Electronic Supplementary Material (ESM)
Rat and human pancreatic islet culture
Rat pancreatic islets of Langerhans were isolated from 3-month-old Sprague–Dawley rats (Skanbur BK, Solna, Sweden) by a collagenase digestion procedure as previously described 1. Islets were hand-picked and further cultured in groups of 150/50-mm well in RPMI-1640 supplemented with 10% FCS, 2 mmol/l L-glutamine, benzylpenicillin (100 U/ml) and streptomycin (0.1 mg/ml), containing 11.1 mmol/l glucose 1]. Human pancreatic islets kindly provided by O. Korsgren (Dept. of Clinical
Immunology, Uppsala University, Sweden) were cultured in the above-mentioned medium, containing 5.6 mmol/l glucose, at 37°C in humidified air with 5% CO2.
Culture medium was replaced every second day. The use of rat and human pancreatic islets was in accordance with international guidelines (NIH publication no. 85-23, revised-1985) and approved by the local ethics committees for Uppsala University.
Viral vectors and in vitro viral transduction
Adenoviruses
We used a serotype 5, E1-deleted/E3-deficient adenoviral vector derived from the widely used AdEasy system 2, which expresses GFP under the control of the cytomegalovirus (CMV) promoter 2. Virus stocks were purified by caesium chloride density-gradient centrifugation (L-80 ultracentrifuge; Beckman Coulter, Fullerton, CA,
USA) and plaque titred by serial dilution and agar overlay on human embryonic kidney (HEK) 293 cell line. Typical titres were 1010 PFU/ml or higher, representing 1 to 5% of the total viral particles as determined by readings of the optical densities.
1 Lentiviruses
Lentiviruses were produced by a five-plasmid transfection procedure. HEK 293 cells were transfected using TransIT 293 (Mirus, Madison, WI, USA) with the backbone vector expressing GFP under the CMV promoter together with four expression vectors encoding the packaging proteins Gag-Pol, Rev, Tat and the G protein of the vesicular stomatitis virus. Viral supernatants were collected starting 24 h after transfection, for four consecutive times every 12 h, pooled and concentrated 100-fold by ultracentrifugation. Using these protocols, titres of 8109 PFUs/ml were obtained 3.
Transduction procedure
For the adenoviral-mediated transduction, rat or human islets were incubated at 37° for
1 h in a minimum volume of 0.1 ml RPMI-1640 supplemented with 2% FCS and containing 20–50 PFU/cell of the adenoviral vector. Transduced islets were washed and further cultured in complete RPMI-1640 medium. The lentiviral-mediated transduction was performed overnight, at 37°C. Rat and human islets were incubated with the lentiviral vector at a concentration of 200 PFUs/cell in complete medium supplemented with 4 g/ml polybrene.
Following transduction, islets were further cultured for 2 to 7 days and analysed for
GFP fluorescence, viability and functionality. Some of the islet groups were incubated for 15 min at 37°C with complete medium containing 2 mmol/l EGTA, washed and then subjected to the transduction procedure.
2 Determination of transduction efficiency
Flow cytometry
The percentage of islet cells successfully transduced was assessed by trypsinising islets to a single-cell suspension as described 4 and analysing GFP expression by flow cytometry in a FACSCalibur flow cytometer (Becton-Dickinson
Immunocytometry Systems, San Jose, CA, USA).
Confocal microscopy
The imaging and analysis of intact isolated islets were performed by confocal laser scanning microscopy. At the time of imaging, rat or human intact islets were transferred from culture to a Petri dish containing PBS. Samples were subjected to optical sectioning at 1-m increments in an axial (z) dimension using a Leica TCS SP confocal laser scanner connected to a Leica DM IRBE microscope (Leica
Microsystems, Heidelberg, Germany). GFP fluorescence was excited at 488 nm and emitted light was collected between 495 and 525 nm. Image z-stacks of the tissue sections were captured, starting and ending at the uppermost- and lowermost- detectable level of GFP fluorescence, respectively. The stacks usually corresponded to a physical distance of ~80 m. All images shown are optical sections of intact islets recorded at 50-m depth.
3 Insulin staining
Islets were dispersed into individual cells by treatment with trypsin (5 mg/ml) in Ca2+-
and Mg2+-free Hanks’ solution at 37°C for 5 min and then applied by a 2-min
centrifugation at 150 g in a Cytospin 3 (Labex Instrument, Helsingborg,
Sweden) on to microscope slides. Islet cells were fixed for 5 min in PBS+4%
paraformaldehyde and further incubated for 10 min at room temperature in
PBS+2% FCS and 0.1% Triton X-100. The slides were then incubated in
PBS+2% FCS+0.1% Triton X-100 solution containing mouse anti-insulin
monoclonal antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room
temperature, washed and further incubated with rhodamine (TRITC)-
conjugated AffiniPure F(ab')2 fragment of goat anti-mouse IgG (H+L)
secondary antibody (Jackson ImmunoResearch Laboratories, West Grove,
PA, USA).
Determination of cell viability
To investigate the effect of adeno- and lentiviruses on islet cell viability, we performed fluorescence microscopy studies at 2, 4 or 7 days of culture after transduction. Rat and human pancreatic islets were incubated in complete medium RPMI-1640, containing 5
g/ml bisbenzimide and 10 g/ml propidium iodide for 10 min at 37°C. The islets were then washed and examined by fluorescence microscopy using a Leitz DMRB microscope (Leica) as previously described 3, 4.
Functional studies
4 Glucose-stimulated insulin secretion was determined for control and for adeno- or lentiviral-transduced rat and human pancreatic islets at day 4 or 7 after transduction.
Ten islets for each experimental group were transferred to flat-bottom multi-well plates containing 100 l KRBH buffer supplemented with 2 mg/ml BSA and 1.67 mmol/l glucose. Islets were incubated at 37°C in an atmosphere of 95% air and 5%
CO2. After 60 min incubation, medium was changed to KRBH supplemented with 16.7 mmol/l glucose followed by another 60 min incubation. The low- and high-glucose media were analysed for insulin content using a rat insulin ELISA according to the manufacturer’s instructions (Mercodia, Uppsala, Sweden). The rat insulin ELISA has a
120% cross-reactivity for human insulin.
Statistical analysis
Data are summarised as meansSEM. The significance of the differences between the groups was determined by Student’s t-test, one-way or two-way ANOVA for repeated measures and the Bonferroni test. Differences were considered significant at p<0.05.
Statistical analyses were performed using Sigma Stat (SPSS Science Software,
Erkrath, Germany).
References
1. Andersson A (1978) Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia 14:397-404 2. Berenjian S, Akusjarvi G (2005) Binary AdEasy vector systems designed for Tet- ON or Tet-OFF regulated control of transgene expression. Virus Res 115:16-23 3. Barbu A, Welsh N, Saldeen J (2002) Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential (ψm) in pancreatic RINm5F cells: prevention by Bcl-2. Mol Cell Endocrinol 190:75-82 4. Welsh N (2000) Assessment of apoptosis and necrosis in isolated islets of Langerhans: methodological considerations. Curr Top Biochem Res 3:189-200
5 ESM figure legends
ESM Fig. 1 Adenoviral-induced islet cell toxicity. a FACS analysis of dispersed islet cells expressing GFP. Intact rat islets were transduced in vitro with different titres of the adenoviral vector and analysed for GFP expression 2 days following transduction. b-e Micrographs showing intact islets transduced with 10 PFUs/cell (b, d) or 1,000
PFUs/cell (c, e) of the adenoviral vector and analysed by fluorescence microscopy on day 2 (b, c) or day 7 (d, e) following transduction. Red, dead cells (propidium iodide- positive nuclei); blue, viable cells (Hoechst-stained nuclei)
ESM Fig. 2 Successful transduction of beta cells using the perfusion-based protocol.
Micrograph showing insulin-positive (red cytoplasm) and GFP-positive (green cytoplasm and/or nuclei) islet cells. Islets were dispersed following transduction, stained for insulin as described and analysed by fluorescence microscopy
ESM Fig. 3 a, b Cellular death induced by adenoviral vectors in rat islets transduced in vitro (black bars) (a) or using the perfusion protocol (hatched bars) (b). Rat islets were transduced with 50 PFUs/cell of adenoviral vector and cell viability was assessed at day 7 after transduction using fluorescence microscopy. Results are means±SEM of six (in vitro) or four (perfusion) experiments. c, d Glucose-stimulated insulin release into the culture buffer of rat islets transduced in vitro (black bars) (c) or using the perfusion protocol (hatched bars) (d). Groups of 10 islets, transduced with 50
PFUs/cell of the adenoviral vector, were incubated for 60 min with 1.67 mmol/l and subsequently with 16.7 mmol/l glucose. Secreted insulin was measured by ELISA.
Results are means of three (in vitro) or four (perfusion) experiments±SEM
6 ESM Fig. 4 a Cellular death induced by the adenoviral vectors and lentiviral vectors in human islets. Human islets were transduced with 100 PFUs/cell of adenoviral vector or 200 transducing infectious units (TIUs)/cell of the lentiviral vector and cell viability was assessed at day 4 after transduction using fluorescence microscopy. Results are means±SEM of four experiments. b Glucose-stimulated insulin release into the culture buffer of human islets transduced by adenoviral or lentiviral vectors. Groups of 10 islets, transduced with 100 PFUs/cell of the adenoviral vector or with 200 TIUs/cell of the lentiviral vector, were incubated for 60 min with 1.67 mmol/l and subsequently with 16.7 mmol/l glucose. Secreted insulin was measured by ELISA. Results are means of two experiments±SEM
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