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Human islet transplantation is a viable treatment option for type 1 diabetes mellitus (T1DM). However, pancreatic islet inflammation after transplantation induced by innate immune responses is likely to hinder graft function. This is mediated by incompatibility between islets and the blood interface, known as instant blood-mediated inflammatory reaction (IBMIR). Herein we hypothesized that portal venous administration of islet cells with human recombinant antithrombin (ATryn®), a serine protease inhibitor (serpin), which plays a central role in the physiological regulation of coagulation and exerts indirect anti-inflammatory activities, may offset coagulation abnormalities such as disseminated intravascular coagulation (DIC) and IBMIR. The current prospective, randomized experiment was conducted using an established preclinical pig model. Three groups were constituted for digested pancreatic tissue transplantation (0.15 ml/kg): control, NaCl 0.9% (n = 7); gold standard, heparin (25 UI/kg) (n = 7); and human recombinant ATryn® (500 UI/kg) (n = 7). Blood samples were collected over time (T0 to 24 h), and biochemical, coagulation, and inflammatory parameters were evaluated. In both the control and heparin groups, one animal died after a portal thrombosis, while no deaths occurred in the ATryn®-treated group. As expected, islet transplantation was associated with an increase in plasma IL-6 or TNF-α levels in all three groups. However, DIC was only observed in the control group, an effect that was suppressed after ATryn® administration. ATryn® administration increased antithrombin activity by 800%, which remained at 200% for the remaining period of the study, without any hemorrhagic complications. These studies suggest that coadministration of ATryn® and pancreatic islets via intraportal transplantation may be a valuable therapeutic approach for DIC without risk for islets and subjects.

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.

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.

The in vitro differentiation of pancreatic stem cells has recently been shown to represent a new source of beta cells for cell therapy in diabetes. Human ductal cell differentiation, in vitro, has been documented in three-dimensional (3D) culture and recently substantiated. Although encouraging, the optimization of the ductal cell source, expansion and differentiation ex vivo are mandatory for clinical relevance. We compared three sources of human ductal cells (hDC) (method A1-2, B, and C). The classical main duct isolation of hDC by explant (A1), or enzymatic digestion (A2), was compared with two indirect methods: from 3D cultured human islet/duct-enriched fractions (B) and dedifferentiated exocrine fractions (C). Method A: few viable hDC were obtained from the main duct. Method B: embedding islet/duct rich fraction in 3D collagen gels expands the cytokeratin 19 (CK19)-positive ductal component in the form of ductal cysts, as we described previously; monolayers derived from digested cysts were 80% ductal (CK19). Method C: initially adherent amylase-positive exocrine clusters contained 12% (CK19) to 22% (CK7) ductal cells. One-week exocrine cultures were amylase negative and 46% (CK19) to 63% (CK7) ductal. Cell viability varied: <20% (A1), 81+/-12% (B), 91+/-2% (C). Extrapolating total yields we obtained (+/-SEM): 10.5+/-4.6 x 10(3) (A1), 36+/-18 x 10(3) (A2), 292+/-50 x 10(6) (B), 1696+/-526 x 10(6) (C) viable hDC per pancreas. A secondary monolayer expansion of cyst-derived hDC (method B) was achieved with NuSerum (4.2-fold on plastic, 2.6-fold on 804G matrix; p < 0.05 vs. control cells on plastic). First passage exocrine-derived ductal cells also responded to matrix and to growth factors, albeit not significantly. In conclusion, this study demonstrated that an abundant hDC supply can be obtained from islet/duct or exocrine fractions followed by monolayer expansion with NuSerum. If their differentiation capacity is confirmed, in particular exocrine-derived ductal cells may represent a promising abundant source of islets for allogenic and autologous diabetes cell therapy.