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. Pluripotent stem cells and adult pancreatic stem cells have been explored as possible cell sources. Recently, a system for long-term culture of proposed adult pancreatic stem cells in a form of organoids was developed. Generated organoids partially mimic the architecture and cell-type composition of pancreatic tissue. Here, we review the attempts over the past decade, to utilize the organoid cell culture principles in order to identify, expand, and differentiate the adult pancreatic stem cells from different compartments of mouse and human pancreata. The development of the culture conditions, effects of specific growth factors and small molecules is discussed. The potential utility of the adult pancreatic stem cells is considered in the context of other cell sources.

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. The second method relies on de novo synthesis of the protein of interest inside the target cells utilizing synthetic mRNA (syn-mRNA) as a template. We established a Cre/lox reporter system in three different cell types derived from human (PANC-1, HEK293) and rat (BRIN-BD11) tissues and used Cre recombinase to model a protein of interest. The system allowed constitutive expression of red fluorescence protein (RFP), while green fluorescence protein (GFP) was expressed only after the genomic action of Cre recombinase. The efficiency of protein delivery into cell nuclei was quantified as the frequency of GFP+ cells in the total cell number. The PTD method showed good efficiency only in BRIN-BD11 cells (68%), whereas it failed in PANC-1 and HEK293 cells. By contrast, the syn-mRNA method was highly effective in all three cell types (29-71%). We conclude that using synthetic mRNA is a more robust and less labor-intensive approach than using the PTD-fusion alternative.

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. METHODS: We analyzed the results of 30 patients undergoing ITx and 49 patients treated with PTx. All patients were C-peptide-negative and suffered from hypoglycemia unawareness syndrome. Patients in the ITx group received a mean number of 12,349 (6,387-15,331) IEQ/kg/person administered percutaneously into the portal vein under local anesthesia and radiological control. The islet number was reached by 1-3 applications, as needed. In both groups, we evaluated glycated hemoglobin, insulin dose, fasting and stimulated C-peptide, frequency of severe hypoglycemia, and complications. We used the Mann Whitney test, Wilcoxon signed-rank test, and paired t-test for analysis. We also individually assessed the ITx outcomes for each patient according to recently suggested criteria established at the EPITA meeting in Igls. RESULTS: Most of the recipients showed a significant improvement in metabolic control one and two years after ITx, with a significant decrease in HbA1c, significant elevation of fasting and stimulated C-peptide, and a markedly significant reduction in insulin dose and the frequency of severe hypoglycemia. Seventeen percent of ITx recipients were temporarily insulin-independent. The results in the PTx group were comparable to those in the ITx group, with 73% graft survival and insulin independence in year 1, 68% 2 years and 55% 5 years after transplantation. There was a higher rate of complications related to the procedure in the PTx group. Severe hypoglycemia was eliminated in the majority of both ITx and PTx recipients. CONCLUSION: This report proves the successful initiation of pancreatic islet transplantation in a center with a well-established PTx program. ITx has been shown to be the method of choice for hypoglycemia unawareness syndrome, and may be considered for application in clinical practice if conservative options are exhausted.

[Islet transplantation as a treatment for hypoglycemia unawareness syndrome. Evaluation of the pilot program and comparison with pancreas transplantation]. / Transplantace Langerhansových ostruvku v lécbe syndromu poruseného vnímání hypoglykemie. Vyhodnocení pilotního programu a porovnání s transplantací pankreatu.

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. The results from 22 patients with hypoglycemia unawareness were statistically analyzed. The mean number of transplanted islet equivalents was 12,096 (6,93316,705) IEQ/kg administered percutaneously in local anesthesia under radiological control to the portal vein. 44 patients underwent PTx from 1996. We evaluated glycated hemoglobin(HbA1c), insulin dose, fasting and stimulated C-peptide, frequency of severe hypoglycemia and complications. Medians (interquartile range) were analyzed using the Wilcoxon signed-rank test.One and two years after ITx, HbA1c decreased, C-peptide became significantly positive, insulin dose and frequency of severe hypoglycemia decreased and 18 % of ITx recipients were temporarily insulin-independent. Bleeding was present in 41 % of patients. One year after PTx, 73 % of patients were insulin and hypoglycemia-free, after two years 68 % of patients were insulin and hypoglycemia-free; graftectomy occurred in 20 % of recipients.Both methods led to restoration of insulin secretion and severe hypoglycemia elimination. PTx made more recipients insulin-independent at the cost of serious complications.

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 (n = 61) and failed (n = 98) islet isolations. Samples of donor pancreatic tissue (n = 78) were taken for immunohistochemical examination. Islet isolations with 250000 islet equivalents were considered successful. We confirmed that BMI of less than 25 kg/m2 (P < 0.001), cold ischemia time more than 8 hours (P < 0.01), hospitalization longer than 96 hours (P < 0.05), higher catecholamine doses (P < 0.05), and edematous pancreases (P < 0.01) all unfavorably affected isolation outcome. Subsequent immunohistochemical examination of donor pancreases confirmed significant differences in insulin-positive areas (P < 0.001). ROC analyses then established that the insulin-positive area in the pancreas could be used to predict the likely success of islet isolation (P < 0.001). At the optimal cutoff point (>1.02%), sensitivity and specificity were 89% and 76%, respectively. To conclude, while the insulin-positive area, determined preislet isolation, as a single variable, is sufficient to predict isolation outcome and helps to improve the success of this procedure, its combination with the established donor scoring system might further improve organ selection.

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 ß-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. Administration of synthetic mRNAs encoding three key transcription regulators of ß-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic ß-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

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. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

[Islet transplantation for treatment of type-1 diabetes mellitus]. / Lécba diabetu transplantací izolovaných Langerhansových ostruvku.

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. METHODS AND RESULTS: We isolated the islets from pancreases of cadaveric donors which did not fulfil criteria to perform organ pancreas transplantation. Altogether, 36 islet implantations were performed in 28 C-peptide negative subjects suffering from type-1 diabetes by August 2010. In 15 subjects (21 implantations) the main indication was extremely instable course of diabetes due to the hypoglycaemia unawareness syndrome. In 5 and 3 cases, combined islet and kidney and islet and liver transplants were performed, respectively. In addition, islet autotransplantation was performed in 5 subjects undergoing total pancreatectomy. No patient died during the study period. In all but 1 patient with primary islet afunction, islet transplantation led to a complete cure of the hypoglycemia unawareness syndrome. Out of 15 patients, 11 subjects in this group showed a significant C-peptide production (> 0.2 pmol/ml) after 1 year. The mean insulin dose after allotransplantation decreased from 37 to 14 units per day and in 3 subjects, insulin therapy could be withdrawn. Serious technical complications occurred in 6 subjects, which only in 2 cases required surgical revision and did not cause long-term sequels. CONCLUSIONS: In comparison with organ pancreas transplantation, pancreatic islet transplantation represents a substantially safer method for restitution of endogenous insulin production. Though it eliminates serious hypoglycemic episodes in labile diabetes, complete insulin withdrawal is still often not possible. However, due to continuing progress in the laboratory techniques as well as in the transplant procedure itself, the results are steadily improving.

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. Although at this time already the sufficient superparamagnetic effect was achieved, most of the particles were deposed in islet macrophages and only later were they found in endosomes of endocrine islet cells. The iron content depended on length of culture period. The labeled islets showed an intact ultrastructure, responded normally to glucose stimulation in vitro, and were able to treat experimental diabetes. For purpose of subsequent magnetic resonance imaging, a 24-hr culture with ferucarbotran leads to sufficient labeling with no apparent adverse effect on beta cell morphology or function.

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. Using two independent methods (immunoblotting and N-terminal sequencing), CYP1A1 protein was detected and quantified in all human hepatic microsomes tested in the study. Its levels, ranging from 0.97 to 3.0 pmol/mg protein, correlated with activities catalyzed by this enzyme [7-ethoxyresorufin O-deethylation (EROD) and oxidation of Sudan I], indicating the presence of enzymatically active CYP1A1. Even though levels of CYP1A1 expression are low, <0.7% of total hepatic CYP, the CYP1A1 contribution to oxidation of carcinogenic Sudan I in the test set of human liver microsomes ranges from 12 to 30%.

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. The aim of this article is to review the literature on HUCB and to assess its eventual usability in the treatment of diabetes mellitus. This review presents some recent reports concerning pancreatic endocrine stem cells and their identification, HUCB stem cells and their advantages and, finally, the potential for converting HUCB-derived stem cells into insulin-producing beta-cells.