation of EGF signaling, and that inhibition of EGF receptor kinase blocks the transdifferentiation. These data demonstrate that pancreatic acinar cells can transdifferentiate into insulin-secreting cells with secretory properties similar to those of native pancreatic beta cells, and that activation of EGF signaling is required in such transdifferentiation.

[Tissue distribution of chelants during life]. / Prizhiznennoe raspredelenie khelantov v tkaniakh

Injection of dithizone and quinoline compounds to animals leads to the intravital zinc chelate granule formation detectable on frozen tissue sections. The intensity of intravital histochemical reaction depends on be complexing ability, the dose, the ways of the agent injection, and also on the presence of other ligands lowering the agent's coordination volume use.