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Current Gene Therapy, 2015, 15, 436-446
A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors Carmen María Jiménez-Moreno1, Irene de Gracia Herrera-Gomez1, Livia López-Noriega1, Petra Isabel Lorenzo1, Nadia Cobo-Vuilleumier1, Esther Fuente-Martín1, José Manuel Mellado-Gil1, Géraldine Parnaud2, Domenico Bosco2, Benoit Raymond Gauthier1,* and Alejandro Martín-Montalvo1,* 1
Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine, Avenida Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092 Sevilla, Spain; 2 Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva Rue Michel-Servet 1, 1211 Geneva, Switzerland Abstract: Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.
Keywords: Diabetes Mellitus, Gene transfer, Infection, Lentivirus, Pancreatic islet, Transduction. INTRODUCTION Type 1 Diabetes Mellitus (T1DM) is one of the most common multifactorial endocrine and metabolic diseases in childhood resulting in persistent hyperglycaemia. Currently, approximately 490,000 children have been diagnosed with T1DM and 78,000 children under the age of 15 are estimated to develop T1DM annually worldwide [1]. More alarmingly, a recent epidemiological study has revealed that the incidence rate of T1DM in children in the United Sates has increased dramatically by 29% between 1985 and 2004 surpassing by 18 times the incidence of Type 2 Diabetes Mellitus (T2DM) in the white population [2]. The most common form of T1DM results from the breakdown of β-cell-specific self-tolerance by T-lymphocytes precipitating an autoimmune attack and eradication of insulin-producing cells [3]. Strong genetic and environmental factors contribute to the onset of T1DM [4]. Existing treatments for T1DM are primarily focused on insulin supplementation. However, despite the beneficial effects of life-long insulin therapy on
*Address correspondence to these co-senior authors at the Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine, Avenida Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092 Sevilla, Spain; Tel: 0034 954 468 004; Fax: 0034 954 461 664; E-mails:
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glucose homeostasis, insulin administration does not eliminate severe diabetic complications such as retinopathy, nephropathy as well as cardiovascular and cerebrovascular diseases [5]. In the past 10 years, clinical islet transplantation has gained much attention as a cell replacement therapy for restoring the functional β-cell mass. Unfortunately, the limited supply of islets from donors has failed to meet demands imposed by the ever-growing number of T1DM patients. An additional major hurdle has been the lack of durability of islet function with insulin independency in less than 10% of patients 5 years after transplantation [6, 7]. Furthermore, several post-transplant events, such as instant blood mediated inflammatory reaction and cytokine cascade, seriously affect the long-term functionality of islets [8-11]. Ex vivo genetic modifications of islets to enhance cell function and survival prior to transplantation have been successfully demonstrated in animal models [12, 13]. This strategy can ultimately increase islet viability and performance providing a tangible approach to improve human islet transplantation and long-term insulin independence. Although protocols designed to modulate gene expression have been extensively used in single cells, the complexity of pancreatic islets has impeded successful gene delivery. Indeed, due to its tridimensional structure, β-cells embedded within the core of islets are sequestered from any significant contact with the remote environment [14-19]. During the last years, several © 2015 Bentham Science Publishers
An Efficient Protocol for Intact Islet Infection
non-viral strategies for genetic modification of islet cells, such as electroporation, microporation, gene gun particle bombardment, cationic liposomes and polymeric particles, have been investigated [15, 19-21]. Unfortunately, in most cases those techniques provided low gene transfer efficiencies and the difficulty of reproducing these protocols have hindered their broad use to allow optimized islet gene transfer. More recently, ex vivo infection of islets was proposed in order to conduct mechanistic studies and also to transfer therapeutically promising genes or alleles prior to islet xenotransplantation [22]. Adenoviral vectors have been used with this purpose since the efficiency of infection in nondividing cells is greater than other vectors and their epichromosomal location reduces the probability of conferring insertional mutations. The efficiency of the majority of adenovial-based infection protocols has been found to be limited to only ~7-30 % of islet cells and infected cells were mostly located in the periphery of the islet [14, 15]. Although several studies reported infection of 30-90 % of islet cells throughout the whole islet [14, 23, 24] excessive viral dosage were used which may cause cytotoxicity [14, 25, 26]. Alternatively, genetic modifications of adenoviral vectors such as the inclusion of Arg-Gly-Asp motif were attempted to enhance transduction efficiency up to ~80 % of islet cells at 10 Plaque Forming Units (PFU) per cell [15]. Unfortunately, the drawback for adenoviral transduction was the methodological difficulties of these experimental protocols and the transient modulation of gene expression [23, 27]. The use of lentiviral vectors in gene therapy has become a powerful tool to safely deliver genetic material with the purpose to rectify molecular defects, enhance functional performance or increase viability of cells. Major advantages of lentiviral vectors include the capacity to infect both dividing and non-dividing cells using repeated dosing, genome integration and long-term expression as well as low immunogenicity [28]. Currently, 89 gene therapy clinical trials using lentiviral vectors are ongoing [29] focusing predominantly on the treatment of primary immunedeficiencies [30]. Transduction protocols using lentiviruses have also been developed for islet infection yielding similar efficiency than adenoviral vectors (~3-50 % of β -cells) [14, 16-18, 31-33]. Given the tremendous attributes of lentiviral vectors combined with their current use in clinical trials, we set out to develop a simple and optimal lentiviral transduction protocol for intact human and mouse pancreatic islets with the longterm goal to apply this protocol for gene therapy in islets prior to transplantation without compromising their integrity and functionality. MATERIALS AND METHODS Consumables Reagents and materials used in this study along with reference numbers and companies of purchase are outlined in Table 1. Animals Male mice (c57bl/6, 12 week-old) were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Mice experimentations were approved by the CABIMER Animal Com-
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mittee and performed in accordance with the Spanish law on animal use RD 53/2013. Table 1.
List of reagents and materials used in this study.
Product
Vendor
Catalog Number
50 x 9 mm Petri dishes
BD Falcon
351006
Affi-Gel blue beads
Bio-Rad
153-7301
Bovine Serum Albumin
Sigma-Aldrich
A3294
CalPhos mammalian transfection kit
ClonTech
631312
CMRL-1066
Cellgro
99-663-CV
Collagenase
Roche
C9263
DAKO fluorescent mounting medium
Dako
S3023
DAPI
Sigma-Aldrich
32670
Donkey serum
Sigma-Aldrich
D9663
Fetal Bovine Serum
Sigma-Aldrich
F7524
Formaldehyde
Panreac AppliChem
252931
Gentamycin
Sigma-Aldrich
G1397
Glutamine
Sigma-Aldrich
G7513
Hanks Balanced Salt Solution 1X
Gibco
14170088
HEPES
Gibco
15630-056
HistoGel
Thermo Scientific
R904012
micro-Plate 96 welllibiTreat
IBIDI
89626
Millex-HV filter 0.45 µm
Merck Millipore
SLHV033RS
PBS
Sigma-Aldrich
P5368
Penicillin/Streptomycin
Sigma-Aldrich
P4333
Polystyrene Round-bottom tube
BD Falcon
352058
RPMI-1640
Sigma-Aldrich
R0883
Sodium pyruvate
Sigma-Aldrich
S8636
SuperFrost Plus slides
Menzel-Glaser
J1800AMNZ
Trypsin-EDTA 10 X
Gibco
15400054
β-mercaptoethanol
Gibco
31350-10
Islets Procuration and Culture Mice were sacrificed by cervical dislocation and pancreatic islets were isolated using the collagenase digestion procedure with subsequent handpicking as previously described [34]. Prior to culture islets were washed with Phosphate Buffered Saline (PBS) containing 100 U/ml penicillin and 100 µg/ml streptomycin to minimize post-isolation contami-
438 Current Gene Therapy, 2015, Vol. 15, No. 4
nations. Subsequently islets were cultured in mouse Complete Media (CM) comprised of RPMI 1640 supplemented with 10 % FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 50 µM β mercaptoethanol and 10 mM HEPES. Isolated human islets were either kindly provided by the Cell Isolation and Transplantation Centre (Geneva, Switzerland) or purchased from Tebu-bio (Le Perray En Yvelines, France). Characteristics of human islet preparations are included in Table S1. Islets were cultured in human Complete Media (CM) composed of CMRL-1066 supplemented with 10 % FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 100 µg/ml gentamycin. Lentivirus Generation We opted for the dual-promoter lentivirus, pHRSIN DUAL-GFP also known as pHRSIN-CSGWdINotIpUbEm (kindly supplied by Dr. Pintor-Toro, CABIMER, Spain) to conduct our studies [35]. This vector allows the cloning and expression of a Gene Of Interest (GOI) under the control of the SFFV promoter while the constitutive Ubiquitin (Ubi) promoter regulates expression of the reporter GFP. Lentivirus amplification and purification was performed by seeding 5 × 106 Hek293T cells into a 100 mm Petri dish and subsequently transfected 24 hours later with: 1) 15 µg of vector, 2) 10 µg the HIV packaging plasmids pCMVDR8.91 and 3) 5 µg of HIV packaging plasmids pVSVG (also known as pMDG). Transient DNA transfection was performed using the CalPhos transfection mammalian kit according to the manufacturer’s recommendations. Viral particles were harvested 72 hours posttransfection, purified using a 0.45 µm Millex-HV filter, and concentrated by ultracentrifugation in an OptimaTM L-100K ultracentrifuge at 87300 x g for 90 minutes at 4º C in a swinging bucket rotor SW-28 (Beckman-Coulter, Spain). Virus particles were resuspended in serum-free DMEM (Invitrogen), distributed in aliquots, snapped frozen in liquid nitrogen, and stored at −80 °C. Viral titer was estimated by transducing Hek293T cells with increasing amounts of pHRSIN DUAL-GFP followed by flow cytometry (FACSCalibur, BD Biosciences, Spain) analysis to determine the PFU/ml based on GFP emission. Live Imaging and Flow Cytometry An ImageXpress Micro System (Molecular Devices) was used to monitor GFP fluorescence in living islets. To this end, approximately 20 transduced human or mouse islets were seeded in µ-Plate 96 welllibiTreat plate in a final volume of 200 µl of CM. Islets were cultured for 4 days at 37º C and images (fluorescence or phase contrast) were automatically captured daily and processed using the MetaXpress software. In parallel, islet transduction efficiency was estimated by flow cytometry. In brief, approximately 20 islets were transferred into 5 ml polystyrene Round-bottom tube in a final volume of 50 µl of CM. Islets were disaggregated using 1 X trypsinization for 5 minutes at 37º C and subsequently centrifuged at 200 x g for 5 minutes. Cells were resuspended in 300 µl of PBS and the number of GFP positive cells was estimated as compared to non-infected cells.
Jiménez-Moreno et al.
Islet Processing and Immunocytochemistry Islet embedding was performed according to the protocol developed by Cozar-Castellano et al. [36]. In brief, approximately 200 human or murine islets were fixed in 10 % formaldehyde at room temperature for 48 hours. Islets were then washed three times in distilled water prior to adding warm (70° C) HistoGel containing 100 µl of 150-300 µm diameter Affi-Gel blue beads. After cooling, Histogel containing the islet-bead mixture was embedded in paraffin following the standard procedures of the CABIMER Histology Core Facility. Paraffin blocks were sectioned (5 µm thickness) using a microtome Leica RM 2255 (Leica Microsystems, Spain). Sections were mounted on SuperFrost Plus slides. After every 10 sections, a slide was stained with hematoxylin-eosin to confirm islet integrity and presence of islets. Sections were deparaffinized/rehydrated at 60˚ C for 20 minutes followed by immersion in decreasing concentrations of ethanol (Xylene 5 minutes/2 x; Ethanol 100 % 1 minute/2 x; Ethanol 96 % 1 minute; Ethanol 80 % 1 minute; Ethanol 70 % 1 minute; Distilled water 2 minutes/2 x). After deparaffinization and rehydration, sections were subjected to heat-induced antigen retrieval using 10 mM sodium citrate buffer (pH 6.0) in the microwave in 3 cycles of 3 minutes at 800 W avoiding boiling of the buffer, with 2 minutes at room temperature between heating cycles. Samples were cold down in the same solution for 20 minutes at room temperature. After washing with PBS, samples were incubated in PBS + 0.5 % Triton X-100 and then washed again with PBS. Blocking was performed with PBS + 0.2 % Triton X-100 containing 1 % Bovine Serum Albumin (BSA) and 3 % Donkey serum for 1 hour at room temperature. Primary antibodies (Table 2) at the indicated dilutions were added in PBS + 0.1 % Triton X-100 containing 1 % BSA and 3 % Donkey serum and incubated overnight at 4˚ C in a dark humid chamber. Subsequently, sections were washed with PBS for 5 minutes, PBS + 0.2 % Triton X-100 for 5 minutes and PBS for 5 minutes. Samples were then incubated with secondary antibodies (Table 2) diluted in PBS + 0.2 % Triton X-100 containing 0.1 % BSA for 1 hour at room temperature in a dark humid chamber. Nuclear counterstaining was performed by DAPI staining diluted 1:1000 in PBS for 5 minutes at room temperature. Finally, samples were washed three times with PBS for 5 minutes each and sections were mounted using DAKO fluorescent mounting medium. Wide-field immunofluorescence microscopy was performed using a Leica microscope (AF6000) (Leica, England). Images were taken at 40X magnification. Confocal images were acquired using a Leica confocal microscope (TCS SP5). The images were scanned under a HCX PL APO lambda blue 63X/ 1.4 OIL objective. To analyze the whole section, each sample was analyzed using a spatial series through the Z axis. Each spatial series was composed of approximately 5-7 optical sections with a size of 0.8 µm and a 3D projection of each zstack was performed using three sections. Viability and Functional Assay Islet viability subsequent to transduction was assessed in groups of 35 islets using the Cell Proliferation Kit I (MTT) according to the manufacturer´s recommendations (Roche, Spain). Optical density was determined at 550 nm with a reference wavelength of 650 nm using a Varioskan Flash
An Efficient Protocol for Intact Islet Infection
Table 2.
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List of antibodies used in this study.
Antibody
Dilution
Vendor
Catalog Number
Anti-GFP
1:200
Abcam
Ab6673
Anti-insulin (H-86)
1:500
Santa Cruz
SC9168
Anti-insulin
1:500
Sigma-Aldrich
I2018
Anti-glucagon
1:150
Sigma-Aldrich
G2654
Anti-glucagon
1:200
Cell Signaling
2760S
Anti-cleaved caspase-3
1:150
Cell Signaling
9661
Alexa fluor 488 donkey anti-goat
1:800
Invitrogen
A11055
Alexa fluor 555 donkey anti-mouse
1:800
Life technologies
A31579
Alexa fluor 647 donkey anti-rabbit
1:800
Life technologies
A31573
spectrophotometer (Thermo Scientific, Spain). In parallel, glucose stimulated insulin secretion was performed to assess the functional integrity of islets. Groups of 10 islets were washed in 500 µL of Krebs-Ringer bicarbonate-HEPES buffer (KRBH) (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3 , 10 mM HEPES, 0.1% BSA) and pre-incubated at 37° C for 45 minutes in 300 µl of the same buffer. Islets were then centrifuged and KRBH buffer was discarded. Subsequently, fresh KRBH supplemented with 2.5 mM glucose was added and islets were incubated for 30 minutes. Next, buffer was harvested (basal insulin secretion) and 500 µL of KRBH supplemented with 16.8 mM glucose was added. Islets were incubated for an additional 30 minutes at 37° C and then buffer was harvested (induced insulin secretion). Insulin levels were measured using a mouse or human insulin enzyme immunoassay kit (Mercodia AB, Spain) according to the manufacturer´s instructions. Stimulation index was expressed as the ratio of insulin levels at 16.8 mM glucose divided by insulin levels at 2.5 mM glucose. Statistical Analysis Results are expressed as the mean± SEM. Statistical differences were estimated by two-tailed unpaired student’s ttest. *Indicates statistical significance, p value
