Abstract
Aims/hypothesis
Successful gene transfer to pancreatic islets might be a powerful tool for dissecting the biological pathways involved in the functional impairment and destruction of beta cells in type 1 diabetes. In the long run, such an approach may also prove useful for promoting islet graft survival after transplantation in diabetic patients. However, efficient genetic modification of primary insulin-producing cells is limited by the specific compact structure of the pancreatic islet. We present here a whole-pancreas perfusion-based transduction procedure for genetic modification of intact pancreatic islets.
Materials and methods
We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion.
Results
In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure.
Conclusions/interpretation
These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research.
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Introduction
Much work is currently directed to finding highly efficient and non-toxic vectors for gene transfer in primary insulin-producing cells. Among these, viral vectors have emerged as first choice for engineering beta cells, mostly due to their capacity to mediate efficient gene transfer in non-dividing cells. Adenoviral vectors in particular offer the advantage of a high gene transfer efficiency as well as relatively long-term gene expression in primary beta cells [1]. However, one important limitation of the adenoviral-mediated gene transfer in beta cell research is the reduced ability to reach and transduce the inner cellular mass of the intact pancreatic islet. This is an important drawback for experimental designs that involve gene therapy approaches in which the final target for genetic modification is the insulin-producing cell, since, at least in rodent models, insulin-producing cells are centrally located in the core of the islet, surrounded by a mantle of non-beta cells at the periphery.
Materials and methods
Sprague-Dawley rats (Skanbur BK, Solna, Sweden) were anaesthetised with an i.p. injection of thiobutabarbital sodium (120 mg/kg body weight). A catheter (inner diameter 1.40 mm) connected to a peristaltic pump was placed in the abdominal aorta so that the perfusion medium could flow freely into the pancreas. The gland was removed from the animals [2] and placed in a funnel at a constant temperature (30°C). The gland was perfused for 45 to 60 min at a flow rate of 1 ml/min with a continuously gassed (95% O2–5% CO2) Krebs–Ringer bicarbonate (KRBH) buffer, supplemented with 20-mg/ml BSA, 20-mg/ml dextran T70 and 0.3-mg/ml glucose, and containing ∼109 plaque-forming units (PFUs) per pancreas of the green fluorescent protein (GFP)-expressing adenoviral vector, which corresponds to 20 to 50 PFUs/islet cell. In some of the experiments, the capillary endothelium was disrupted before administering the viral vector by pre-perfusion for 40 s with medium containing 0.1% Triton X-100, followed by a 10-min wash with KRBH buffer only. Following transduction, islets were isolated and subsequently cultured for 2 to 7 days.
All other experimental procedures have been previously described [3, 4] or are given in the Electronic supplementary material (ESM).
Results
Adenoviral-mediated gene transfer to intact rat pancreatic islets
High-titre in vitro adenoviral-mediated transduction of intact islets reached efficiencies above 50% (ESM Fig. 1). This approach, however, was paralleled by extensive cellular death [5] (ESM Fig. 1). Low-titre-mediated gene transfer into intact islets, on the other hand, resulted in 31% GFP-positive islet cells and no major increase in cell death (Fig. 1 and ESM Fig. 1). This percentage was increased up to 48% if, prior to the transduction procedure, cell-to-cell contact of the intact islets was transiently disturbed by EGTA treatment. Confocal laser-scanning microscopy sections through adenovirus-transduced isolated rat islets showed that the cells transduced by the in vitro technique were located in the outermost cell layer, while the cells within the core of the islet were almost exclusively GFP-negative (Fig. 1). A similar GFP-positive cell distribution pattern was seen in the EGTA-pretreated rat islets (Fig. 1d).
EGTA-enhanced adenoviral- and lentiviral-mediated transduction of intact human islets in vitro
Having observed a modest increase in gene transfer efficiency using EGTA, we next investigated whether a similar approach affected transduction of intact human islets. Adenoviruses and lentiviruses were equally efficient in transducing intact human islets: 28% positive cells with the adenoviral vector and 30% with the lentiviral vector (Fig. 2a). However, analysis of optical sections revealed that lentiviral vectors were more efficient in reaching the cells located in the inner layers of the islets (Fig. 2d), while adenoviral vectors promoted gene transfer only in cells located at the periphery (Fig. 2b). Disruption of cell-to-cell contact by EGTA pretreatment resulted in a significantly improved transduction efficiency mediated by adenoviral vectors but did not increase the outcome of lentiviral-mediated transduction (Fig. 2a).
Perfusion-based adenoviral transduction of rat pancreas
As the majority of the islet cells cannot be reached by viral vectors, even though cell contacts have been disrupted by EGTA treatment, we next hypothesised that viral vectors delivered via the vascular system would more efficiently reach the islet core cells. When intact rat pancreases were perfused with KRBH buffer containing ∼109 PFUs, only 11% of the islet cells were GFP-positive (Fig. 1a,b). However, as demonstrated by optical sectioning (Fig. 1e), the GFP-positive cells were dispersed throughout the islet rather than being confined to the periphery as in the in vitro approach (Fig. 1c,d). Disruption of the endothelium within the islet capillaries by Triton X-100 resulted in a significantly increased transduction efficiency, reaching more than 70% (Fig. 1a,b), accompanied by an even distribution of the positive cells both in the islet core and at the periphery (Fig. 1f). Additional insulin staining of islets isolated from the Triton X-100-perfused pancreases demonstrated that 65% of the GFP-positive cells were insulin-producing cells (ESM Fig. 2). The perfusion procedure had no influence on the number or size of the isolated islets. All islet preparations transduced by the perfusion-based technique and all islet sizes were characterised by a uniform distribution of GFP-positive cells. Moreover, the function and viability of perfusion-transduced islets were no different from the control islets (ESM Fig. 3). Nor was the viability and function of the in vitro virus-transduced human islets affected by the different treatments (ESM Fig. 4).
Discussion
Gene transfer to pancreatic islets is an attractive approach for elucidating basic mechanisms of beta cell function and destruction and could also provide a potential tool for improving islet graft survival following islet transplantation in type 1 diabetes [6]. Considering the compact structure of the pancreatic islet, a successful vector for gene delivery would have to be able to breach the outer surface of the islet and penetrate to the core. This is particularly important, since the rodent insulin-producing cell, the most obvious targets for gene transfer in diabetes research, is preferentially located to the centre of the islet.
Here we show that in vitro transduction of intact rat and human islets using adenoviral vectors promoted GFP expression in only 30% of the cells. Moreover, by confocal sectioning of the intact islet, we also demonstrated that only the cells located in the outermost layer are successfully targeted. For some applications, such as immunoprotection of transplanted islets [6], low transfer efficiencies could suffice, provided that the transgene encodes a soluble protein. However, significantly higher transfection efficiencies are probably required for the study of specific mechanisms involved in the functional impairment/destruction of beta cells or when the induction of an intracellular protein (as anti-apoptotic proteins) is envisaged [1].
One option for increasing the transfection efficiency of intact islets is to dramatically increase the viral vector concentrations. However, both in dispersed islet cells [5] and in intact islets (present results), exposure to adenoviral vectors at high concentrations resulted in significant induction of cell death. In gene-therapy-based in vivo studies on type 1 diabetes, the toxicity exhibited by high concentrations of the viral vectors may be especially unfavourable because the vector itself could induce a virus-specific immune response or exacerbate an ongoing autoimmune attack.
Therefore, we evaluated alternative ways of exposing the inner islet cell mass to the gene transfer vectors. By transiently disturbing cell-to-cell contact and therefore increasing paracellular permeability, we observed an increase of the transfection efficiency. However, the distribution of GFP-positive cells within the islet was not dramatically modified, as the cells located in the core of the islet were still essentially GFP-negative. Instead, we next delivered adenoviral particles via the pancreatic arteries to the perfused pancreas ex vivo. Although the perfusion-based protocol resulted in rather low transduction efficiencies, the GFP-positive cells were distributed evenly in the core of the islet as well as at the periphery. We also observed that dilation of the pancreatic capillaries, promoted by glucose or adenosine, was not sufficient to allow efficient adenovirus passage from the blood vessels to the islet cells (results not shown). However, the number of islet cells expressing GFP was dramatically increased when the endogenous endothelium was disrupted by Triton X-100. This substance is known to selectively destroy the endothelium of the isolated heart, without affecting the smooth muscle layer [7]. Also in our system, the viability and function of islet cells exposed to Triton X-100 was unaffected.
Other recent publications have reported in vivo transduction of islet cells using i.v. or intraductal injections of viral particles in combination with clamping the pancreatic blood flow for 20 to 30 min [8, 9]. These novel and interesting developments allow pancreatic transgene expression in vivo, but only at low levels, as the endothelium was not disrupted in these protocols. To the best of our knowledge, Triton X-100 and other endothelium-disrupting treatments are not tolerated in vivo, making highly efficient in vivo transduction not yet feasible.
The transduction protocol described here could provide a significantly improved yield of successfully ex vivo engineered beta cells, while keeping the specific pancreatic islet structure intact. This is of major importance, as preservation of the structural and functional integrity of genetically engineered pancreatic islets is a prerequisite for performing meaningful islet cell research. By ex vivo perfusion of intact human pancreases, followed by islet isolation and subsequent islet transplantation, a similar approach might be used for ex vivo gene transfer into human islet cells, as a possible tool for a successful application of gene therapy as a potential cure for type 1 diabetes.
Abbreviations
- ESM:
-
Electronic supplementary material
- GFP:
-
green fluorescent protein
- KRBH:
-
Krebs–Ringer bicarbonate
- PFU:
-
plaque-forming unit
References
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Acknowledgements
We thank G. Akusjärvi, Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden for providing the adenoviral vector, and G. Mostoslavsky, Department of Genetics, Harvard Medical School and Molecular Medicine, Boston, MA, USA for providing the lentiviral vector. This work was supported by grants from the Swedish Research Council, the Swedish Diabetes Association, the Family Ernfors Fund, Barndiabetesfonden, Samariten Foundation, Lars Hiertas Minne Foundation, the European Foundation for the Study of Diabetes (Lilly) and the Novo Nordisk Fund.
Duality of interest The authors have stated that they have no dualities of interest.
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Supplementary Fig. S1
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) (JPEG 161 kb)
Supplementary Fig. S2
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 (JPEG 66 kb)
Supplementary Fig. S3
a, b Cellular death induced by adenoviral vectors in rat islets transduced in vitro (black barsblack bars) (a) or using the perfusion protocol (hatch 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) experiment±SEM (JPEG 137 kb)
Supplementary Fig. S4
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 (JPEG 96 kb)
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Barbu, A.R., Bodin, B., Welsh, M. et al. A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans. Diabetologia 49, 2388–2391 (2006). https://doi.org/10.1007/s00125-006-0390-5
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DOI: https://doi.org/10.1007/s00125-006-0390-5