CLINICAL ISLET TRANSPLANTATION

T. Itoh¹, H. Noguchi¹, M. Takita¹, K. Sugimoto¹, M. Shimoda², B. Naziruddin³, M.F. Levy³, Y. Yasunami⁴, S. Matsumoto^1
1Islet Cell Laboratory, Baylor Research Institute Fort Worth Campus, Fort Worth/UNITED STATES OF AMERICA, ²Division Of Cardiology, Department Of Internal Medicine, Baylor University Medical Center, Dallas/UNITED STATES OF AMERICA, ³Islet Cell Transplant Program, Baylor Regional Transplant Institute, Dallas/UNITED STATES OF AMERICA, ⁴Regenarative Medicine & Transplantation, Faculty of Medicine, Fukuoka University, Fukuoka/JAPAN

Body: Introduction: Pancreatic islet transplantation is a promising therapy for type 1 diabetes mellitus. However, there are still major issues that prevent the use of this treatment as a standard therapy. Limited ability of current viability assay is one of those issues. Real time, convenient and accurate assessment of islet viability is critical for avoiding transplantation of non-therapeutic preparations. High-mobility group box 1 (HMGB1) protein has been initially found as a DNA-binding protein and present in almost all eukaryotic cells to stabilize nucleosome formation and acts as a nuclear factor that enhances transcription. Recently we discovered that islets contain high levels of HMGB1 and damaged islets can release HMGB1 to outside the cell in mice model (J Clin Invest. in press). In this study we examined whether the HMGB1 levels of islet culture medium could reflect outcome of islet transplantation for the potency assay of isolated islets. Methods: Mice islets (C57 BL/6) were cultured under normoxic condition (air with 5% CO₂) for 24 hours (normoxia group) or hypoxic condition (1% O₂, 5% CO₂ and 94% N₂) for 6 hours (hypoxia-6hr group) or 24 hours (hypoxia-24hr group) at 37 degrees Celsius. These three groups of islets were transplanted beneath the kidney capsule of streptozotocin (STZ)-diabetic mice (C57 BL/6) as in vivo assay. To assess the viability of those islets, propidium iodide (PI)⁺ area assay, glucose-stimulated insulin release test (GSIRT), ATP/DNA assay and medium HMGB1/islet DNA assay were performed. PI⁺ and islet area (n=20 in each group from two individual experiments) were calculated by digital image soft.ATP, DNA, insulin and HMGB1 levels (n=10 in each group from two individual experiments) were measured by ELISA.Statistical significance was determined by one-way ANOVA and Dunnett post hoc test. Results: All of STZ-diabetic mice (n=3) received with 200 normoxia islets became normoglycemic after transplantation. However, all of diabetic mice received with same number of either hypoxia-6hr or hypoxia-24hr islets (n=5, each) remained hyperglycemic after transplantation. PI⁺ area assay showed the significant difference between normoxia (PI⁺ area/islet area; 2.0±1.4%) and hypoxia-6hr groups (23.3±8.7%, p<0.0001). In the GSIRT, stimulation index (SI) showed the significant difference between normoxia (22.0±13.9) and hypoxia-6hr groups (2.7±0.8, p<0.01). However, the actual insulin secretion in response to high glucose (20.0mM) showed no significant difference among normoxia (4.1±3.2ng/μg DNA), hypoxia-6hr (5.2±3.3 ng/μg DNA) and hypoxia-24hr groups (3.4±0.7 ng/μg DNA). ATP/DNA assay showed no significant difference between normoxia (18.3±9.2pmol/μg DNA) and hypoxia-6hr groups (20.1±14.0pmol/μg DNA). Medium HMGB1 levels showed the significant difference between normoxia (0.14±0.03ng/μg DNA) and hypoxia-6hr groups (1.10±0.81ng/μg DNA, p<0.05). Conclusion: These findings demonstrated that medium HMGB1 assay, PI⁺ area assay and SI could distinguish normoxia (cured) group from hypoxia-6hr (non-cured) group but not in ATP/DNA assay and insulin secretion levels in response to high glucose. Measuring medium concentration of HMGB1 is easy and well-established method. Therefore medium HMGB1 assay can be a new convenient potency assay to predict outcome of islet transplantation.

Disclosure: All authors have declared no conflicts of interest.