Efficient gene delivery and silencing of mouse and human pancreatic islets.

BACKGROUND: In view of the importance of beta cells in glucose homeostasis and the profound repercussions of beta cell pathology on human health, the acquisition of tools to study pancreatic islet function is essential for the design of alternative novel therapies for diabetes. One promising approach toward this goal involves the modification of gene expression profile of beta cells. RESULTS: This study describes a new method of gene and siRNA delivery into human pancreatic islets by microporation technology. We demonstrated that mild islet distention with accutase greatly enhanced the transfection efficiency without compromising in vitro function (secretion, apoptosis and viability). As an example, the recently identified gene involved in type 2 diabetes, ZnT8, can be over-expressed or silenced by RNA interference using this technology. Microporation can also be used on rodent islets. CONCLUSIONS: Taken together, our results demonstrate that microporation technology can be used to modify gene expression in whole rodent and human islets without altering their in vitro function and will be key to the elucidation of the factors responsible for proper islet function.

PPARgamma-dependent and -independent effects of rosiglitazone on lipotoxic human pancreatic islets.

We explored the in vitro effects of Rosiglitazone (RZG), a PPARgamma agonist, on human pancreatic islet dysfunctions induced by chronic free fatty acid exposure. We demonstrated that RZG beneficial effects on insulin secretion and apoptosis did not imply PDX-1 or insulin gene modulation. It rather involved, through a PPARgamma-dependent mechanism, a reduction of iNOS overexpressed in lipotoxic islets. This reduction likely led to the restoration of ATP level and insulin secretion as well as the decrease in apoptosis. More interestingly, we also demonstrated that RZG beneficial effects involved PPARgamma-independent mechanisms. RZG treatment led to a limitation of oxidative stress exemplified by an increase of GPx and SOD expression. It also increased UCP2 expression that seemed to display antioxidant action in this model. Thus, RZG did not appear to exert a direct action on insulin expression but rather an indirect action on insulin secretion and apoptosis, through PPARgamma-dependent and -independent mechanisms, via regulation of nitrogen and oxygen reactive species injury.

Quantitative in vivo islet potency assay in normoglycemic nude mice correlates with primary graft function after clinical transplantation.

Reliable assays are critically needed to monitor graft potency in islet transplantation (IT). We tested a quantitative in vivo islet potency assay (QIVIPA) based on human C-peptide (hCP) measurements in normoglycemic nude mice after IT under the kidney capsule. QIVIPA was initially tested by transplanting incremental doses of human islets. hCP levels in mice were correlated with the number of transplanted islet equivalents (r(2) = 0.6, P<0.01). We subsequently evaluated QIVIPA in eight islet preparations transplanted in type 1 diabetic patients. Conversely to standard criteria including islet mass, viability, purity, adenosine triphosphate content, or glucose stimulated insulin secretion, hCP in mice receiving 1% of the final islet product was correlated to primary graft function (hCP increase) after IT (r(2)=0.85, P<0.01). QIVIPA appears as a reliable test to monitor islet graft potency, applicable to validate new methods to produce primary islets or other human insulin secreting cells.

Expression of progenitor cell markers during expansion of sorted human pancreatic beta cells.

Functional pancreatic beta cell mass is dynamic and although fully differentiated, beta cells are capable of reentering the cell cycle upon appropriate stimuli. Stimulating regeneration-competent cells in situ is clearly the most desirable way to restore damaged tissue. Regeneration by dedifferentiation and transdifferentiation is a potential source of cells exhibiting a more developmentally immature phenotype and a wide differentiation potential. In this context and to gain a better understanding of the transformation induced in human beta cells during forced in vitro expansion, we focused on identifying differences in gene expression along with phenotypical transformation between proliferating and quiescent human beta cells. FACS-purified beta cells from three different human pancreata were cultured during 3-4 months (8-10 subcultures) on HTB-9 cell matrix with hepatocyte growth factor. Gene expression profiling was performed on cells from each subculture on "in-house" pancreas-specific microarrays consisting of 218 genes and concomitant morphological transformations were studied by immunocytochemistry. Immunocytochemical studies indicated a shift from epithelial to neuroepithelial cell phenotype, including progenitor cell features such as protein gene product 9.5 (PGP 9.5), Reg, vimentin, and neurogenin 3 protein expression. The expression of 49 genes was downregulated, including several markers of endocrine differentiation while 76 were induced by cell expansion including several markers of progenitor cells. Their pattern also argues for the transdifferentiation of beta cells into progenitor cells, demonstrating neuroepithelial features and overexpressing both PBX1, a homeodomain protein that can bind as a heterodimer with PDX1 and could switch the nature of its transcriptional activity, and neurogenin 3, a key factor for the generation of endocrine islet cells. Our study of the machinery that regulates human beta cell expansion and dedifferentiation may help elucidate some of the critical genes that control the formation of adult pancreatic progenitor cells and hence design targets to modify their expression in view of the production of insulin-secreting cells.

Automated digital image analysis of islet cell mass using Nikon's inverted eclipse Ti microscope and software to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

Reliable assessment of islet viability, mass, and purity must be met prior to transplanting an islet preparation into patients with type 1 diabetes. The standard method for quantifying human islet preparations is by direct microscopic analysis of dithizone-stained islet samples, but this technique may be susceptible to inter-/intraobserver variability, which may induce false positive/negative islet counts. Here we describe a simple, reliable, automated digital image analysis (ADIA) technique for accurately quantifying islets into total islet number, islet equivalent number (IEQ), and islet purity before islet transplantation. Islets were isolated and purified from n = 42 human pancreata according to the automated method of Ricordi et al. For each preparation, three islet samples were stained with dithizone and expressed as IEQ number. Islets were analyzed manually by microscopy or automatically quantified using Nikon's inverted Eclipse Ti microscope with built-in NIS-Elements Advanced Research (AR) software. The AIDA method significantly enhanced the number of islet preparations eligible for engraftment compared to the standard manual method (p < 0.001). Comparisons of individual methods showed good correlations between mean values of IEQ number (r(2) = 0.91) and total islet number (r(2) = 0.88) and thus increased to r(2) = 0.93 when islet surface area was estimated comparatively with IEQ number. The ADIA method showed very high intraobserver reproducibility compared to the standard manual method (p < 0.001). However, islet purity was routinely estimated as significantly higher with the manual method versus the ADIA method (p < 0.001). The ADIA method also detected small islets between 10 and 50 µm in size. Automated digital image analysis utilizing the Nikon Instruments software is an unbiased, simple, and reliable teaching tool to comprehensively assess the individual size of each islet cell preparation prior to transplantation. Implementation of this technology to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

1,25-dihydroxyvitamin D3 protects RINm5F and human islet cells against cytokine-induced apoptosis: implication of the antiapoptotic protein A20.

Transplantation of islets of Langerhans is a potential cure for type 1 diabetes, but its success is hampered by destruction of the islets. The data presented herein suggest that the active metabolite of vitamin D3 [1,25-(OH)2D3] may promote islet cell survival by modulating the effects of inflammatory cytokines, which contribute to beta-cell demise. We investigated some of the mechanisms triggering the apoptotic machinery in rat insulinoma RINm5F cells and human islets treated with IL-1beta plus interferon-gamma plus TNFalpha and assessed the effects of 1,25-(OH)2D3 in these processes. Mitochondrial transmembrane permeability and apoptotic features, determined by percentage of sub-G1 cells, quantitation of DNA strand breaks, and Hoechst staining, were significantly increased by cytokines and reverted toward control values by 1,25-(OH)2D3 cotreatment. The cytoprotection of cells correlated with the abrogation of cytokine-induced nitric oxide production. The activation of nuclear factor-kappaB plays a key role in the different pathways implicated in nitric oxide generation. We demonstrated for the first time, in both RINm5F cells and human islets, that 1,25-(OH)2D3 was able to induce and maintain high levels of A20, an antiapoptotic protein known to block nuclear factor-kappaB activation. Our study showed a clear efficiency of 1,25-(OH)2D3 on the apoptotic machinery triggered by cytokines in beta-cells and suggests that 1,25-(OH)2D3 could help overcome a major obstacle encountered in the cellular therapy of diabetes, such as nonfunction in the immediate posttransplantation period.

Modulation of specific beta cell gene (re)expression during in vitro expansion of human pancreatic islet cells.

The need for transplantable beta cells with a stable phenotype has given rise to several strategies including the expansion of existing pancreatic islets and/or growth of new ones. In vitro studies of beta cell proliferation on extracellular matrices plus growth factors have highlighted a possible cell expansion technique; however, the technique was accompanied with loss of insulin secretion. Herein we showed that human islet cell proliferation was marked by a decreased expression of specific differentiation markers, particularly insulin, insulin promoting factor-1 (IPF-1), and glucokinase. After a 6-day expansion period, we tried to reexpress the beta cell differentiation markers with compounds known for their differentiation and/or insulin-secreting properties. Sodium butyrate was a potent factor of IPF-1, insulin, and glucokinase gene reexpression; it also clearly induced secretion of gastrin, a known neogenic factor. Other compounds, namely TGF-beta, calcitriol, GLP-1, and activin A, efficiently enhanced the glucose sensor machinery, particularly Glut-1 and glucokinase, thus triggering glucose responsiveness. Our results indicate that specific beta cell gene expression may be induced after expansion and dedifferentiation. This rekindles interest in human beta cell expansion. The possible stabilization of specialized genes needed by beta cells to fulfill their role as nutrient sensors and metabolic regulators may also be of interest to ensure graft maintenance and efficiency.

Upgrading pretransplant human islet culture technology requires human serum combined with media renewal.

BACKGROUND.: The original Edmonton protocol used fresh islets, but for obvious logistic advantages most transplant centers have implemented pretransplant culture in human albumin. The aim of this study was to improve current pretransplant human islet culture techniques. METHODS.: Clinical-grade purified human islets from a total of 24 donors were directly resuspended after isolation in CMRL 1066-based media at 37 degrees C, and media additions and renewal were tested. At days 1 and 5 of culture, in vitro quality controls included islet viability, insulin content and function, apoptosis, and in vivo islet potency assay in nude mice. RESULTS.: Replacing human albumin with human AB serum improved 1- and 5-day preservation of islet function and viability which was further enhanced with antioxidant Stem Ease, leading to the iCulture medium (enriched CMRL: pyruvate, zinc sulfate, insulin, transferrin, selenium, 2.5% human AB serum and Stem Ease). Major damage occurs in the first day of culture and frequent media renewal (25% vol/hr) in this period further improved viability, apoptosis, islet recovery, and function in vitro and in vivo, compared with only changing medium after overnight culture. CONCLUSIONS.: The described human islet culture technique (iCulture medium+renewal) seems to be the best choice for clinical human islet culture when short (1 day) or long (5 days) periods are used. Media choice and dilution play a major role in the function and survival of human islets in culture.

Rapid purification of human ductal cells from human pancreatic fractions with surface antibody CA19-9.

Generating human insulin-secreting cells for cell therapy of diabetes represents a highly competitive world challenge. Human ductal cells can give rise to islets in vivo and in vitro. The goal of this study was to devise a rapid sorting method to highly purify human ductal cells from pancreatic tissue using a pan-ductal membrane antibody carbohydrate antigen 19-9 (CA19-9). Human pancreatic sections confirmed antibody specificity. The human exocrine fraction (30% ductal cells) was sorted with magnetic bead technology or by FACS. Immunocytochemistry post-sorting determined ductal cell content. The manual magnetic bead technique resulted in 74%+/-2 (n = 4) CA19 positive cells. Whereas the automated AutoMACS technique (n = 5) yielded 92.6%+/-0.5 CA19-9 positive cells with only a minor beta cell contamination (0.2%+/-0.03); cell yield post-sorting was 12.9%+/-2.5 (1.69+/-0.41 x 10(6) cells) with 51.7%+6.5 (n = 5) viability post-sorting. The FACS (n = 6) resulted in 97.1%+/-0.82 CA19-9 positive cells, a cell yield of 25.5%+/-5.6 (5.03+/-1.0 x 10(6)), with 72.1%+/-6.1 viability post-sorting.