A Preliminary Characterization of a Novel Recombinant Clostridial Collagenase Blend.

Clostridial collagenases are essential biotechnological tissue dissociation agents owing to their ability to cleave different types of collagen. Standardization of collagenase-based protocols has been hampered by impurities in products manufactured from Clostridium histolyticum. To enhance the purification process, we produced recombinant collagenase classes G and H, taking advantage of the Escherichia coli expression system.

The Potential of Pancreatic Organoids for Diabetes Research and Therapy.

The success of clinical transplantation of pancreas or isolated pancreatic islets supports the concept of cell-based cure for diabetes. One limitation is the shortage of cadaver human pancreata. The demand-supply gap could potentially be bridged by harnessing the self-renewal capacity of stem cells.

Reprogramming of Human Pancreatic Organoid Cells into Insulin-Producing β-Like Cells by Small Molecules and in Vitro Transcribed Modified mRNA Encoding Neurogenin 3 Transcription Factor.

Reprogramming of non-endocrine pancreatic cells into insulin-producing cells represents a promising therapeutic approach for the restoration of endogenous insulin production in diabetic patients. In this paper, we report that human organoid cells derived from the pancreatic tissue can be reprogrammed into the insulin-producing cells (IPCs) by the combination of in vitro transcribed modified mRNA encoding transcription factor neurogenin 3 and small molecules modulating the epigenetic state and signalling pathways. Upon the reprogramming, IPCs formed 4.

Synthetic mRNA is a more reliable tool for the delivery of DNA-targeting proteins into the cell nucleus than fusion with a protein transduction domain.

Cell reprogramming requires efficient delivery of reprogramming transcription factors into the cell nucleus. Here, we compared the robustness and workload of two protein delivery methods that avoid the risk of genomic integration. The first method is based on fusion of the protein of interest to a protein transduction domain (PTD) for delivery across the membranes of target cells.

Benefits of Islet Transplantation as an Alternative to Pancreas Transplantation: Retrospective Study of More Than 10 Ten Years of Experience in a Single Center.

Background: Pancreas transplantation (PTx) represents the method of choice in type 1 diabetic patients with conservatively intractable hypoglycemia unawareness syndrome. In 2005, the Institute for Clinical and Experimental Medicine (IKEM) launched a program to investigate the safety potential of islet transplantation (ITx) in comparison to PTx.

Aim: This study aims to compare the results of PTx and ITx regarding severe hypoglycemia elimination, metabolic control, and complication rate.

[Islet transplantation as a treatment for hypoglycemia unawareness syndrome. Evaluation of the pilot program and comparison with pancreas transplantation].

Islet transplantation (ITx) started in 2005 in IKEM as a potentially safer alternative to pancreas transplantation (PTx), which so far had represented the method of choice in type-1 diabetic patients with conservatively intractable hypoglycemia unawareness syndrome. The aim of the study was to compare these two methods with regard to severe hypoglycemia elimination and to frequency of complications.Up to November 2015 a total number of 48 patients underwent ITx.

Combining Donor Characteristics with Immunohistological Data Improves the Prediction of Islet Isolation Success.

Variability of pancreatic donors may significantly impact the success of islet isolation. The aim of this study was to evaluate donor factors associated with isolation failure and to investigate whether immunohistology could contribute to organ selection. Donor characteristics were evaluated for both successful ( = 61) and failed ( = 98) islet isolations.

Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors.

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors.

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression.

Formation of Cholangiogenic Cysts Following Intrahepatic Islet Transplantation in Streptozotocin Diabetic Rats.

Permanent hyperinsulinemia and the resulting overstimulation of the insulin receptor signaling pathway is suspected as a trigger of cancer genesis in the livers of type 2 diabetic patients. Liver tissue (LT) surrounding transplanted pancreatic islets (PI) can be permanently exposed to insulin in even higher concentrations than in type 2 diabetic patients. Therefore, this study examines the effect of PI transplantation (Tx) on LT in animals with streptozotocin (STZ)-induced diabetes mellitus.

Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor stimulates trans-differentiation of human non-endocrine pancreatic cells into insulin-producing cells.

Differentiation of pancreatic β-cells is regulated by a wide range of signalling pathways. The aim of our current work was to evaluate the effect of the Jak/Stat signalling pathway on the differentiation of human non-endocrine pancreatic cells into insulin-producing cells. Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor (LIF) stimulated differentiation of C-peptide-negative human non-endocrine pancreatic cells into insulin-producing cells in 6.

Dynamic contrast-enhanced magnetic resonance imaging as a tool to monitor the blood supply to an artificial cavity used as a site for islet transplantation in rats.

Background: The transplantation of islets of Langerhans isolated from one donor pancreas can rarely release a diabetic recipient from insulin injections. The major reason is the destruction of 50%-60% of the transplanted tissue, which proceeds typically within a few hours after the insertion of the islets into the portal vein. Therefore, several groups have focused on development of an artificial site for islet transplantation.

The effect of epigenetic factors on differentiation of pancreatic progenitor cells into insulin-producing cells.

Differentiation of pancreatic progenitors into insulin-producing β cells is regulated by various transcription factors. To be expressed the genes coding these transcription factors need to be in accessible DNA. Whether a particular gene is present in a form of active euchromatin structure with accessible DNA or in an inactive heterochromatin structure with inaccessible DNA is determined by various epigenetic modifications.

[Islet transplantation for treatment of type-1 diabetes mellitus].

Background: Organ pancreas transplantation represents the only method enabling long-term normalization of glucose metabolism in type-1 diabetic subjects so far. Unfortunately, surgical complications of this kind of therapy are still frequent. As a safer alternative, transplantation of isolated pancreatic islets was introduced at the Institute for Clinical and Experimental Medicine as a clinical experiment in the year 2005.

An acidic pH and activation of phosphoinositide 3-kinase stimulate differentiation of pancreatic progenitors into insulin-producing cells.

Adult pancreatic nonendocrine cells represent a potential alternative source of insulin-producing tissue for the treatment of diabetes. Differentiation of these cells is regulated by various signaling pathways including the phosphoinositide 3-kinase (PI3K) pathway. Therefore, we evaluated the effect of PI3K on this process.

In vivo differentiation of human umbilical cord blood-derived cells into insulin-producing beta cells.

In our study we confirmed the potential of human umbilical cord blood cells to differentiate into insulin-producing cells following transplantation into immunocompromised mice. The average number of C-peptide-positive human cells per animal was 18 +/- 13 as assessed by immunofluorescence staining and fluorescence in situ hybridization specific for human ALU sequence. Differentiation into insulin-producing cells was further confirmed by reverse transcription-polymerase chain reaction specific for human insulin mRNA.

Differentiation of CD133-positive pancreatic cells into insulin-producing islet-like cell clusters.

Adult pancreatic stem and progenitor cells could represent an alternative source of insulin-producing tissue for diabetes treatment. In order to identify these cells, we have focused on the human pancreatic cells expressing cell surface molecule CD133, a marker of adult stem cells. We found that population of human CD133-positive pancreatic cells contains endocrine progenitors expressing neurogenin-3 and cells expressing human telomerase, ABCG2, Oct-3/4, Nanog, and Rex-1, markers of pluripotent stem cells.

Labeling of pancreatic islets with iron oxide nanoparticles for in vivo detection with magnetic resonance.

In vitro labeling of pancreatic islets with iron nanoparticles enables their direct posttransplant visualization by magnetic resonance; however, there is still a discrepancy in the fate of iron nanoparticles. This study was performed to detail the labeling process, consequently to improve the labeling efficacy and to confirm safety for islet cells. The islets were visible on T2*-weighted magnetic resonance images as hypointense spots immediately after 1-hr cultivation.

The application of umbilical cord blood cells in the treatment of diabetes mellitus.

In recent years, human umbilical cord blood (HUCB) has emerged as an attractive tool for cell-based therapy. Although at present the clinical application of HUCB is limited to the fields of hematology and oncology, a rising number of studies show potential for further application in the treatment of non-hematopoietic diseases. HUCB, with its real abundance, simple collection procedure and no serious ethical dilemmas, represents a valuable alternative to the use of other stem cell sources.

Isolation and characterization of human CXCR4-positive pancreatic cells.

The existence of an adult PSC that may be used in the treatment of diabetes is still a matter of scientific debate as conclusive evidence of such a stem cell in the adult pancreas has not yet been presented. The main reason why putative PSC has not yet been identified is the lack of specific markers that may be used to isolate and purify them. In order to increase the list of potential PSC markers we have focused on the human pancreatic cells that express cell surface receptor CXCR4, a marker of stem cells derived from different adult tissues.

Vitality of pancreatic islets labeled for magnetic resonance imaging with iron particles.

We previously described an in vivo method for pancreatic islet visualization using magnetic resonance imaging with the aid of superparamagnetic nanoparticles of iron oxide (Resovist) or by magnetic beads precoated with antibodies (Dynabeads). The aim of this study was to investigate the in vitro effect of islet labeling on their quality. Isolated rat islets were cultivated for 48 hours with a contrast agent or, in the case of magnetic antibody-coated beads, for only 2 hours.

Magnetic resonance imaging of intrahepatically transplanted islets using paramagnetic beads.

Superparamagnetic agents can be reliably used for magnetic resonance imaging (MRI) of pancreatic islets located in the liver sinusoids. However, the main disadvantages seemed to be the rather long culture time necessary for islet labeling and the low specificity of these agents. In the present study we investigated a more specific approach with a shorter labeling time using immunomagnetic particles.

In vitro assessment of pancreatic islet vitality by oxymetry.

In order to assess the quality of freshly isolated and cultivated pancreatic islets designed for experimental transplantation in rats we combined the vitality staining test, in vitro measurement of insulin secretion capacity, and assessment of islet respiration. Oxygen consumption was measured using the respirometer Oxygraph 2K equipped with polarographic oxygen sensors. The results of oxymetry demonstrated a linear correlation between islet number and oxygen consumption.

Expression of cytochrome P450 1A1 and its contribution to oxidation of a potential human carcinogen 1-phenylazo-2-naphthol (Sudan I) in human livers.

Cytochrome P450 1A1 (CYP1A1) is one of the most important enzymes implicated in the metabolic activation of carcinogens. To date, there is still conflicting evidence for the expression of enzymatically functional CYP1A1 in human liver. In the present work, we clearly demonstrate that CYP1A1 capable of metabolizing a carcinogen 1-phenylazo-2-naphthol (Sudan I) is expressed in livers of eight American Caucasian donors.