Human islet donor characteristics
Islets from 12 non-diabetic human cadaveric donors were obtained from the Islet Cell Resource Center (ICRC; http://icr.coh.org/, accessed 7 September 2010) (Table 1). Donors ranged in age from 27 to 76 years; islets from one 7-year-old donor were initially included in the study, but were subsequently excluded from analyses because of the marked biological differences between juvenile and adult islets . Of the remaining 11 donors, four were female, six male and one unrecorded. BMI ranged from low-normal (19.6 kg/m2) to severely obese (43.8 kg/m2). ICRC-determined islet purity was between 70 and 95%, viability was between 80 and 95%.
Human islet transplantation
All animal handling was in accordance with approved Institutional Animal Care and Use Committee protocols at the University of Pittsburgh; use of human islets was approved by the University of Pittsburgh Institutional Review Board. Human islets were transplanted under the kidney capsule of 2- to 3-month-old, streptozotocin-induced diabetic male NOD–severe combined immunodeficiency (SCID) mice (Jackson laboratory, Bar Harbor, ME, USA) as previously described . Briefly, NOD–SCID mice were rendered diabetic by intraperitoneal injection of 125 mg/kg streptozotocin for two consecutive days. Diabetes was determined by the presence of hyperglycaemia (>16.7 mmol/l), polyuria and weight loss. Random non-fasted blood glucose was measured from tail snip using a portable glucometer. After at least 3 days of hyperglycaemia, mice were transplanted with 2,500 to 4,000 islet equivalents (IEQ) beneath the kidney capsule. IEQ was defined as: 125 μm diameter islet = 1 IEQ. The initial two transplants were 4,000 IEQ, but subsequently the number of IEQ transplanted per donor was reduced until it became clear that 2,500 IEQ were sufficient to reverse hyperglycaemia [28, 29]. Recipients receiving >2,500 IEQ were not different from those receiving 2,500 IEQ with respect to blood glucose, plasma insulin, age or BMI. Multiple mice transplanted from the same donor received the same number of IEQ. Blood glucose levels were measured on days 1, 3, 5, 10 and 14 after transplant, before surgical catheterisation and infusion.
Mouse catheterisation and infusions
Detailed protocols on surgical catheterisation, tether system, housing, catheter maintenance, arterial blood sampling, erythrocyte return and techniques for venous infusion can be found in the online supplement of a previous publication . Transplanted mice with free access to food and water were catheterised 14 days after transplant, in the femoral artery and vein, using a sterile technique. After 3 days recovery, mice were infused for 4 days with 0.9% saline or 50% glucose (both wt/vol.) at 100 μl/h (approximately 2 g kg−1 h−1). Mice with elevated blood glucose 14 days after transplant (>11.1 mmol/l; n = 5) received saline; mice with blood glucose <11.1 mmol/l received either saline (n = 6) or glucose (n = 8). Three grafts, all from donor 10, did not have blood glucose measured on day 14 after transplant; all three had hyperglycaemia during the infusion period (14.2, 24.2 and 29.3 mmol/l) and were put in the >11 mmol/l blood glucose group. When several normoglycaemic grafted mice were available from one donor (donors 4, 5, 7 and 9 [Table 1]), one mouse received saline and the other(s) received glucose. Grafts from donors 1, 8 and 10, and one graft from donor 11, had blood glucose >11 mmol/l. Infusates contained 250 μg/ml BrdU, for continuous exposure of 25 μg/h over the 4 days. Morning (postprandial) arterial blood was sampled from the unhandled mice via catheter at 0, 24, 48, 72 and 96 h of infusion. Following infusion, the mice were killed, and pancreases and engrafted kidneys fixed for histological analysis.
Blood glucose was measured using a portable glucometer (Precision QID; Abbot Laboratories, Abbot Park, IL, USA) before and a glucometer (Ascencia Elite XL; Bayer, Tarrytown, NY, USA) after catheterisation. Plasma mouse and human insulin were measured by radioimmunoassay (Millipore/Linco, St Charles, MO, USA); cross-reactivity of the human assay with human proinsulin is <0.2% and with rodent insulin <0.1%. Cross-reactivity of the rodent insulin assay with human insulin is high; mouse insulin values were obtained by subtracting measured human insulin from rodent (total) insulin.
Engrafted kidneys were fixed in Bouin’s solution (Sigma, St Louis, MO, USA) for 4 h at room temperature, dehydrated, embedded in paraffin and sectioned (5 μm). For BrdU/insulin immunofluorescence, sections were rehydrated, incubated in 1 mol/l HCl for 30 min at 37°C, blocked in PBS with 5% vol./vol. goat serum and 1% (wt/vol.) BSA, and exposed overnight to anti-BrdU (1:200; Abcam, Cambridge, MA, USA) and anti-insulin (1:50; Invitrogen, Carlsbad, CA, USA) antibodies at 4°C, followed by exposure to fluorescent secondary antibodies (Alexa 488 and 594 conjugated, 1:200, and Hoechst, 1:1,000; all from Invitrogen). For macrophage marker F4/80–insulin–BrdU–DAPI co-staining, paraffin sections were rehydrated, digested with pepsin (Digest-All; Invitrogen), permeabilised in 0.1% vol./vol. Triton X-100 in PBS, blocked and stained exactly as above using primary antibodies for insulin (1:500; Invitrogen), F4/80 (1:100; Abcam) and BrdU (1:5; Amersham, Piscataway, NJ, USA), and an additional secondary antibody conjugated to Cy5 (1:200; Invitrogen). TUNEL staining was performed using a kit (Dead End kit; Promega, Madison, WI, USA) followed by immunofluorescence staining for insulin and Hoechst as above.
Microscopy and image analysis
For BrdU and TUNEL quantification, stained sections were imaged using an upright fluorescence microscope (Provis; Olympus, Center Valley, PA, USA) at ×400, blinded and insulin-positive and TUNEL- or BrdU-positive beta cells manually counted. For BrdU, 3,034 ± 246 beta cells were counted per graft; for TUNEL 2,618 ± 317 beta cells were counted per graft. To verify BrdU and insulin colocalisation in the same cell and to determine whether insulin-positive BrdU-positive cells represented macrophages engulfing dying beta cells, confocal microscopy was performed (Fluoview 1000; Olympus) at ×400.
All data are presented as mean ± SEM or as individual values. Statistical significance was calculated by Prism 4 (GraphPad Software, La Jolla, CA, USA), using Student’s t tests. One-way ANOVA was used when comparing multiple groups, with Tukey’s Multiple Comparison Test. Two-way ANOVA was used when comparing blood glucose differences over time between infusion groups. Linear regressions were calculated by GraphPad Prism, with reported p values signifying whether the slope of the regression line was significantly different from zero. For all analyses, p < 0.05 was considered significant.